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1.
Braz J Med Biol Res ; 53(5): e9021, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32348423

RESUMO

Lung cancer is the most common malignancy worldwide and is characterized by rapid progression, aggressive behavior, frequent recurrence, and poor prognosis. The TCGA database indicates that chondroitin polymerizing factor (CHPF) is overexpressed in human lung cancer tissues compared with normal tissues and this overexpression corresponds to shorter overall survival in lung cancer patients. In this study, to investigate the function of CHPF in lung cancer, lentiviral vectors expressing CHPF shRNA were stably transduced into A549 and H1299 cells. Compared to shCtrl cells, CHPF knockdown cells had significantly reduced proliferation. Furthermore, the silencing of CHPF in A549 and H1299 cells resulted in apoptotic induction, which led to decreased colony formation. Wound healing and transwell invasion assays revealed that CHPF could positively regulate the migration of lung cancer cells. The tumorigenic role of CHPF was also validated in nude mouse xenograft models. Affymetrix gene chip analysis indicated that CHPF regulated the proliferation and invasion of lung cancer cells through CDH1, RRM2, MKI67, and TNFRSF10B. We thus highlight CHPF as a novel target for lung cancer treatment.


Assuntos
Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares/metabolismo , N-Acetilgalactosaminiltransferases/metabolismo , Animais , Western Blotting , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Neoplasias Pulmonares/genética , Camundongos , Camundongos Endogâmicos BALB C , Análise em Microsséries , N-Acetilgalactosaminiltransferases/genética , Reação em Cadeia da Polimerase em Tempo Real
2.
Sci Rep ; 10(1): 1199, 2020 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-31988291

RESUMO

ß-1,4-N-Acetyl-Galactosaminyltransferase 1 (B4GALNT1) encodes the key enzyme B4GALNT1 to generate gangliosides GM2/GD2. GM2/GD2 gangliosides are surface glycolipids mainly found on brain neurons as well as peripheral nerves and skin melanocytes and are reported to exacerbate the malignant potential of melanomas. In order to elucidate the mechanism, we performed functional analyses of B4GALNT1-overexpressing cells. We analyzed ganglioside pattern on four melanoma and two neuroblastoma cell lines by high performance liquid chromatography (HPLC). We overexpressed B4GALNT1 in GM2/GD2-negative human melanoma cell line (SH4) and confirmed production of GM2/GD2 by HPLC. They showed higher anchorage independence growth (AIG) in colony formation assay, and exhibited augmented motility. In vitro, cell proliferation was not affected by GM2/GD2 expression. In vivo, GM2/GD2-positive SH4 clones showed significantly higher tumorigenesis in NOD/Scid/IL2Rγ-null mice, and immunostaining of mouse CD31 revealed that GM2/GD2 induced remarkable angiogenesis. No differences were seen in melanoma stem cell and Epithelial-Mesenchymal Transition markers between GM2/GD2-positive and -negative SH4 cells. We therefore concluded that B4GALNT1, and consequently GM2/GD2, enhanced tumorigenesis via induction of angiogenesis, AIG, and cell motility. RNA-Seq suggested periostin as a potential key factor for angiogenesis and AIG. These findings may lead to development of novel therapy for refractory melanoma.


Assuntos
Carcinogênese/metabolismo , Movimento Celular/genética , Proliferação de Células/genética , Gangliosídeo G(M2)/metabolismo , Melanoma/metabolismo , N-Acetilgalactosaminiltransferases/metabolismo , Neovascularização Patológica/metabolismo , Neoplasias Cutâneas/metabolismo , Animais , Linhagem Celular Tumoral , Feminino , Xenoenxertos , Humanos , Masculino , Melanoma/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , N-Acetilgalactosaminiltransferases/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , RNA-Seq , Neoplasias Cutâneas/patologia , Transfecção , Carga Tumoral/genética
3.
Carbohydr Polym ; 232: 115822, 2020 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-31952617

RESUMO

Chondroitin sulfate is a linear glycosaminoglycan widely distributed as an important extracellular matrix component of mammalian cells. It participates in numerous pathological processes, however, illustration of its diverse biological roles is hampered by the unavailability of structurally defined chondroitin polymers and their derivatives. Herein, we report a novel homogeneous chondroitin polymers synthetic strategy which combines stepwise oligosaccharides synthesis with one-pot homogeneous chondroitin chain polymerization. Exogenous trisaccharide was proved to be the necessary acceptor for PmCS-catalyzed homogeneous chondroitin polymers synthetic reactions. The strategy exhibited a well-controlled relationship between the final sugar chain length and the molar ratios of reaction substrates that could synthesize homogenous chondroitin polymers with unprecedented narrow molecular weight distribution. More importantly, the strategy was further expanded to synthesis of unnatural zwitterionic and N-sulfonated chondroitin polymers by incorporation of sugar nucleotide derivatives into the synthetic approach.


Assuntos
Condroitina/biossíntese , N-Acetilgalactosaminiltransferases/metabolismo , Polímeros/metabolismo , Configuração de Carboidratos , Condroitina/análogos & derivados , Condroitina/química , Pasteurella multocida/enzimologia , Polimerização , Polímeros/química
4.
Nat Chem Biol ; 16(3): 351-360, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31932717

RESUMO

Polypeptide GalNAc-transferase T3 (GalNAc-T3) regulates fibroblast growth factor 23 (FGF23) by O-glycosylating Thr178 in a furin proprotein processing motif RHT178R↓S. FGF23 regulates phosphate homeostasis and deficiency in GALNT3 or FGF23 results in hyperphosphatemia and familial tumoral calcinosis. We explored the molecular mechanism for GalNAc-T3 glycosylation of FGF23 using engineered cell models and biophysical studies including kinetics, molecular dynamics and X-ray crystallography of GalNAc-T3 complexed to glycopeptide substrates. GalNAc-T3 uses a lectin domain mediated mechanism to glycosylate Thr178 requiring previous glycosylation at Thr171. Notably, Thr178 is a poor substrate site with limiting glycosylation due to substrate clashes leading to destabilization of the catalytic domain flexible loop. We suggest GalNAc-T3 specificity for FGF23 and its ability to control circulating levels of intact FGF23 is achieved by FGF23 being a poor substrate. GalNAc-T3's structure further reveals the molecular bases for reported disease-causing mutations. Our findings provide an insight into how GalNAc-T isoenzymes achieve isoenzyme-specific nonredundant functions.


Assuntos
Fatores de Crescimento de Fibroblastos/química , N-Acetilgalactosaminiltransferases/metabolismo , Animais , Células CHO , Cricetulus , Fatores de Crescimento de Fibroblastos/metabolismo , Glicopeptídeos/química , Glicosilação , Humanos , Isoenzimas/metabolismo , Lectinas/metabolismo , N-Acetilgalactosaminiltransferases/fisiologia , Treonina/metabolismo
5.
Cell Mol Life Sci ; 77(4): 719-733, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31302752

RESUMO

Cytidine base editors (CBEs) have been demonstrated to be useful for precisely inducing C:G-to-T:A base mutations in various organisms. In this study, we showed that the BE4-Gam system induced the targeted C-to-T base conversion in porcine blastocysts at an efficiency of 66.7-71.4% via the injection of a single sgRNA targeting a xeno-antigen-related gene and BE4-Gam mRNA. Furthermore, the efficiency of simultaneous three gene base conversion via the injection of three targeting sgRNAs and BE4-Gam mRNA into porcine parthenogenetic embryos was 18.1%. We also obtained beta-1,4-N-acetyl-galactosaminyl transferase 2, alpha-1,3-galactosyltransferase, and cytidine monophosphate-N-acetylneuraminic acid hydroxylase deficient pig by somatic cell nuclear transfer, which exhibited significantly decreased activity. In addition, a new CBE version (termed AncBE4max) was used to edit genes in blastocysts and porcine fibroblasts (PFFs) for the first time. While this new version demonstrated a three genes base-editing rate of 71.4% at the porcine GGTA1, B4galNT2, and CMAH loci, it increased the frequency of bystander edits, which ranged from 17.8 to 71.4%. In this study, we efficiently and precisely mutated bases in porcine blastocysts and PFFs using CBEs and successfully generated C-to-T and C-to-G mutations in pigs. These results suggest that CBEs provide a more simple and efficient method for improving economic traits, reducing the breeding cycle, and increasing disease tolerance in pigs, thus aiding in the development of human disease models.


Assuntos
Citidina/genética , Edição de Genes/métodos , Suínos/genética , Animais , Blastocisto/metabolismo , Sistemas CRISPR-Cas , Galactosiltransferases/genética , Vetores Genéticos/genética , Oxigenases de Função Mista/genética , Mutagênese , N-Acetilgalactosaminiltransferases/genética , RNA Guia/genética , Suínos/embriologia
6.
Genet Epidemiol ; 44(1): 104-116, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31830326

RESUMO

Single genome-wide studies may be underpowered to detect trait-associated rare variants with moderate or weak effect sizes. As a viable alternative, meta-analysis is widely used to increase power by combining different studies. The power of meta-analysis critically depends on the underlying association patterns and heterogeneity levels, which are unknown and vary from locus to locus. However, existing methods mainly focus on one or only a few combinations of the association pattern and heterogeneity level, thus may lose power in many situations. To address this issue, we propose a general and unified framework by combining a class of tests including and beyond some existing ones, leading to high power across a wide range of scenarios. We demonstrate that the proposed test is more powerful than some existing methods in simulation studies, then show their performance with the NHLBI Exome-Sequencing Project (ESP) data. One gene (B4GALNT2) was found by our proposed test, but not by others, to be statistically significantly associated with plasma triglyceride. The signal was driven by African-ancestry subjects but it was previously reported to be associated with coronary artery disease among European-ancestry subjects. We implemented our method in an R package aSPUmeta, publicly available at https://github.com/ytzhong/metaRV and will be on CRAN soon.


Assuntos
Doença da Artéria Coronariana/genética , Estudo de Associação Genômica Ampla/métodos , Metanálise como Assunto , Modelos Genéticos , N-Acetilgalactosaminiltransferases/genética , Triglicerídeos/sangue , Grupo com Ancestrais do Continente Africano/genética , Grupo com Ancestrais do Continente Europeu/genética , Exoma/genética , Humanos , Fenótipo
8.
Mol Cell Biol ; 40(3)2020 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-31767633

RESUMO

Clear cell renal cell carcinoma (ccRCC) is regarded as the most aggressive subtype of RCC, with high rates of metastasis and recurrence. An extensive body of studies had proved long noncoding RNAs (lncRNAs) play pivotal parts in the development and evolution of diverse malignant tumors. However, the potential of LINC01094 in ccRCC tumorigenesis is still unexplored. In the present research, with the aid of the TCGA database, we found that LINC01094 was highly expressed in ccRCC tissues. Upregulation of LINC01094 was also confirmed in ccRCC cell lines, and functional experiments delineated that LINC01094 knockdown led to inhibition on ccRCC cell growth and metastasis. Moreover, LINC01094 was activated by FOXM1 at the transcriptional level. Further assay demonstrated that LINC01094 worked as a sponge of microRNA 224-5p (miR-224-5p) and CHSY1 was a miR-224-5p-targeted mRNA. Further, we verified that LINC01094 acted as a competing endogenous RNA in ccRCC to regulate CHSY1 expression via competitively bind to miR-224-5p. Lastly, our results expounded that LINC01094 exerted its tumor-promoting performance in ccRCC development through miR-224-5p/CHSY1 regulatory axis, which shed light on the molecular mechanism underlying LINC01094 in ccRCC and opened a new prospective for the treatment of ccRCC.


Assuntos
Carcinoma de Células Renais/genética , Proteína Forkhead Box M1/genética , Neoplasias Renais/genética , MicroRNAs/genética , RNA Longo não Codificante/genética , Animais , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Glucuronosiltransferase/genética , Humanos , Neoplasias Renais/patologia , Camundongos Endogâmicos BALB C , Camundongos Nus , Enzimas Multifuncionais/genética , N-Acetilgalactosaminiltransferases/genética
9.
Cancer Immunol Immunother ; 69(2): 175-187, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31853576

RESUMO

High grade ovarian serous cancer (HGSC) is a malignant disease with high mortality. Glycosylation plays important roles in tumor invasion and immune evasion, but its effect on the immune microenvironment of HGSC remains unclear. This study examined the association of glycosyltransferase expression with HGSC prognosis and explored the underlying mechanism using clinical specimens and integrated bioinformatic analyses. We identified a cluster of 15 glycogenes associated with reduced overall survival, and GALNT10 was found to be an independent predictor of HGSC prognosis. The high GALNT10 expression was associated with increased regulatory CD4+ T cells infiltration and decreased granzyme B expression in CD8+ T cells. The expression of GALNT10 and its product, Tn antigen, in HGSC specimens was associated with the increased infiltration of M2 macrophages and neutrophils, and the decreased infiltration of CD3+ T cells, NK cells, and B cells. Taken collectively, high GALNT10 expression confers with immunosuppressive microenvironment to promote tumor progression and predicts poor clinical outcomes in HGSC patients.


Assuntos
Cistadenocarcinoma Seroso/genética , Cistadenocarcinoma Seroso/mortalidade , Expressão Gênica , N-Acetilgalactosaminiltransferases/genética , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/mortalidade , Microambiente Tumoral/genética , Biomarcadores Tumorais , Biologia Computacional/métodos , Cistadenocarcinoma Seroso/patologia , Bases de Dados Genéticas , Feminino , Humanos , Imuno-Histoquímica , Imunomodulação/genética , Gradação de Tumores , Neoplasias Ovarianas/patologia , Prognóstico , Estudos Retrospectivos
10.
Insect Biochem Mol Biol ; 115: 103254, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31655162

RESUMO

Silkworm Bombyx mori is one of the insect hosts for recombinant protein production at academic and industrial levels. B. mori and other insect cells can produce mammalian proteins with proper posttranslational modifications, such as N-glycosylation, but the structures of N-glycans in B. mori are mainly high mannose- and paucimannose-type, while mammals also produce hybrid- and complex-type glycans. Recently, complex-type N-glycans whose structures are different from mammalian ones have been identified in some insect cell N-glycomes at very low levels compared with levels of high mannose- and paucimannose-type glycans. However, their functions and the enzymes involved in the biosynthesis of insect complex-type N-glycans are not clear, and complex-type N-glycans, except for N-acetylglucosamine-terminated glycans, are still not identified in the B. mori N-glycome. Here, we focused on the ß-1,4-galactosyltransferase family (also known as glycosyltransferase family 7, GT7) that contains mammalian ß-1,4-galactosyltransferase and insect ß-1,4-N-acetylgalactosaminyltransferase. A gene for a GT7 protein (BmGalNAcT) from B. mori was cloned, expressed in a soluble form using a silkworm expression system, and the gene product showed strict ß-1,4-N-acetylgalactosaminyltransferase activity but not ß-1,4-galactosyltransferase activity. A mutation in Ile298 or Ile310, which are predicted to be located in the active site, reduced its glycosyltransferase activity, suggesting that these residues and the corresponding residues are responsible for substrate specificity of GT7. These results suggested that BmGalNAcT may be involved in the complex-type N-glycans, and moreover, bioinformatics analysis revealed that B. mori might have an extra gene for a GT7 enzyme with different specificity in addition to the known insect GT7 glycosyltransferases.


Assuntos
Bombyx/enzimologia , N-Acetilgalactosaminiltransferases/metabolismo , Animais , Bombyx/genética , Feminino , Masculino , Mutagênese Sítio-Dirigida , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/isolamento & purificação , Especificidade por Substrato
11.
J Biotechnol ; 306: 159-168, 2019 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-31604106

RESUMO

Human chorionic gonadotropin (hCG) is a glycoprotein hormone that exists as a heterodimer comprised of an α subunit and ß subunit linked with disulfide bridges. The ß subunit contains four O-glycosylation sites. Previous studies have found that the translation of mRNA to polypeptides of the ß subunit was a severely limiting step for the expression of recombinant hCG protein in Chinese hamster ovary (CHO) cells. The effects of O-glycosylation on recombinant hCG protein expression were assessed by adding O-glycan precursors and overexpressing and knocking down key regulatory genes of O-glycan precursor synthesis and O-glycan sugar chain synthesis or hydrolases. The results indicated that O-glycosylation was indeed limiting in the expression of recombinant hCG protein, and N-acetylgalactosamine (GalNAc) was the major limiting precursor. Glutamine-fructose-6-phosphate transaminase 2 (Gfat2) and Uridine diphosphate-glucose pyrophosphorylase 2 (Ugp2), key regulatory genes of O-glycan precursor synthesis, were overexpressed. Ugp2 overexpression significantly increased the recombinant hCG protein level by 1.92 times compared to that of the control. The LC-MS/MS analysis and Phaseolus vulgaris leucoagglutinin (PHA-L) lectin blot analysis showed that Ugp2 overexpression significantly increased the total galactosylation levels of intracellular proteins and the O-glycosylation of recombinant hCG protein. The stability of the hCG protein to trypsin digestion was also enhanced. Ugp2 is the major limiting enzyme of the O-glycan precursor synthesis in recombinant hCG protein production. Furthermore, the effects and mechanisms of the key genes of O-glycan sugar chain synthesis and hydrolases such as polypeptide N-acetylgalactosaminyltransferase1 (Galnt1), Core 1 synthase, glycoprotein-N-acetylgalactosamine 3-beta-galactosyltransferase (C1galt1), O-linked N-acetylglucosamine transferase (Ogt) and Hexosaminidase (Hex), were evaluated. The results indicated that Galnt1 overexpression increased the recombinant hCG protein level by 1.57 times and improved the total galactosylation of intracellular proteins, O-glycosylation and the stability of recombinant hCG protein. Galnt1 is the major limiting enzyme of O-glycan sugar chain synthesis. Overexpression of Ugp2 and Galnt1 simultaneously improved the recombinant hCG protein level by 2.44 times, and both had synergistic effects. Based on the results of overexpression of Galnt1, the major limiting gene of O-Glycan chain synthesis, the precursors GalNAc and Gal were added and increased the recombinant hCG protein level by 3.68 times. This study revealed the major limiting factors of O-glycosylation of recombinant hCG protein in CHO cells and proposed an effective expression regulation strategy.


Assuntos
Gonadotropina Coriônica/química , Gonadotropina Coriônica/metabolismo , Processamento de Proteína Pós-Traducional/genética , Acetilglucosamina/metabolismo , Animais , Células CHO , Gonadotropina Coriônica/genética , Cricetinae , Cricetulus , Meios de Cultura , Expressão Gênica , Técnicas de Inativação de Genes , Glicosilação , Humanos , N-Acetilgalactosaminiltransferases/genética , Nucleotidiltransferases/genética , Polissacarídeos/genética , Polissacarídeos/metabolismo , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
12.
Turk J Pediatr ; 61(1): 130-133, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31559735

RESUMO

Albaramki J, Dmour H, Shboul M, Bonnard C, Venkatesh B, Odeh R. Recessive mutation in GALNT3 causes hyperphosphatemic familial tumoral calcinosis associated with chronic recurrent multifocal osteomyelitis. Turk J Pediatr 2019; 61: 130-133. Hyperphosphatemic familial tumoral calcinosis is a rare autosomal recessive disorder that is characterized by persistent hyperphosphatemia and extra-articular calcifications. Three cases were previously reported with hyperphosphatemic familial tumoral calcinosis that were associated with chronic recurrent multifocal osteomyelitis, an autoinflammatory disorder that is characterized by recurrent episodes of bone pain. We describe here an 11-year-old child who was diagnosed with these two conditions and was found to carry a splice site mutation c.1524+1G > A in the GALNT3 gene.


Assuntos
Calcinose/genética , Hiperostose Cortical Congênita/genética , Hiperfosfatemia/genética , Mutação , N-Acetilgalactosaminiltransferases/genética , Osteomielite/genética , Criança , Humanos , Masculino
13.
Emerg Microbes Infect ; 8(1): 1428-1437, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31560252

RESUMO

Switching of receptor binding preference has been widely considered as one of the necessary mutations for avian influenza viruses, enabling efficient transmissions between human hosts. By stably overexpressing B4GalNT2 gene in MDCK cells, surface α2,3-siallylactose receptors were modified without affecting α2,6-receptor expression. The cell line MDCK-B4GalNT2 was used as a tool to screen for α2,3-receptor requirements in a panel of influenza viruses with previously characterized glycan array data. Infection of viruses with α2,3-receptor binding capability was inhibited in MDCK-B4GalNT2 cells, with the exception of A/WSN/33 (WSN). Infection with the 2009 pandemic H1N1 strains, A/California/04/2009 (Cal04) and A/Hong Kong/415742/2009 (HK09), despite showing α2,6-receptor binding, was also found to be inhibited. Further investigation showed that viral inhibition was due to a reduction in viral entry rate and viral attachment. Recombinant WSN virus with the neuraminidase (NA) gene swapped to A/Puerto Rico/8/1934 (PR8) and Cal04 resulted in a significant viral inhibition in MDCK-B4GalNT2 cells. With oseltamivir, the NA active site was found to be important for the replication results of WSN, but not Cal04.


Assuntos
Vírus da Influenza A Subtipo H1N1/fisiologia , N-Acetilgalactosaminiltransferases/genética , Ácido N-Acetilneuramínico/metabolismo , Neuraminidase/metabolismo , Ligação Viral , Internalização do Vírus , Animais , Antivirais/farmacologia , Linhagem Celular , Cães , Vírus da Influenza A Subtipo H1N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H1N1/genética , Células Madin Darby de Rim Canino , N-Acetilgalactosaminiltransferases/metabolismo , Neuraminidase/genética , Oseltamivir/farmacologia , Replicação Viral/efeitos dos fármacos
14.
Proc Natl Acad Sci U S A ; 116(41): 20404-20410, 2019 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-31548401

RESUMO

Polypeptide N-acetylgalactosaminyl transferases (GalNAc-Ts) initiate mucin type O-glycosylation by catalyzing the transfer of N-acetylgalactosamine (GalNAc) to Ser or Thr on a protein substrate. Inactive and partially active variants of the isoenzyme GalNAc-T12 are present in subsets of patients with colorectal cancer, and several of these variants alter nonconserved residues with unknown functions. While previous biochemical studies have demonstrated that GalNAc-T12 selects for peptide and glycopeptide substrates through unique interactions with its catalytic and lectin domains, the molecular basis for this distinct substrate selectivity remains elusive. Here we examine the molecular basis of the activity and substrate selectivity of GalNAc-T12. The X-ray crystal structure of GalNAc-T12 in complex with a di-glycosylated peptide substrate reveals how a nonconserved GalNAc binding pocket in the GalNAc-T12 catalytic domain dictates its unique substrate selectivity. In addition, the structure provides insight into how colorectal cancer mutations disrupt the activity of GalNAc-T12 and illustrates how the rules dictating GalNAc-T12 function are distinct from those for other GalNAc-Ts.


Assuntos
Neoplasias Colorretais/metabolismo , N-Acetilgalactosaminiltransferases/química , N-Acetilgalactosaminiltransferases/metabolismo , Proteínas de Neoplasias/química , Sequência de Aminoácidos , Humanos , Modelos Moleculares , Conformação Proteica
15.
G3 (Bethesda) ; 9(11): 3891-3906, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31554716

RESUMO

AUTS2 was originally discovered as the gene disrupted by a translocation in human twins with Autism spectrum disorder, intellectual disability, and epilepsy. Since that initial finding, AUTS2-linked mutations and variants have been associated with a very broad array of neuropsychiatric disorders, sugg esting that AUTS2 is required for fundamental steps of neurodevelopment. However, genotype-phenotype correlations in this region are complicated, because most mutations could also involve neighboring genes. Of particular interest is the nearest downstream neighbor of AUTS2, GALNT17, which encodes a brain-expressed N-acetylgalactosaminyltransferase of unknown brain function. Here we describe a mouse (Mus musculus) mutation, T(5G2;8A1)GSO (abbreviated 16Gso), a reciprocal translocation that breaks between Auts2 and Galnt17 and dysregulates both genes. Despite this complex regulatory effect, 16Gso homozygotes model certain human AUTS2-linked phenotypes very well. In addition to abnormalities in growth, craniofacial structure, learning and memory, and behavior, 16Gso homozygotes display distinct pathologies of the cerebellum and hippocampus that are similar to those associated with autism and other types of AUTS2-linked neurological disease. Analyzing mutant cerebellar and hippocampal transcriptomes to explain this pathology, we identified disturbances in pathways related to neuron and synapse maturation, neurotransmitter signaling, and cellular stress, suggesting possible cellular mechanisms. These pathways, coupled with the translocation's selective effects on Auts2 isoforms and coordinated dysregulation of Galnt17, suggest novel hypotheses regarding the etiology of the human "AUTS2 syndrome" and the wide array of neurodevelopmental disorders linked to variance in this genomic region.


Assuntos
Proteínas do Citoesqueleto/genética , N-Acetilgalactosaminiltransferases/genética , Fatores de Transcrição/genética , Animais , Comportamento Animal , Cerebelo/metabolismo , Cerebelo/patologia , Proteínas do Citoesqueleto/metabolismo , Feminino , Hipocampo/metabolismo , Hipocampo/patologia , Humanos , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Mutação , Transtornos do Neurodesenvolvimento/genética , Transtornos do Neurodesenvolvimento/patologia , Fenótipo , Crânio/anatomia & histologia , Síndrome , Fatores de Transcrição/metabolismo
16.
Biochimie ; 165: 108-114, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31336136

RESUMO

Oridonin is a diterpenoid isolated from the Rabdosia rubescens and has multiple biological effects, such as anti-inflammation and anti-tumor activities. In present study, we revealed that the sensitizing effect of oridonin on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in several cancer cells, but not in normal cells. Oridonin enhanced death-signaling inducing complexes (DISC) formation and DR5 glycosylation without affecting expression of downstream intracellular apoptosis-related proteins. Oridonin upregulated peptidyl O-glycosyltransferase GALNT14 in a dose- and time-dependent manner. Knockdown of GALNT14 by siRNA and Endo H treatment reduced oridonin-induced DR5 glycosylation. Furthermore, treatment with inhibitor of glycosylation (benzyl-α-GalNAc) blocked oridonin plus TRAIL-induced apoptosis. Collectively, our results suggest that oridonin-induced DR5 glycosylation contributes to TRAIL-induced apoptotic cell death in cancer cells.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Apoptose/efeitos dos fármacos , Diterpenos de Caurano/farmacologia , N-Acetilgalactosaminiltransferases/metabolismo , Receptores do Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Ligante Indutor de Apoptose Relacionado a TNF/fisiologia , Linhagem Celular , Glicosilação , Humanos , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia
17.
Genes (Basel) ; 10(6)2019 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-31181852

RESUMO

Assistance dog training programs can see as many as 60% of their trainees dismissed. Many training programs utilize behavioral assays prior to admittance to identify likely successful candidates, yet such assays can be insconsistent. Recently, four canine retrotransposon mobile element insertions (MEIs) in or near genes WBSCR17 (Cfa6.6 and Cfa6.7), GTF2I (Cfa6.66) and POM121 (Cfa6.83) were identified in domestic dogs and gray wolves. Variations in these MEIs were significantly associated with a heightened propensity to initiate prolonged social contact or hypersociability. Using our dataset of 837 dogs, 228 of which had paired survey-based behavioral data, we discovered that one of the insertions in WBSCR17 is the most important predictor of dog sociable behaviors related to human proximity, measured by the Canine Behavioral Assessment Research Questionnaire (C-BARQ©). We found a positive correlation between insertions at Cfa6.6 and dog separation distress in the form of restlessness when about to be left alone by the owner. Lastly, assistance dogs showed significant heterozygosity deficiency at locus Cfa6.6 and higher frequency of insertions at Cfa6.6 and Cfa6.7. We suggest that training programs could utilize this genetic survey to screen for MEIs at WBSCR17 to identify dogs with sociable traits compatible with successful assistance dog performance.


Assuntos
Comportamento Animal , Sequências Repetitivas Dispersas/genética , N-Acetilgalactosaminiltransferases/genética , Animais , Cães , Educação , Feminino , Homozigoto , Humanos , Masculino , Fenótipo , Lobos/genética
18.
Neuroscience ; 411: 1-10, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31129200

RESUMO

Parkinson's disease (PD) is a progressive aging disorder that affects millions worldwide, thus, disease-modifying-therapies are urgently needed. PD pathology includes α-synuclein (aSyn) accumulation as synucleinopathy. Loss of GM1 gangliosides occurs in PD brain, which is modeled in GM2 synthase transgenic mice. GM2+/- mice have low, not absent GM1 and develop age-onset motor deficits, making them an excellent PD drug testing model. FTY720 (fingolimod) reduces synucleinopathy in A53T aSyn mice and motor dysfunction in 6-OHDA and rotenone PD models, but no one has tested FTY720 in mice that develop age-onset PD-like motor problems. We confirmed that GM2+/-mice had equivalent rotarod, hindlimb reflexes, and adhesive removal functions at 9 mo. From 11 mo, GM2+/- mice received oral FTY720 or vehicle 3x/week to 16 mo. As bladder problems occur in PD, we also assessed GM2+/- bladder function. This allowed us to demonstrate improved motor and bladder function in GM2+/- mice treated with FTY720. By immunoblot, FTY720 reduced levels of proNGF, a biomarker of bladder dysfunction. In humans with PD, arm swing becomes abnormal, and brachial plexus modulates arm swing. Ultrastructure of brachial plexus in wild type and GM2 transgenic mice confirmed abnormal myelination and axons in GM2 transgenics. FTY720 treated GM2+/- brachial plexus sustained myelin associated protein levels and reduced aggregated aSyn and PSer129 aSyn levels. FTY720 increases brain derived neurotrophic factor (BDNF) and we noted increased BDNF in GM2+/- brachial plexus and cerebellum, which contribute to rotarod performance. These findings provide further support for testing low dose FTY720 in patients with PD.


Assuntos
Comportamento Animal/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Cloridrato de Fingolimode/farmacologia , Doença de Parkinson Secundária/tratamento farmacológico , Moduladores do Receptor de Esfingosina 1 Fosfato/farmacologia , alfa-Sinucleína/metabolismo , Animais , Encéfalo/metabolismo , Cloridrato de Fingolimode/uso terapêutico , Camundongos , Camundongos Transgênicos , Destreza Motora/efeitos dos fármacos , N-Acetilgalactosaminiltransferases/genética , N-Acetilgalactosaminiltransferases/metabolismo , Doença de Parkinson Secundária/metabolismo , Teste de Desempenho do Rota-Rod , Moduladores do Receptor de Esfingosina 1 Fosfato/uso terapêutico
19.
Dev Cell ; 50(1): 57-72.e6, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31105009

RESUMO

The mammalian glycocalyx is a heavily glycosylated extramembrane compartment found on nearly every cell. Despite its relevance in both health and disease, studies of the glycocalyx remain hampered by a paucity of methods to spatially classify its components. We combine metabolic labeling, bioorthogonal chemistry, and super-resolution localization microscopy to image two constituents of cell-surface glycans, N-acetylgalactosamine (GalNAc) and sialic acid, with 10-20 nm precision in 2D and 3D. This approach enables two measurements: glycocalyx height and the distribution of individual sugars distal from the membrane. These measurements show that the glycocalyx exhibits nanoscale organization on both cell lines and primary human tumor cells. Additionally, we observe enhanced glycocalyx height in response to epithelial-to-mesenchymal transition and to oncogenic KRAS activation. In the latter case, we trace increased height to an effector gene, GALNT7. These data highlight the power of advanced imaging methods to provide molecular and functional insights into glycocalyx biology.


Assuntos
Carcinoma Ductal Pancreático/patologia , Glicocálix/metabolismo , Microscopia/métodos , N-Acetilgalactosaminiltransferases/metabolismo , Neoplasias Ovarianas/patologia , Neoplasias Pancreáticas/patologia , Polissacarídeos/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Transição Epitelial-Mesenquimal , Feminino , Glicosilação , Humanos , Neoplasias Ovarianas/metabolismo , Neoplasias Pancreáticas/metabolismo , Prognóstico , Taxa de Sobrevida , Células Tumorais Cultivadas
20.
Mol Cell Biol ; 39(14)2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31036568

RESUMO

GALGT2 (also B4GALNT2) encodes a glycosyltransferase that is normally confined to the neuromuscular and myotendinous junction in adult skeletal muscle. GALGT2 overexpression in muscle can inhibit muscular dystrophy in mouse models of the disease by inducing the overexpression of surrogate muscle proteins, including utrophin, agrin, laminins, and integrins. Despite its well-documented biological properties, little is known about the endogenous regulation of muscle GALGT2 expression. Here, we demonstrate that epidermal growth factor receptor (EGFR) ligands can activate the human GALGT2 promoter. Overexpression of one such ligand, soluble heparin-binding EGF-like growth factor (sHB-EGF), also stimulated mouse muscle Galgt2 gene expression and expression of GALGT2-inducible surrogate muscle genes. Deletion analysis of the GALGT2 promoter identified a 45-bp region containing a TFAP4-binding site that was required for sHB-EGF activation. sHB-EGF increased TFAP4 binding to this site in muscle cells and increased endogenous Tfap4 gene expression. sHB-EGF also increased muscle EGFR protein expression and activated EGFR-Akt signaling. sHB-EGF expression was concentrated at the neuromuscular junction, and Hbegf deletion reduced Galgt2-dependent synaptic glycosylation. Hbegf deletion also mimicked Galgt2-dependent neuromuscular and muscular dystrophy phenotypes. These data demonstrate that sHB-EGF is an endogenous regulator of muscle Galgt2 gene expression and can mimic Galgt2-dependent muscle phenotypes.


Assuntos
Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Músculo Esquelético/metabolismo , N-Acetilgalactosaminiltransferases/genética , Junção Neuromuscular/metabolismo , Animais , Sítios de Ligação , Células CHO , Linhagem Celular , Cricetulus , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Glicosilação , Células HEK293 , Humanos , Masculino , Camundongos , N-Acetilgalactosaminiltransferases/química , N-Acetilgalactosaminiltransferases/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
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