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1.
PLoS Biol ; 18(3): e3000631, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32150533

RESUMO

Endocytic recycling of internalized transmembrane proteins is essential for many important physiological processes. Recent studies have revealed that retromer-related Sorting Nexin family (SNX)-Bin/Amphiphysin/Rvs (BAR) proteins can directly recognize cargoes like cation-independent mannose 6-phosphate receptor (CI-MPR) and Insulin-like growth factor 1 receptor (IGF1R); however, it remains poorly understood how SNX-BARs select specific cargo proteins and whether they recognize additional ligands. Here, we discovered that the binding between SNX-BARs and CI-MPR or IGF1R is mediated by the phox-homology (PX) domain of SNX5 or SNX6 and a bipartite motif, termed SNX-BAR-binding motif (SBM), in the cargoes. Using this motif, we identified over 70 putative SNX-BAR ligands, many of which play critical roles in apoptosis, cell adhesion, signal transduction, or metabolite homeostasis. Remarkably, SNX-BARs could cooperate with both SNX27 and retromer in the recycling of ligands encompassing the SBM, PDZ-binding motif, or both motifs. Overall, our studies establish that SNX-BARs function as a direct cargo-selecting module for a large set of transmembrane proteins transiting the endosome, in addition to their roles in phospholipid recognition and biogenesis of tubular structures.


Assuntos
Proteoma/metabolismo , Receptor IGF Tipo 2/metabolismo , Nexinas de Classificação/química , Nexinas de Classificação/metabolismo , Motivos de Aminoácidos , Sítios de Ligação , Transporte Biológico , Simulação por Computador , Técnicas de Inativação de Genes , Células HeLa , Humanos , Domínios Proteicos , Proteoma/química , Receptor IGF Tipo 2/química , Semaforinas/metabolismo , Nexinas de Classificação/genética
2.
Nat Commun ; 11(1): 806, 2020 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-32041945

RESUMO

The co-evolution and co-existence of viral pathogens with their hosts for millions of years is reflected in dynamic virus-host protein-protein interactions (PPIs) that are intrinsic to the spread of infections. Here, we investigate the system-wide dynamics of protein complexes throughout infection with the herpesvirus, human cytomegalovirus (HCMV). Integrating thermal shift assays and mass spectrometry quantification with virology and microscopy, we monitor the temporal formation and dissociation of hundreds of functional protein complexes and the dynamics of host-host, virus-host, and virus-virus PPIs. We establish pro-viral roles for cellular protein complexes and translocating proteins. We show the HCMV receptor integrin beta 1 dissociates from extracellular matrix proteins, becoming internalized with CD63, which is necessary for virus production. Moreover, this approach facilitates characterization of essential viral proteins, such as pUL52. This study of temporal protein complex dynamics provides insights into mechanisms of HCMV infection and a resource for biological and therapeutic studies.


Assuntos
Infecções por Citomegalovirus/metabolismo , Infecções por Citomegalovirus/virologia , Citomegalovirus/fisiologia , Linhagem Celular , Citomegalovirus/metabolismo , Ácidos Graxos/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Fatores Imunológicos/metabolismo , Integrina beta1/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Agregação Patológica de Proteínas , Biossíntese de Proteínas , Mapas de Interação de Proteínas , Estabilidade Proteica , Proteômica , Receptor IGF Tipo 2/metabolismo , Transdução de Sinais , Tetraspanina 30/metabolismo , Proteínas Virais/metabolismo , Replicação Viral
3.
Nucleic Acids Res ; 48(3): 1372-1391, 2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31840180

RESUMO

Release of phosphorothioate antisense oligonucleotides (PS-ASOs) from late endosomes (LEs) is a rate-limiting step and a poorly defined process for productive intracellular ASO drug delivery. Here, we examined the role of Golgi-endosome transport, specifically M6PR shuttling mediated by GCC2, in PS-ASO trafficking and activity. We found that reduction in cellular levels of GCC2 or M6PR impaired PS-ASO release from endosomes and decreased PS-ASO activity in human cells. GCC2 relocated to LEs upon PS-ASO treatment, and M6PR also co-localized with PS-ASOs in LEs or on LE membranes. These proteins act through the same pathway to influence PS-ASO activity, with GCC2 action preceding that of M6PR. Our data indicate that M6PR binds PS-ASOs and facilitates their vesicular escape. The co-localization of M6PR and of GCC2 with ASOs is influenced by the PS modifications, which have been shown to enhance the affinity of ASOs for proteins, suggesting that localization of these proteins to LEs is mediated by ASO-protein interactions. Reduction of M6PR levels also decreased PS-ASO activity in mouse cells and in livers of mice treated subcutaneously with PS-ASO, indicating a conserved mechanism. Together, these results demonstrate that the transport machinery between LE and Golgi facilitates PS-ASO release.


Assuntos
Endossomos/genética , Proteínas da Matriz do Complexo de Golgi/genética , Oligonucleotídeos Antissenso/genética , Receptor IGF Tipo 2/genética , Animais , Endocitose/genética , Endossomos/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Proteínas da Matriz do Complexo de Golgi/metabolismo , Células HeLa , Humanos , Camundongos , Oligonucleotídeos Fosforotioatos/genética , Transporte Proteico/genética , Receptor IGF Tipo 2/metabolismo
4.
Environ Toxicol ; 35(2): 145-151, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31714667

RESUMO

The use of herbs as alternative cardiovascular disease treatment has attracted a great deal of attention owing to their lower toxicity. Whether Carthamus tinctorius extract prevent cardiomyoblast cell hypertrophy remains unclear. The present study was performed to investigate the effect of C tinctorius extract (CTF) on rat cardiomyoblast cell H9c2 and the possible molecular mechanisms. H9c2 cells were treated with lipopolysaccharide (LPS; 2 µg/mL) for 12 hours, subsequently treated with CTF (1-25 µg/mL) The incubation continued for another 24 hours, and the cells were analyzed with actin staining assay, western blot analysis, and siRNA transfection assays. In the present study, the increased cell size induced by LPS was significantly decreased by pretreating at a concentration of 1-25 µg/mL CTF. It was found that CTF could inhibit cardiac hypertrophy induced by LPS and decrease hypertrophic proteins calcineurin, p-GATA-4, GATA-4, atrial natriuretic peptide, and B-type natriuretic peptide levels in H9c2 cells. Additionally, LPS-induced insulin-like growth factor-II receptor (IGF-IIR) hypertrophy pathway was downregulated by CTF. Moreover, IGF-IR siRNA or inhibitors both reversed the CTF effects, confirming that CTF activates IGF-1R to prevent LPS-induced H9c2 cardiomyoblast cell hypertrophy. The current findings indicate that CTF activates IGF-IR to inhibit IGF-IIR signaling pathway which resulted in reducing H9c2 cardiomyoblast cell hypertrophy induced by LPS.


Assuntos
Carthamus tinctorius/química , Lipopolissacarídeos/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Extratos Vegetais/farmacologia , Receptor IGF Tipo 1/metabolismo , Receptor IGF Tipo 2/metabolismo , Animais , Cardiomegalia/prevenção & controle , Tamanho Celular , Relação Dose-Resposta a Droga , Técnicas de Silenciamento de Genes , Miócitos Cardíacos/metabolismo , Extratos Vegetais/isolamento & purificação , Ratos , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 2/genética , Transdução de Sinais
5.
Nat Cell Biol ; 21(10): 1219-1233, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31576058

RESUMO

Protein trafficking requires coat complexes that couple recognition of sorting motifs in transmembrane cargoes with biogenesis of transport carriers. The mechanisms of cargo transport through the endosomal network are poorly understood. Here, we identify a sorting motif for endosomal recycling of cargoes, including the cation-independent mannose-6-phosphate receptor and semaphorin 4C, by the membrane tubulating BAR domain-containing sorting nexins SNX5 and SNX6. Crystal structures establish that this motif folds into a ß-hairpin, which binds a site in the SNX5/SNX6 phox homology domains. Over sixty cargoes share this motif and require SNX5/SNX6 for their recycling. These include cargoes involved in neuronal migration and a Drosophila snx6 mutant displays defects in axonal guidance. These studies identify a sorting motif and provide molecular insight into an evolutionary conserved coat complex, the 'Endosomal SNX-BAR sorting complex for promoting exit 1' (ESCPE-1), which couples sorting motif recognition to the BAR-domain-mediated biogenesis of cargo-enriched tubulo-vesicular transport carriers.


Assuntos
Endossomos/metabolismo , Proteínas de Membrana/metabolismo , Nexinas de Classificação/química , Nexinas de Classificação/metabolismo , Motivos de Aminoácidos/genética , Animais , Drosophila melanogaster , Células HEK293 , Células HeLa , Humanos , Domínios Proteicos/genética , Transporte Proteico/fisiologia , Receptor IGF Tipo 2/química , Receptor IGF Tipo 2/metabolismo , Semaforinas/genética , Semaforinas/metabolismo , Nexinas de Classificação/genética
6.
Int J Mol Sci ; 20(17)2019 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-31480672

RESUMO

Cardiovascular diseases have a high prevalence worldwide and constitute the leading causes of mortality. Recently, malfunctioning of ß-catenin signaling has been addressed in hypertensive heart condition. Ang-II is an important mediator of cardiovascular remodeling processes which not only regulates blood pressure but also leads to pathological cardiac changes. However, the contribution of Ang-II/ß-catenin axis in hypertrophied hearts is ill-defined. Employing in vitro H9c2 cells and in vivo spontaneously hypertensive rats (SHR) cardiac tissue samples, western blot analysis, luciferase assays, nuclear-cytosolic protein extracts, and immunoprecipitation assays, we found that under hypertensive condition ß-catenin gets abnormally induced that co-activated LEF1 and lead to cardiac hypertrophy changes by up-regulating the IGF-IIR signaling pathway. We identified putative LEF1 consensus binding site on IGF-IIR promoter that could be regulated by ß-catenin/LEF1 which in turn modulate the expression of cardiac hypertrophy agents. This study suggested that suppression of ß-catenin expression under hypertensive condition could be exploited as a clinical strategy for cardiac pathological remodeling processes.


Assuntos
Cardiomegalia/induzido quimicamente , Cardiomegalia/metabolismo , Receptor IGF Tipo 2/metabolismo , Transdução de Sinais , beta Catenina/metabolismo , Angiotensina II , Animais , Biomarcadores/metabolismo , Cardiomegalia/patologia , Núcleo Celular/metabolismo , Fator de Transcrição GATA4/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Miócitos Cardíacos/metabolismo , Fatores de Transcrição NFATC/metabolismo , Regiões Promotoras Genéticas/genética , Proteína Quinase C-alfa/metabolismo , Ratos Endogâmicos SHR
7.
Int J Mol Sci ; 20(11)2019 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-31181759

RESUMO

The aim of the present work is the development of highly efficient targeting molecules to specifically address mesoporous silica nanoparticles (MSNs) designed for the photodynamic therapy (PDT) of prostate cancer. We chose the strategy to develop a novel compound that allows the improvement of the targeting of the cation-independent mannose 6-phosphate receptor, which is overexpressed in prostate cancer. This original sugar, a dimannoside-carboxylate (M6C-Man) grafted on the surface of MSN for PDT applications, leads to a higher endocytosis and thus increases the efficacy of MSNs.


Assuntos
Fotoquimioterapia/métodos , Neoplasias da Próstata/metabolismo , Receptor IGF Tipo 2/metabolismo , Linhagem Celular Tumoral , Endocitose , Humanos , Masculino , Manosefosfatos/administração & dosagem , Manosefosfatos/química , Manosefosfatos/farmacologia , Nanopartículas/administração & dosagem , Nanopartículas/química , Nanopartículas/metabolismo , Dióxido de Silício/química
8.
Am J Physiol Cell Physiol ; 317(2): C235-C243, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31116582

RESUMO

Doxorubicin (DOX) is an anthracycline antibiotic commonly employed for the treatment of various cancers. However, its therapeutic uses are hampered by side effects associated with cumulative doses during the course of treatment. Whereas deregulation of autophagy in the myocardium has been involved in a variety of cardiovascular diseases, the role of autophagy in DOX-induced cardiomyopathy remains debated. Our earlier studies have shown that DOX treatment in a rat animal model leads to increased expression of the novel stress-inducible protein insulin-like growth factor II receptor-α (IGF-IIRα) in cardiac tissues, which exacerbated the cardiac injury by enhancing oxidative stress and p53-mediated mitochondria-dependent cardiac apoptosis. Through this study, we investigated the contribution of IGF-IIRα to dysregulation of autophagy in heart using both in vitro H9c2 cells (DOX treated, 1 µM) and in vivo transgenic rat models (DOX treated, 5 mg/kg ip for 6 wk) overexpressing IGF-IIRα specifically in the heart. We found that IGF-IIRα primarily localized to mitochondria, causing increased mitochondrial oxidative stress that was severely aggravated by DOX treatment. This was accompanied by a significant perturbation in mitochondrial membrane potential and increased leakage of cytochrome c, causing increased cleaved caspase-3 activity. There were significant alterations in phosphorylated AMP-activated protein kinase (p-AMPK), phosphorylated Unc-51 like kinase-1 (p-ULK1), PARKIN, PTEN-induced kinase 1 (PINK1), microtubule-associated protein 1 light chain 3 (LC3), and p62 proteins, which were more severely disrupted under the combined effect of IGF-IIRα overexpression plus DOX. Finally, LysoTracker Red staining showed that IGF-IIRα overexpression causes lysosomal impairment, which was rescued by rapamycin treatment. Taken together, we found that IGF-IIRα leads to mitochondrial oxidative stress, decreased antioxidant levels, disrupted mitochondrial membrane potential, and perturbed mitochondrial autophagy contributing to DOX-induced cardiomyopathy.


Assuntos
Antibióticos Antineoplásicos/toxicidade , Doxorrubicina/toxicidade , Cardiopatias/induzido quimicamente , Mitocôndrias Cardíacas/efeitos dos fármacos , Mitofagia/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Receptor IGF Tipo 2/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Cardiotoxicidade , Linhagem Celular , Cardiopatias/genética , Cardiopatias/metabolismo , Cardiopatias/patologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Ratos Sprague-Dawley , Ratos Transgênicos , Receptor IGF Tipo 2/genética , Transdução de Sinais/efeitos dos fármacos
9.
Hum Gene Ther ; 30(7): 855-864, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30803275

RESUMO

Gene therapy for Pompe disease with adeno-associated virus (AAV) vectors has advanced into early phase clinical trials; however, the paucity of cation-independent mannose-6-phosphate receptor (CI-MPR) in skeletal muscle, where it is needed to take up acid α-glucosidase (GAA), has impeded the efficacy of Pompe disease gene therapy. Long-acting selective ß2 receptor agonists previously enhanced the CI-MPR expression in muscle. In this study we have evaluated the selective ß2 agonist salmeterol in GAA knockout mice in combination with an AAV vector expressing human GAA specifically in the liver. Quadriceps glycogen content was significantly decreased by administration of the AAV vector with salmeterol, in comparison with the AAV vector alone (p < 0.01). Importantly, glycogen content of the quadriceps was reduced to its lowest level by the combination of AAV vector and salmeterol administration. Rotarod testing revealed significant improvement following treatment, in comparison with untreated mice, and salmeterol improved wirehang performance. Salmeterol treatment decreased abnormalities of autophagy in the quadriceps, as shown be lower LC3 and p62. Vector administration reduced the abnormal vacuolization and accumulation of nuclei in skeletal muscle. Thus, salmeterol could be further developed as adjunctive therapy to improve the efficacy of liver depot gene therapy for Pompe disease.


Assuntos
Terapia Genética , Doença de Depósito de Glicogênio Tipo II/genética , Doença de Depósito de Glicogênio Tipo II/terapia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/metabolismo , Xinafoato de Salmeterol/farmacologia , Animais , Dependovirus/genética , Modelos Animais de Doenças , Ativação Enzimática , Expressão Gênica , Técnicas de Transferência de Genes , Terapia Genética/métodos , Vetores Genéticos/genética , Doença de Depósito de Glicogênio Tipo II/metabolismo , Camundongos , Camundongos Knockout , Receptor IGF Tipo 2/genética , Receptor IGF Tipo 2/metabolismo , Transdução Genética
10.
Bioessays ; 41(3): e1800146, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30706963

RESUMO

It has long been believed that membrane proteins present in degradative compartments such as endolysosomes or vacuoles would be destined for destruction. Now however, it appears that mechanisms and machinery exist in simple eukaryotes such as yeast and more complex organisms such as mammals that can rescue potentially "doomed" membrane proteins by retrieving them from these "late" compartments and recycling them back to the Golgi complex. In yeast, a sorting nexin dimer containing Snx4p can recognize and retrieve the Atg27p membrane protein while in mammals, the AP5 complex (with SPG11 and SPG15) directs the recycling of Golgi-localized proteins along with the cation-independent mannose 6-phosphate receptor (CIMPR). Although the respective machinery is different, there is much commonality between yeast and mammals regarding the mechanisms of retrieval and the physiological importance of these late recycling pathways.


Assuntos
Endossomos/metabolismo , Lisossomos/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico/fisiologia , Vacúolos/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Autofagia , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Transporte/metabolismo , Complexo de Golgi/metabolismo , Mamíferos/metabolismo , Proteínas/metabolismo , Receptor IGF Tipo 2/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Nexinas de Classificação/metabolismo
11.
Oncol Rep ; 41(4): 2299-2310, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30720132

RESUMO

Hepatocellular carcinoma (HCC) is one of the most common human malignancies, the incidence of which is growing worldwide. The prognosis of HCC is very poor and it is often accompanied by a high rate of recurrence. Conventional chemotherapeutic approaches are largely inefficient. In order to develop novel effective methods for the early detection and prognosis of HCC, novel markers and therapeutic targets are urgently required. The present study focused on the effects of the expression of the tumor suppressor gene insulin­like growth factor­2 receptor (IGF2R) on patient survival and tumor recurrence in patients with HCC; this study paid specific attention to the influence of transarterial chemoembolization (TACE) prior to surgery. The mRNA expression levels of IGF2R were measured in primary human HCC and corresponding non­neoplastic tumor­surrounding tissue (TST) by reverse transcription­polymerase chain reaction (RT­PCR) (n=92). Subsequently, the associations between IGF2R expression and clinicopathological parameters, outcomes of HCC and TACE pretreatment prior to surgery were determined. Furthermore, the effects of the IGF2R gene polymorphisms rs629849 and rs642588 on susceptibility and on clinicopathological features of HCC were investigated. RT­PCR demonstrated that the mRNA expression levels of IGF2R were downregulated in HCC compared with in TST samples (P=0.004), which was associated with a worse recurrence­free survival of patients with HCC (P=0.002) and a lower occurrence of cirrhosis (P=0.05). TACE­pretreated patients with HCC (n=26) exhibited significantly higher IGF2R mRNA expression in tumor tissues (P=0.019). In addition, significantly more patients with HCC in the TACE­pretreated group exhibited upregulated IGF2R mRNA expression compared with in the non­treated patients (P=0.032). The IGF2R SNPs rs629849 and rs642588 were not significantly associated with HCC risk, whereas a homozygous IGF2R rs629849 GG genotype was associated with a significantly elevated risk of non­viral liver cirrhosis (P=0.05). In conclusion, these data suggested an important role for IGF2R expression in HCC, particularly with regards to TACE treatment prior to surgery.


Assuntos
Carcinoma Hepatocelular/patologia , Quimioembolização Terapêutica , Cirrose Hepática/patologia , Neoplasias Hepáticas/patologia , Recidiva Local de Neoplasia/diagnóstico , Receptor IGF Tipo 2/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/terapia , Estudos de Casos e Controles , Intervalo Livre de Doença , Feminino , Seguimentos , Predisposição Genética para Doença , Humanos , Estimativa de Kaplan-Meier , Cirrose Hepática/epidemiologia , Cirrose Hepática/genética , Cirrose Hepática/terapia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/terapia , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/prevenção & controle , Polimorfismo de Nucleotídeo Único , Prognóstico , Receptor IGF Tipo 2/genética
12.
Cell Biol Int ; 43(11): 1234-1244, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30791178

RESUMO

GCC88 is a golgin coiled-coil protein at the trans-Golgi (TGN) that functions as a tethering factor for the endosome-derived retrograde transport vesicles. Here, we demonstrate that GCC88 is required for the endosome-to-TGN retrograde transport of the cation-independent mannose 6-phosphate receptor (CI-M6PR). The knockout of GCC88 perturbs the retrieval of CI-M6PR and decreases its cellular level at the steady state, which causes the improper processing of newly synthesized cathepsin-D, a lysosomal hydrolase dependent on CI-M6PR for its delivery to lysosomes. At the whole cell level, the knockout of GCC88 reduces the lysosomal proteolytic capacity but does not impair of the efficiency of autophagy within these cells.


Assuntos
Catepsina D/metabolismo , Endossomos/metabolismo , Proteínas da Matriz do Complexo de Golgi/fisiologia , Lisossomos/metabolismo , Receptor IGF Tipo 2/metabolismo , Rede trans-Golgi/metabolismo , Autofagia , Proteínas da Matriz do Complexo de Golgi/genética , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Transporte Proteico
13.
PLoS One ; 14(2): e0211908, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30735526

RESUMO

Three artificially selected duck populations (AS), higher lean meat ratios (LTPD), higher fat ratios (FTPD) and higher quality meat (CMD), have been developed in China, providing excellent populations for investigation of artificial selection effects. However, the genetic signatures of artificial selection are unclear. In this study, we sequenced the genome sequences of these three artificially selected populations and their ancestral population (mallard, M). We then compared the genome sequences between AS and M and between LTPD and FTPD using integrated strategies such as anchoring scaffolds to pseudo-chromosomes, mutation detection, selective screening, GO analysis, qRT-PCR, and protein multiple sequences alignment to uncover genetic signatures of selection. We anchored duck scaffolds to pseudo-chromosomes and obtained 28 pseudo-chromosomes, accounting for 84% of duck genome in length. Totally 78 and 99 genes were found to be under selection between AS and M and between LTPD and FTPD. Genes under selection between AS and M mainly involved in pigmentation and heart rates, while genes under selection between LTPD and FTPD involved in muscle development and fat deposition. A heart rate regulator (HCN1), the strongest selected gene between AS and M, harbored a GC deletion in AS and displayed higher mRNA expression level in M than in AS. IGF2R, a regulator of skeletal muscle mass, was found to be under selection between FTPD and LTPD. We also found two nonsynonymous substitutions in IGF2R, which might lead to higher IGF2R mRNA expression level in FTPD than LTPD, indicating the two nonsynonymous substitutions might play a key role for the regulation of duck skeletal muscle mass. Taken together, these results of this study provide valuable insight for the genetic basis of duck artificial selection.


Assuntos
Proteínas Aviárias/genética , Composição Corporal/genética , Patos/genética , Genoma , Carne , Seleção Genética , Tecido Adiposo/metabolismo , Sequência de Aminoácidos , Animais , Proteínas Aviárias/metabolismo , Cruzamento/métodos , Mapeamento Cromossômico , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Frequência Cardíaca/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Masculino , Anotação de Sequência Molecular , Músculo Esquelético/metabolismo , Pigmentação/genética , Receptor IGF Tipo 2/genética , Receptor IGF Tipo 2/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
14.
J Leukoc Biol ; 105(3): 519-530, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30657605

RESUMO

The plasminogen system is harnessed in a wide variety of physiological processes, such as fibrinolysis, cell migration, or efferocytosis; and accordingly, it is essential upon inflammation, tissue remodeling, wound healing, and for homeostatic maintenance in general. Previously, we identified a plasminogen receptor in the mannose 6-phosphate/insulin-like growth factor 2 receptor (M6P/IGF2R, CD222). Here, we demonstrate by means of genetic knockdown, knockout, and rescue approaches combined with functional studies that M6P/IGF2R is up-regulated on the surface of macrophages, recognizes plasminogen exposed on the surface of apoptotic cells, and mediates plasminogen-induced efferocytosis. The level of uptake of plasminogen-coated apoptotic cells inversely correlates with the TNF-α production by phagocytes indicating tissue clearance without inflammation by this mechanism. Our results reveal an up-to-now undetermined function of M6P/IGF2R in clearance of apoptotic cells, which is crucial for tissue homeostasis.


Assuntos
Fagocitose/efeitos dos fármacos , Plasminogênio/farmacologia , Receptor IGF Tipo 2/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Técnicas de Inativação de Genes , Humanos , Células Jurkat , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Células THP-1 , Fator de Necrose Tumoral alfa/metabolismo
15.
PLoS One ; 14(1): e0207836, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30657762

RESUMO

Sanfilippo syndrome type B (Sanfilippo B; Mucopolysaccharidosis type IIIB) occurs due to genetic deficiency of lysosomal alpha-N-acetylglucosaminidase (NAGLU) and subsequent lysosomal accumulation of heparan sulfate (HS), which coincides with devastating neurodegenerative disease. Because NAGLU expressed in Chinese hamster ovary cells is not mannose-6-phosphorylated, we developed an insulin-like growth factor 2 (IGF2)-tagged NAGLU molecule (BMN 250; tralesinidase alfa) that binds avidly to the IGF2 / cation-independent mannose 6-phosphate receptor (CI-MPR) for glycosylation independent lysosomal targeting. BMN 250 is currently being developed as an investigational enzyme replacement therapy for Sanfilippo B. Here we distinguish two cellular uptake mechanisms by which BMN 250 is targeted to lysosomes. In normal rodent-derived neurons and astrocytes, the majority of BMN250 uptake over 24 hours reaches saturation, which can be competitively inhibited with IGF2, suggestive of CI-MPR-mediated uptake. Kuptake, defined as the concentration of enzyme at half-maximal uptake, is 5 nM and 3 nM in neurons and astrocytes, with a maximal uptake capacity (Vmax) corresponding to 764 nmol/hr/mg and 5380 nmol/hr/mg, respectively. Similar to neurons and astrocytes, BMN 250 uptake in Sanfilippo B patient fibroblasts is predominantly CI-MPR-mediated, resulting in augmentation of NAGLU activity with doses of enzyme that fall well below the Kuptake (5 nM), which are sufficient to prevent HS accumulation. In contrast, uptake of the untagged recombinant human NAGLU (rhNAGLU) enzyme in neurons, astrocytes and fibroblasts is negligible at the same doses tested. In microglia, receptor-independent uptake, defined as enzyme uptake resistant to competition with excess IGF2, results in appreciable lysosomal delivery of BMN 250 and rhNAGLU (Vmax = 12,336 nmol/hr/mg and 5469 nmol/hr/mg, respectively). These results suggest that while receptor-independent mechanisms exist for lysosomal targeting of rhNAGLU in microglia, BMN 250, by its IGF2 tag moiety, confers increased CI-MPR-mediated lysosomal targeting to neurons and astrocytes, two additional critical cell types of Sanfilippo B disease pathogenesis.


Assuntos
Acetilglucosaminidase/metabolismo , Endocitose , Fator de Crescimento Insulin-Like II/uso terapêutico , Mucopolissacaridose III/tratamento farmacológico , Mucopolissacaridose III/patologia , Proteínas Recombinantes de Fusão/uso terapêutico , Acetilglucosaminidase/farmacocinética , Acetilglucosaminidase/uso terapêutico , Animais , Astrócitos/metabolismo , Axônios/metabolismo , Cátions , Fibroblastos/metabolismo , Heparitina Sulfato/metabolismo , Hipocampo/patologia , Humanos , Fator de Crescimento Insulin-Like II/farmacocinética , Lisossomos/enzimologia , Microglia/metabolismo , Ratos , Receptor IGF Tipo 2/metabolismo , Proteínas Recombinantes de Fusão/farmacocinética
16.
Eur J Pharm Sci ; 127: 161-174, 2019 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-30366077

RESUMO

The Cancer-Immunity Cycle is a series of anticancer immune responses initiated and allowed to proceed and expand iteratively. Paclitaxel (PTX) is a classic chemotherapeutic agent, which could induce immunogenic cell death (ICD) to trigger the Cancer-Immunity Cycle. However, the Cycle is severely impaired by tumor cell immunosuppression of host T cell antitumor activity through the programmed cell death receptor 1 (PD-1) and programmed cell death ligand 1 (PD-L1) (PD-1/PD-L1) immune checkpoint pathway. Here, we demonstrated that PTX mediated the Cancer-Immunity Cycle could be enhanced by PD-L1 knockdown (KD) and followed mTOR pathway inhibition in tumor cells. PD-L1 siRNA (siP) and the hydrophobic chemotherapy drug PTX were co-delivered with a rationally designed hybrid micelle (HM). We showed clear evidence that the HM-siP/PTX is capable of delivering siP and PTX simultaneously to the B16F10 cells both in vitro and in vivo. We demonstrated that HM-PTX/siP reduced the expression of PD-L1 and p-S6K (a marker of mTOR pathway activation) both in vitro and in melanoma-bearing mice and attenuated synergistically tumor growth by chemical toxicity, promoting cytotoxic T-cell immunity and suppressing the mTOR pathway.


Assuntos
Antineoplásicos Fitogênicos/administração & dosagem , Antígeno B7-H1/genética , Melanoma Experimental/terapia , Micelas , Paclitaxel/administração & dosagem , RNA Interferente Pequeno/administração & dosagem , Animais , Antineoplásicos Fitogênicos/farmacocinética , Antígeno B7-H1/metabolismo , Calreticulina/metabolismo , Linhagem Celular Tumoral , Melanoma Experimental/imunologia , Melanoma Experimental/metabolismo , Camundongos Endogâmicos C57BL , Paclitaxel/farmacocinética , RNA Interferente Pequeno/farmacocinética , Receptor IGF Tipo 2/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia
17.
Transl Res ; 205: 33-43, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30392876

RESUMO

Metabolic syndrome (MetS) is characterized by a cluster of metabolic disorders including obesity, dyslipidemia, hyperglycemia, and hypertension. Here, we report that 27 microRNAs were found to be expressed differently in serum and urine samples of MetS patients compared to control subjects on microarray analysis. Further qualitative real time- polymerase chain reaction analyses confirmed that circulating levels of miR-143-3p were significantly elevated in MetS patients compared with controls, both in serum and urine samples. After accounting for confounding factors, high levels of miR-143-3p remained an independent risk factor for insulin resistance. Inhibition of miR-143-3p expression in mice protected against development of obesity-associated insulin resistance. Furthermore, we demonstrated that insulin-like growth factor 2 receptor (IGF2R) was among the target genes of miR-143-3p by searching 3 widely used bioinformatics databases and preliminary validation. Our experiments suggest that knockdown of circulating miR-143-3p may protect against insulin resistance in the setting of MetS via targeting of IGF2R and activation of the insulin signaling pathway. Our results characterize the miR-143-3p-IGF2R pathway as a potential target for the treatment of obesity-associated insulin resistance.


Assuntos
Resistência à Insulina , Síndrome Metabólica/fisiopatologia , MicroRNAs/sangue , Receptor IGF Tipo 2/metabolismo , Células 3T3-L1 , Adulto , Idoso , Animais , Estudos de Casos e Controles , Estudos Transversais , Regulação para Baixo , Feminino , Inativação Gênica , Células HEK293 , Humanos , Insulina/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Pessoa de Meia-Idade , Obesidade/fisiopatologia , Processamento Pós-Transcricional do RNA , Transdução de Sinais
18.
J Virol ; 93(5)2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30541844

RESUMO

Human herpesvirus 8 (HHV-8) viral interleukin-6 (vIL-6) localizes largely to the endoplasmic reticulum (ER) and here associates functionally with both the gp130 signal transducer and the novel ER membrane protein vitamin K epoxide reductase complex subunit 1 variant-2 (VKORC1v2). The latter interaction contributes to the viability of latently infected primary effusion lymphoma (PEL) cells and to HHV-8 productive replication, in part via promotion of ER-associated degradation (ERAD) of nascent pro-cathepsin D (pCatD) and consequent suppression of lysosome-localized proapoptotic mature CatD. Here we report that VKORC1v2 associates with insulin-like growth factor 2 receptor (IGF2R), also known as cation-independent mannose-6-phosphate receptor, which is involved in trafficking of mannose-6-phosphate-conjugated glycoproteins to lysosomes. VKORC1v2 effected reduced IGF2R expression in a manner dependent on VKORC1v2-IGF2R interaction, while vIL-6, which could inhibit VKORC1v2-IGF2R interaction, effected increased expression of IGF2R. These effects were independent of changes in IGF2R mRNA levels, indicating likely posttranslational mechanisms. In kinetic analyses involving labeling of either newly synthesized or preexisting IGF2R, vIL-6 promoted accumulation of the former while having no detectable effect on the latter. Furthermore, vIL-6 led to decreased K48-linked ubiquitination of IGF2R and suppression of ERAD proteins effected increased IGF2R expression and loss of IGF2R regulation by vIL-6. Depletion-based experiments identified IGF2R as a promoter of PEL cell viability and virus yields from lytically reactivated cultures. Our findings identify ER-transiting nascent IGF2R as an interaction partner of VKORC1v2 and target of vIL-6 regulation and IGF2R as a positive contributor to HHV-8 biology, thereby extending understanding of the mechanisms of VKORC1v2-associated vIL-6 function.IMPORTANCE HHV-8 vIL-6 promotes productive replication in the context of reactivated lytic replication in primary effusion lymphoma (PEL) and endothelial cells and sustains latently infected PEL cell viability. Viral IL-6 is also considered to contribute significantly to HHV-8-associated pathogenesis, since vIL-6 can promote cell proliferation, cell survival, and angiogenesis that are characteristic of HHV-8-associated Kaposi's sarcoma, PEL and multicentric Castleman's disease (MCD), in addition to proinflammatory activities observed in MCD-like "Kaposi's sarcoma-associated herpesvirus-induced cytokine syndrome." We show in the present study that vIL-6 can promote productive replication and latent PEL cell viability through upregulation of the mannose-6-phosphate- and peptide hormone-interacting receptor IGF2R, which is a positive factor in HHV-8 biology via these activities. VKORC1v2-enhanced ER-associated degradation of IGF2R and vIL-6 promotion of IGF2R expression through prevention of its interaction with VKORC1v2 and consequent rescue from degradation represent newly recognized activities of VKOCR1v2 and vIL-6.


Assuntos
Células Endoteliais/virologia , Herpesvirus Humano 8/metabolismo , Interleucina-6/metabolismo , Linfoma de Efusão Primária/virologia , Receptor IGF Tipo 2/metabolismo , Vitamina K Epóxido Redutases/metabolismo , Catepsina D/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Receptor gp130 de Citocina/metabolismo , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Precursores Enzimáticos/metabolismo , Células HEK293 , Humanos , Manosefosfatos/metabolismo , Receptor IGF Tipo 2/biossíntese , Receptor IGF Tipo 2/genética , Ubiquitinação , Ativação Viral/genética , Latência Viral/genética , Replicação Viral/genética
19.
J Cell Biol ; 218(2): 615-631, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30559172

RESUMO

Retromer is a peripheral membrane protein complex that coordinates multiple vesicular trafficking events within the endolysosomal system. Here, we demonstrate that retromer is required for the maintenance of normal lysosomal morphology and function. The knockout of retromer subunit Vps35 causes an ultrastructural alteration in lysosomal structure and aberrant lysosome function, leading to impaired autophagy. At the whole-cell level, knockout of retromer Vps35 subunit reduces lysosomal proteolytic capacity as a consequence of the improper processing of lysosomal hydrolases, which is dependent on the trafficking of the cation-independent mannose 6-phosphate receptor (CI-M6PR). Incorporation of CI-M6PR into endosome transport carriers via a retromer-dependent process is restricted to those tethered by GCC88 but not golgin-97 or golgin-245. Finally, we show that this retromer-dependent retrograde cargo trafficking pathway requires SNX3, but not other retromer-associated cargo binding proteins, such as SNX27 or SNX-BAR proteins. Therefore, retromer does contribute to the retrograde trafficking of CI-M6PR required for maturation of lysosomal hydrolases and lysosomal function.


Assuntos
Endossomos/metabolismo , Lisossomos/metabolismo , Complexos Multiproteicos/metabolismo , Receptor IGF Tipo 2/metabolismo , Autoantígenos/genética , Autoantígenos/metabolismo , Transporte Biológico Ativo/fisiologia , Endossomos/genética , Proteínas da Matriz do Complexo de Golgi/genética , Proteínas da Matriz do Complexo de Golgi/metabolismo , Células HeLa , Humanos , Lisossomos/genética , Complexos Multiproteicos/genética , Receptor IGF Tipo 2/genética , Nexinas de Classificação/genética , Nexinas de Classificação/metabolismo
20.
PLoS One ; 13(11): e0207205, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30419003

RESUMO

Sorting nexin 5 (SNX5), a member of sorting nexin family, plays an important role in membrane trafficking, including the retrograde trafficking of the cation independent mannose 6-phosphate receptor (CI-M6PR) and macropinocytosis. Using ESI-LCMS/MS analysis, we confirmed that SNX5 serine 226 is phosphorylated. Since SNX5 forms heterodimers with SNX1 or SNX2, we examined the effect of phosphorylation at S226 on the heterodimer formations. Wild-type and mutants of SNX5, in which S226 was mutated to a glutamic acid or an alanine, were expressed in 8505C cells. In pull-down assays using SNX5 as bait, only the S226E mutant failed to precipitate both SNX1 and SNX2. Confocal microscopy data indicated that the wild type and S226A mutant were colocalized with SNX1 and SNX2 in endosomes, but the S226E was not. SNX5 and SNX6 support each other's functions and are involved with CI-M6PR retrograde trafficking. In SNX5 and SNX6 double knockdown cells, CI-M6PR was dispersed and colocalized with the endosomal marker EEA1. In a rescue experiment using SNX5 mutants, the S226A rescued CI-M6PR localization, similar to control cells, but S226E did not. Furthermore, the decrease in the uptake of dextran by macropinocytosis in SNX5 knockdown cells was recovered by the expression of rescue-wild type or S226A mutant, but not by the rescue-S226E mutant. These observations indicate that SNX5 constitutive phosphorylation that mimics the mutant S226E decreases the active SNX5 in these cells. The phosphorylation of SNX5 regulates the dimerization with SNX1 or SNX2, and this suggests that it controls membrane trafficking and protein sorting.


Assuntos
Transporte Biológico/fisiologia , Pinocitose/fisiologia , Nexinas de Classificação/genética , Nexinas de Classificação/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Dextranos/metabolismo , Endossomos/metabolismo , Humanos , Mutação , Fosforilação , Multimerização Proteica , Receptor IGF Tipo 2/metabolismo
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