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1.
Int J Mol Sci ; 22(15)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34361094

RESUMO

Protein-protein interactions between G protein-coupled receptors (GPCRs) can augment their functionality and increase the repertoire of signaling pathways they regulate. New therapeutics designed to modulate such interactions may allow for targeting of a specific GPCR activity, thus reducing potential for side effects. Dopamine receptor (DR) heteromers are promising candidates for targeted therapy of neurological conditions such as Parkinson's disease since current treatments can have severe side effects. To facilitate development of such therapies, it is necessary to identify the various DR binding partners. We report here a new interaction partner for DRD2 and DRD3, the orphan receptor G protein-coupled receptor 143 (GPR143), an atypical GPCR that plays multiple roles in pigment cells and is expressed in several regions of the brain. We previously demonstrated that the DRD2/ DRD3 antagonist pimozide also modulates GPR143 activity. Using confocal microscopy and two FRET methods, we observed that the DRs and GPR143 colocalize and interact at intracellular membranes. Furthermore, co-expression of wildtype GPR143 resulted in a 57% and 67% decrease in DRD2 and DRD3 activity, respectively, as determined by ß-Arrestin recruitment assay. GPR143-DR dimerization may negatively modulate DR activity by changing affinity for dopamine or delaying delivery of the DRs to the plasma membrane.


Assuntos
Dopamina/metabolismo , Proteínas do Olho/metabolismo , Glicoproteínas de Membrana/metabolismo , Domínios e Motivos de Interação entre Proteínas , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/metabolismo , beta-Arrestinas/metabolismo , Proteínas do Olho/genética , Humanos , Glicoproteínas de Membrana/genética , Mutação , Ligação Proteica , Receptores de Dopamina D2/genética , Receptores de Dopamina D3/genética , Transdução de Sinais
2.
Int J Mol Sci ; 22(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34445325

RESUMO

Blue cone monochromatism (BCM) is an X-linked recessive cone dysfunction disorder caused by mutations in the OPN1LW/OPN1MW gene cluster, encoding long (L)- and middle (M)-wavelength-sensitive cone opsins. Here, we report on the unusual clinical presentation of BCM caused by a novel mutation in the OPN1LW gene in a young man. We describe in detail the phenotype of the proband, and the subclinical morpho-functional anomalies shown by his carrier mother. At a clinical level, the extensive functional evaluation demonstrated in the proband the M/L cone affection and the sparing of S-cone function, distinctive findings of BCM. Interestingly, spectral-domain optical coherence tomography showed the presence of foveal hypoplasia with focal irregularities of the ellipsoid layer in the foveal area, reported to be associated with some cases of cone-rod dystrophy and achromatopsia. At a molecular level, we identified the novel mutation c.427T > C p.(Ser143Pro) in the OPN1LW gene and the common missense mutation c.607T > C (p.Cys203Arg) in the OPN1MW gene. In addition, we discovered the c.768-2_769delAGTT splicing variant in the GPR143 gene. To our knowledge, this is the first case of foveal hypoplasia in a BCM patient and of mild clinical affection in a female carrier caused by the concomitant effect of variants in OPN1LW/OPN1MW and GPR143 genes, thus as the result of the simultaneous action of two independent genetic defects.


Assuntos
Defeitos da Visão Cromática/genética , Proteínas do Olho/genética , Fóvea Central/anormalidades , Glicoproteínas de Membrana/genética , Opsinas de Bastonetes/genética , Adulto , Defeitos da Visão Cromática/patologia , Humanos , Masculino , Mutação , Linhagem
3.
Int J Mol Sci ; 22(15)2021 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-34360710

RESUMO

A presynaptic active zone organizer protein Bassoon orchestrates numerous important functions at the presynaptic active zone. We previously showed that the absence of Bassoon exclusively in forebrain glutamatergic presynapses (BsnEmx1cKO) in mice leads to developmental disturbances in dentate gyrus (DG) affecting synaptic excitability, morphology, neurogenesis and related behaviour during adulthood. Here, we demonstrate that hyperexcitability of the medial perforant path-to-DG (MPP-DG) pathway in BsnEmx1cKO mice emerges during adolescence and is sustained during adulthood. We further provide evidence for a potential involvement of tropomyosin-related kinase B (TrkB), the high-affinity receptor for brain-derived neurotrophic factor (BDNF), mediated signalling. We detect elevated TrkB protein levels in the dorsal DG of adult mice (~3-5 months-old) but not in adolescent (~4-5 weeks-old) mice. Electrophysiological analysis reveals increased field-excitatory-postsynaptic-potentials (fEPSPs) in the DG of the adult, but not in adolescent BsnEmx1cKO mice. In line with an increased TrkB expression during adulthood in BsnEmx1cKO, blockade of TrkB normalizes the increased synaptic excitability in the DG during adulthood, while no such effect was observed in adolescence. Accordingly, neurogenesis, which has previously been found to be increased in adult BsnEmx1cKO mice, was unaffected at adolescent age. Our results suggest that Bassoon plays a crucial role in the TrkB-dependent postnatal maturation of the hippocampus.


Assuntos
Hipocampo/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Prosencéfalo/metabolismo , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , Transmissão Sináptica , Animais , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas Tirosina Quinases/genética
4.
J Immunol ; 207(5): 1448-1455, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34362834

RESUMO

Identification of the receptors involved in innate immune recognition of Staphylococcus aureus, a major cause of morbidity and mortality in humans, is essential to develop alternative strategies to treat infections caused by antibiotic-resistant strains. In the current study, we examine the role of endosomal TLRs, which sense the presence of prokaryotic-type nucleic acids, in anti-staphylococcal host defenses using infection models involving genetically defective mice. Single deficiencies in TLR7, 9, or 13 resulted in mild or no decrease in host defenses. However, the simultaneous absence of TLR7, 9, and 13 resulted in markedly increased susceptibility to cutaneous and systemic S. aureus infection concomitantly with decreased production of proinflammatory chemokines and cytokines, neutrophil recruitment to infection sites, and reduced production of reactive oxygen species. This phenotype was significantly more severe than that of mice lacking TLR2, which senses the presence of staphylococcal lipoproteins. Notably, the combined absence of TLR7, 9, and 13 resulted in complete abrogation of IL-12 p70 and IFN-ß responses to staphylococcal stimulation in macrophages. Taken together, our data highlight the presence of a highly integrated endosomal detection system, whereby TLR7, 9, and 13 cooperate in sensing the presence of staphylococcal nucleic acids. We demonstrate that the combined absence of these receptors cannot be compensated for by cell surface-associated TLRs, such as TLR2, or cytosolic receptors. These data may be useful to devise strategies aimed at stimulating innate immune receptors to treat S. aureus infections.


Assuntos
Endossomos/metabolismo , Glicoproteínas de Membrana/metabolismo , Neutrófilos/imunologia , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/fisiologia , Receptor 7 Toll-Like/metabolismo , Receptor Toll-Like 9/metabolismo , Receptores Toll-Like/metabolismo , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Suscetibilidade a Doenças , Feminino , Humanos , Imunidade Inata , Mediadores da Inflamação/metabolismo , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor 7 Toll-Like/genética , Receptor Toll-Like 9/genética , Receptores Toll-Like/genética
5.
Nat Commun ; 12(1): 4502, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301937

RESUMO

Cells in many tissues, such as bone, muscle, and placenta, fuse into syncytia to acquire new functions and transcriptional programs. While it is known that fused cells are specialized, it is unclear whether cell-fusion itself contributes to programmatic-changes that generate the new cellular state. Here, we address this by employing a fusogen-mediated, cell-fusion system to create syncytia from undifferentiated cells. RNA-Seq analysis reveals VSV-G-induced cell fusion precedes transcriptional changes. To gain mechanistic insights, we measure the plasma membrane surface area after cell-fusion and observe it diminishes through increases in endocytosis. Consequently, glucose transporters internalize, and cytoplasmic glucose and ATP transiently decrease. This reduced energetic state activates AMPK, which inhibits YAP1, causing transcriptional-reprogramming and cell-cycle arrest. Impairing either endocytosis or AMPK activity prevents YAP1 inhibition and cell-cycle arrest after fusion. Together, these data demonstrate plasma membrane diminishment upon cell-fusion causes transient nutrient stress that may promote transcriptional-reprogramming independent from extrinsic cues.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Transporte Biológico , Fusão Celular , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Células Gigantes/metabolismo , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Camundongos , RNA-Seq/métodos , Transdução de Sinais/genética , Fatores de Transcrição/genética , Proteínas do Envelope Viral/genética
6.
Int J Mol Sci ; 22(14)2021 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-34299025

RESUMO

Several studies have demonstrated that melanoma-derived extracellular vesicles (EVs) are involved in lymph node metastasis; however, the molecular mechanisms involved are not completely defined. Here, we found that EMILIN-1 is proteolyzed and secreted in small EVs (sEVs) as a novel mechanism to reduce its intracellular levels favoring metastasis in mouse melanoma lymph node metastatic cells. Interestingly, we observed that EMILIN-1 has intrinsic tumor and metastasis suppressive-like properties reducing effective migration, cell viability, primary tumor growth, and metastasis. Overall, our analysis suggests that the inactivation of EMILIN-1 by proteolysis and secretion in sEVs reduce its intrinsic tumor suppressive activities in melanoma favoring tumor progression and metastasis.


Assuntos
Movimento Celular/genética , Proliferação de Células/genética , Vesículas Extracelulares/metabolismo , Melanoma/metabolismo , Glicoproteínas de Membrana/metabolismo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Biologia Computacional , Metástase Linfática/genética , Masculino , Espectrometria de Massas , Melanoma/genética , Melanoma/patologia , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Proteólise , RNA-Seq , Reação em Cadeia da Polimerase em Tempo Real , Estatísticas não Paramétricas , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
7.
Anticancer Res ; 41(8): 4047-4052, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34281873

RESUMO

BACKGROUND/AIM: Tropomyosin-related kinase B (TrkB)/brain-derived neurotrophic factor (BDNF) signaling plays a role in inducing malignant phenotypes in several aggressive types of cancers. To create a conclusive therapy targeting TrkB/BDNF signaling in solid refractory cancers, the biological significance of TrkB/BDNF signaling was analyzed in pancreatic ductal adenocarcinoma (PDAC) cells. MATERIALS AND METHODS: Three PDAC cell lines were used as target cells to investigate proliferation and invasiveness. Small interfering RNA (siRNA) and the TrkB tyrosine kinase inhibitor k252a were used as TrkB/BDNF signaling inhibitors. RESULTS: All PDAC cell lines expressed TrkB and BDNF. When TrkB and BDNF were inhibited by siRNA or k252a, the invasiveness of PANC-1 and SUIT-2 cells significantly decreased. When TrkB was inhibited by siRNA or k252a, proliferation was significantly inhibited in PDAC cells. CONCLUSION: TrkB/BDNF signaling may be a new therapeutic target for PDAC. Therapies targeting TrkB/BDNF signaling may be a conclusive cancer therapy for refractory solid cancer.


Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Glicoproteínas de Membrana/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptor trkB/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Carbazóis/farmacologia , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Alcaloides Indólicos/farmacologia , Glicoproteínas de Membrana/antagonistas & inibidores , Glicoproteínas de Membrana/genética , Neoplasias Pancreáticas/patologia , Inibidores de Proteínas Quinases/farmacologia , Interferência de RNA , RNA Interferente Pequeno/genética , Receptor trkB/antagonistas & inibidores , Receptor trkB/genética , Transdução de Sinais/efeitos dos fármacos
8.
Molecules ; 26(12)2021 Jun 09.
Artigo em Inglês | MEDLINE | ID: mdl-34207849

RESUMO

The ancient paralogs premelanosome protein (PMEL) and glycoprotein nonmetastatic melanoma protein B (GPNMB) have independently emerged as intriguing disease loci in recent years. Both proteins possess common functional domains and variants that cause a shared spectrum of overlapping phenotypes and disease associations: melanin-based pigmentation, cancer, neurodegenerative disease and glaucoma. Surprisingly, these proteins have yet to be shown to physically or genetically interact within the same cellular pathway. This juxtaposition inspired us to compare and contrast this family across a breadth of species to better understand the divergent evolutionary trajectories of two related, but distinct, genes. In this study, we investigated the evolutionary history of PMEL and GPNMB in clade-representative species and identified TMEM130 as the most ancient paralog of the family. By curating the functional domains in each paralog, we identified many commonalities dating back to the emergence of the gene family in basal metazoans. PMEL and GPNMB have gained functional domains since their divergence from TMEM130, including the core amyloid fragment (CAF) that is critical for the amyloid potential of PMEL. Additionally, the PMEL gene has acquired the enigmatic repeat domain (RPT), composed of a variable number of imperfect tandem repeats; this domain acts in an accessory role to control amyloid formation. Our analyses revealed the vast variability in sequence, length and repeat number in homologous RPT domains between craniates, even within the same taxonomic class. We hope that these analyses inspire further investigation into a gene family that is remarkable from the evolutionary, pathological and cell biology perspectives.


Assuntos
Melanócitos/metabolismo , Glicoproteínas de Membrana/metabolismo , Mutação , Doenças Neurodegenerativas/patologia , Antígeno gp100 de Melanoma/metabolismo , Sequência de Aminoácidos , Proteínas Amiloidogênicas/metabolismo , Animais , Biologia Computacional/métodos , Humanos , Glicoproteínas de Membrana/genética , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/metabolismo , Filogenia , Pigmentação , Domínios Proteicos , Homologia de Sequência , Antígeno gp100 de Melanoma/genética
9.
Cell Prolif ; 54(8): e13094, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34312932

RESUMO

OBJECTIVES: Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the progressive and selective degeneration of dopaminergic neurons. Microglial activation and neuroinflammation are associated with the pathogenesis of PD. However, the relationship between microglial activation and PD pathology remains to be explored. MATERIALS AND METHODS: An acute regimen of MPTP was administered to adult C57BL/6J mice with normal, much reduced or repopulated microglial population. Damages of the dopaminergic system were comprehensively assessed. Inflammation-related factors were assessed by quantitative PCR and Multiplex immunoassay. Behavioural tests were carried out to evaluate the motor deficits in MPTP-challenged mice. RESULTS: The receptor for colony-stimulating factor 1 inhibitor PLX3397 could effectively deplete microglia in the nigrostriatal pathway of mice via feeding a PLX3397-formulated diet for 21 days. Microglial depletion downregulated both pro-inflammatory and anti-inflammatory molecule expression at baseline and after MPTP administration. At 1d post-MPTP injection, dopaminergic neurons showed a significant reduction in PLX3397-fed mice, but not in control diet (CD)-fed mice. However, partial microglial depletion in mice exerted little effect on MPTP-induced dopaminergic injuries compared with CD mice at later time points. Interestingly, microglial repopulation brought about apparent resistance to MPTP intoxication. CONCLUSIONS: Microglia can inhibit PD development at a very early stage; partial microglial depletion has little effect in terms of the whole process of the disease; and microglial replenishment elicits neuroprotection in PD mice.


Assuntos
Intoxicação por MPTP/patologia , Microglia/metabolismo , 1-Metil-4-Fenil-1,2,3,6-Tetra-Hidropiridina/administração & dosagem , Aminopiridinas/farmacologia , Animais , Comportamento Animal/efeitos dos fármacos , Ciclo-Oxigenase 2/metabolismo , Citocinas/metabolismo , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Mediadores da Inflamação/metabolismo , Intoxicação por MPTP/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Microglia/citologia , Microglia/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Pirróis/farmacologia , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo
10.
J Biol Chem ; 297(1): 100855, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34097876

RESUMO

In the last several years, NAD+ supplementation has emerged as an innovative and safe therapeutic strategy for a wide spectrum of disorders, including diabetes and neuropathy. However, critical questions remain as to how NAD+ and its precursors are taken up by cells, as well as the effects of long-lasting intracellular NAD+ (iNAD+) increases. Here, we investigated the kinetics of iNAD+ levels in different cell types challenged with prolonged exposure to extracellular NAD+ (eNAD+). Surprisingly, we found that after the initial increase, iNAD+ contents decreased back to control levels (iNAD+ resetting). Focusing our attention on HeLa cells, we found that oxygen and ATP consumption occurred with similar temporal kinetics after eNAD+ exposure. Using [3H]NAD+ and [14C]NAD+, we determined that NAD+ resetting was not due to increased dinucleotide extrusion but rather due to reduced uptake of cleaved NAD+ products. Indeed, eNAD+ exposure reduced the expression of the ecto-5'-nucleotidase CD73, the nicotinamide adenine mononucleotide transporter solute carrier family 12 member 8, and the nicotinamide riboside kinase. Interestingly, silencing the NAD+-sensor enzyme sirtuin 1 prevented eNAD+-dependent transcriptional repression of ecto-5'-nucleotidase, solute carrier family 12 member 8, and nicotinamide riboside kinase, as well as iNAD+ resetting. Our findings provide the first evidence for a sirtuin 1-mediated homeostatic response aimed at maintaining physiological iNAD+ levels in conditions of excess eNAD+ availability. These data may be of relevance for therapies designed to support the NAD+ metabolome via extracellular supplementation of the dinucleotide or its precursors.


Assuntos
5'-Nucleotidase/genética , ADP-Ribosil Ciclase 1/genética , Metabolismo Energético/genética , Glicoproteínas de Membrana/genética , NAD/metabolismo , Sirtuína 1/genética , Trifosfato de Adenosina/metabolismo , Transporte Biológico/genética , Células HeLa , Homeostase/genética , Humanos , Cinética , Oxigênio/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Transdução de Sinais/genética
11.
Int J Biol Macromol ; 185: 696-707, 2021 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-34174316

RESUMO

The inspection of variations in the proteomic aspects conspire the biomarker discovery in diagnostics of peculiar diseases. Recent developments in high-throughput proteomic techniques have provided leverage in the discovery of biomarkers during the etiology of various diseases. We identified potential biomarkers by utilizing proteomics, bioinformatics and gene expression studies. Meticulous assessment of collagen and hydroxyproline levels along with the glycogen and protein carbonyl levels exhibited deterioration in the N' - Nitrosodiethylamine (NDEA) administered rat livers and subsequent salubrious effect of pomegranate juice. The immunohistochemical inspection of iNOS and nitrite estimation indicated the peccant fibrotic alterations. 2D proteome profiling and MALDI-TOF MS/MS furthered the significant biomarkers to be analyzed for the gene ontology by PANTHER, cluster analysis by DAVID and network simulation by STRING 10.0. Several genes found relevant after MALDI analysis were evaluated by real-time PCR (RTPCR). Our data revealed CYP2b15, HSP70, TRFE, HPT, Il1rl2, Ric8a, Krt18, Hsp90b1 and iNOS as novel biomarkers for the mechanism of pomegranate against liver fibrosis. It can be inferred that NDEA-induced liver fibrosis actuates various biological pathways by the identified biomarkers and pomegranate juice modifies them.


Assuntos
Biomarcadores/metabolismo , Dimetilnitrosamina/efeitos adversos , Cirrose Hepática/tratamento farmacológico , Extratos Vegetais/administração & dosagem , Romã (Fruta)/química , Animais , Suplementos Nutricionais , Modelos Animais de Doenças , Sucos de Frutas e Vegetais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Queratina-18/genética , Queratina-18/metabolismo , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Extratos Vegetais/farmacologia , Proteômica , Ratos , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Espectrometria de Massas em Tandem
13.
Nat Commun ; 12(1): 3516, 2021 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-34112782

RESUMO

Profiling studies have revealed considerable phenotypic heterogeneity in cancer-associated fibroblasts (CAFs) present within the tumour microenvironment, however, functional characterisation of different CAF subsets is hampered by the lack of specific markers defining these populations. Here we show that genetic deletion of the Endo180 (MRC2) receptor, predominantly expressed by a population of matrix-remodelling CAFs, profoundly limits tumour growth and metastasis; effects that can be recapitulated in 3D co-culture assays. This impairment results from a CAF-intrinsic contractility defect and reduced CAF viability, which coupled with the lack of phenotype in the normal mouse, demonstrates that upregulated Endo180 expression by a specific, potentially targetable CAF subset is required to generate a supportive tumour microenvironment. Further, characterisation of a tumour subline selected via serial in vivo passage for its ability to overcome these stromal defects provides important insight into, how tumour cells adapt to a non-activated stroma in the early stages of metastatic colonisation.


Assuntos
Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Glicoproteínas de Membrana/metabolismo , Receptores de Superfície Celular/metabolismo , Esferoides Celulares/metabolismo , Células Estromais/metabolismo , Microambiente Tumoral/genética , Animais , Neoplasias da Mama/patologia , Neoplasias da Mama/secundário , Fibroblastos Associados a Câncer/citologia , Proliferação de Células/genética , Sobrevivência Celular/genética , Células Cultivadas , Técnicas de Cocultura , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Feminino , Humanos , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Células NIH 3T3 , Metástase Neoplásica , Receptores de Superfície Celular/genética , Ensaio Tumoral de Célula-Tronco
14.
PLoS Pathog ; 17(6): e1009687, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34181691

RESUMO

COVID-19 outbreak is the biggest threat to human health in recent history. Currently, there are over 1.5 million related deaths and 75 million people infected around the world (as of 22/12/2020). The identification of virulence factors which determine disease susceptibility and severity in different cell types remains an essential challenge. The serine protease TMPRSS2 has been shown to be important for S protein priming and viral entry, however, little is known about its regulation. SPINT2 is a member of the family of Kunitz type serine protease inhibitors and has been shown to inhibit TMPRSS2. Here, we explored the existence of a co-regulation between SPINT2/TMPRSS2 and found a tightly regulated protease/inhibitor expression balance across tissues. We found that SPINT2 negatively correlates with SARS-CoV-2 expression in Calu-3 and Caco-2 cell lines and was down-regulated in secretory cells from COVID-19 patients. We validated our findings using Calu-3 cell lines and observed a strong increase in viral load after SPINT2 knockdown, while overexpression lead to a drastic reduction of the viral load. Additionally, we evaluated the expression of SPINT2 in datasets from comorbid diseases using bulk and scRNA-seq data. We observed its down-regulation in colon, kidney and liver tumors as well as in alpha pancreatic islets cells from diabetes Type 2 patients, which could have implications for the observed comorbidities in COVID-19 patients suffering from chronic diseases.


Assuntos
COVID-19/metabolismo , Glicoproteínas de Membrana/metabolismo , SARS-CoV-2/metabolismo , Internalização do Vírus , Células A549 , COVID-19/genética , Células CACO-2 , Humanos , Glicoproteínas de Membrana/genética , SARS-CoV-2/genética , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Índice de Gravidade de Doença
15.
Nat Commun ; 12(1): 3416, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099706

RESUMO

APOE and Trem2 are major genetic risk factors for Alzheimer's disease (AD), but how they affect microglia response to Aß remains unclear. Here we report an APOE isoform-specific phospholipid signature with correlation between human APOEε3/3 and APOEε4/4 AD brain and lipoproteins from astrocyte conditioned media of APOE3 and APOE4 mice. Using preclinical AD mouse models, we show that APOE3 lipoproteins, unlike APOE4, induce faster microglial migration towards injected Aß, facilitate Aß uptake, and ameliorate Aß effects on cognition. Bulk and single-cell RNA-seq demonstrate that, compared to APOE4, cortical infusion of APOE3 lipoproteins upregulates a higher proportion of genes linked to an activated microglia response, and this trend is augmented by TREM2 deficiency. In vitro, lack of TREM2 decreases Aß uptake by APOE4-treated microglia only, suggesting TREM2-APOE interaction. Our study elucidates phenotypic and transcriptional differences in microglial response to Aß mediated by APOE3 or APOE4 lipoproteins in preclinical models of AD.


Assuntos
Doença de Alzheimer/patologia , Apolipoproteína E3/metabolismo , Apolipoproteína E4/metabolismo , Encéfalo/patologia , Microglia/patologia , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Apolipoproteína E3/administração & dosagem , Apolipoproteína E3/genética , Apolipoproteína E4/administração & dosagem , Apolipoproteína E4/genética , Encéfalo/citologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Fosfolipídeos/metabolismo , Presenilina-1/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA-Seq , Receptores Imunológicos/genética , Receptores Imunológicos/metabolismo
16.
Cell Mol Life Sci ; 78(13): 5275-5301, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34023917

RESUMO

For a long time, PLS3 (plastin 3, also known as T-plastin or fimbrin) has been considered a rather inconspicuous protein, involved in F-actin-binding and -bundling. However, in recent years, a plethora of discoveries have turned PLS3 into a highly interesting protein involved in many cellular processes, signaling pathways, and diseases. PLS3 is localized on the X-chromosome, but shows sex-specific, inter-individual and tissue-specific expression variability pointing towards skewed X-inactivation. PLS3 is expressed in all solid tissues but usually not in hematopoietic cells. When escaping X-inactivation, PLS3 triggers a plethora of different types of cancers. Elevated PLS3 levels are considered a prognostic biomarker for cancer and refractory response to therapies. When it is knocked out or mutated in humans and mice, it causes osteoporosis with bone fractures; it is the only protein involved in actin dynamics responsible for osteoporosis. Instead, when PLS3 is upregulated, it acts as a highly protective SMN-independent modifier in spinal muscular atrophy (SMA). Here, it seems to counteract reduced F-actin levels by restoring impaired endocytosis and disturbed calcium homeostasis caused by reduced SMN levels. In contrast, an upregulation of PLS3 on wild-type level might cause osteoarthritis. This emphasizes that the amount of PLS3 in our cells must be precisely balanced; both too much and too little can be detrimental. Actin-dynamics, regulated by PLS3 among others, are crucial in a lot of cellular processes including endocytosis, cell migration, axonal growth, neurotransmission, translation, and others. Also, PLS3 levels influence the infection with different bacteria, mycosis, and other pathogens.


Assuntos
Glicoproteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/metabolismo , Neurônios Motores/fisiologia , Atrofia Muscular Espinal/fisiopatologia , Osteoclastos/fisiologia , Osteoporose/fisiopatologia , Animais , Humanos , Glicoproteínas de Membrana/genética , Proteínas dos Microfilamentos/genética , Osteoclastos/citologia
17.
Cancer Med ; 10(10): 3403-3412, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33934535

RESUMO

A primary factor in tumor morbidity and mortality, lung adenocarcinoma (LUAD) is known to be a major subtype of lung cancer, having the lowest survival rate among all other cancers. Using The Cancer Genome Atlas (TCGA) database the relationship between the immune infiltrate and the NUP62CL was explored and the value of the NUP62CL expression in the prognosis and diagnosis LUAD was examined. Using the logistic regression and the Wilcoxon signed-rank test the relationship between the NUP62CL and the clinico-pathological features was analyzed. There was a significant correlation between the clinical stage (p = 0.005), the N (p = 0.004), and the decreased expression of NUP62CL. The prognosis of LUAD with high NUP62CL expression was revealed to be worse than that with low NUP62CL expression (p < 0.001) by the Kaplan-Meier survival analysis. The potentiality of NUP62CL to be a significant factor of prognosis for LUAD was indicated by the analyses of the multivariate and the univariate Cox regression models. In LUAD, the crucial role of recombination and maintenance of telomere as a significant pathway for NUP62CL was suggested by the Gene Set Enrichment Analysis (GSEA). To analyze the correlation between the genes and the tumor infiltrating immune cells the CIBERSORT was used. Moreover the positive correlation with the NUP62CL expression in LUAD of the infiltration level of the tumor infiltrating B lymphocytes and memory CD4+ T cells was exhibited by CIBERSORT. Therefore, NUP62CL may be a new valuable prognostic indicator for LUAD.


Assuntos
Adenocarcinoma de Pulmão/genética , Neoplasias Pulmonares/genética , Linfócitos do Interstício Tumoral/patologia , Glicoproteínas de Membrana/genética , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Adenocarcinoma de Pulmão/patologia , Linfócitos B/patologia , Biomarcadores Tumorais/genética , Linfócitos T CD4-Positivos/patologia , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/patologia , Prognóstico , Modelos de Riscos Proporcionais
18.
Cancer Sci ; 112(8): 3190-3204, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34036684

RESUMO

Alterations of glycosyltransferase expression are often associated with tumor occurrence and progression. Among the many glycosyltransferases, increased expression of fucosyltransferase 8 (FUT8) has been frequently observed to be involved in progression and metastasis of various types of cancer. The regulatory mechanisms of FUT8 expression remain unclear. FUT8 expression was shown, in this study, to be elevated in breast cancer. Systematic analysis revealed that transcription factor activator protein 2γ (AP-2γ) is the target gene of microRNA-10b (miR-10b), which we previously identified as a positive regulator of FUT8. Overexpression of AP-2γ inhibited FUT8 expression, with associated reduction of cell invasiveness and migration ability. AP-2γ was capable of binding to transcription factor STAT3, and phosphorylation of STAT3 induced transcription of the FUT8 gene. On the basis of our findings, we propose that binding of AP-2γ to STAT3 results in formation of the AP-2γ/STAT3 complex and consequent inhibition of STAT3 phosphorylation, thereby preventing entry of p-STAT3 into the nucleus to initiate FUT8 transcription. This study clarifies the molecular mechanisms whereby transcription factor AP-2γ regulates FUT8 expression in breast cancer.


Assuntos
Neoplasias da Mama/patologia , Fucosiltransferases/genética , Glicoproteínas de Membrana/genética , Receptores Imunológicos/genética , Fator de Transcrição AP-2/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Núcleo Celular/metabolismo , Feminino , Fucosiltransferases/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Camundongos , Invasividade Neoplásica , Transplante de Neoplasias , Fosforilação , Fator de Transcrição STAT3/metabolismo , Transcrição Genética
19.
Eur J Neurol ; 28(8): 2603-2613, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33969597

RESUMO

BACKGROUND AND PURPOSE: Nasu-Hakola disease (NHD) is a rare, autosomal recessive disorder characterized by skeletal and neurological symptoms. Behavioral symptoms with cognitive impairment may mimic the behavioral variant of frontotemporal dementia (bvFTD) and other early-onset dementias. Our patients were analyzed and the literature was reviewed to delineate neurological and neuroimaging findings suggestive of NHD. METHOD: Fourteen patients carrying a pathogenic mutation in the TREM2 gene were found in our database. Demographic, clinical, laboratory and radiological data were retrieved and analyzed. RESULTS: The presenting clinical picture was behavioral changes with cognitive decline resembling bvFTD in all patients. The mean age was 37.1 ± 4.97 years and the mean duration of the disease was 8.9 ± 3.51 years. Only two patients had typical bone cysts. Seven patients had bilateral calcification of the basal ganglia in computed tomography of the brain. Magnetic resonance imaging of the brain revealed severe atrophy of the corpus callosum, enlargement of the ventricles, atrophy of the caudate nuclei and periventricular white matter changes in all patients. Symmetrical global atrophy of the brain mainly affecting frontoparietal and lateral temporal regions were observed in all cases, and 13 patients had atrophy of the hippocampus. Cerebrospinal fluid examination of 10 patients showed elevated protein levels in six and the presence of oligoclonal bands in four patients. CONCLUSION: A combination of white matter changes, enlarged ventricles, atrophy of the caudate nuclei and thinning of the corpus callosum in magnetic resonance imaging strongly suggests NHD in patients with FTD syndrome. Molecular genetic analysis should be performed in suspected cases, and families should receive genetic counseling.


Assuntos
Demência Frontotemporal , Lipodistrofia , Glicoproteínas de Membrana/genética , Osteocondrodisplasias , Receptores Imunológicos/genética , Panencefalite Esclerosante Subaguda , Adulto , Encéfalo/diagnóstico por imagem , Demência Frontotemporal/diagnóstico por imagem , Demência Frontotemporal/genética , Humanos , Imageamento por Ressonância Magnética , Neuroimagem
20.
J Cell Sci ; 134(10)2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-34028541

RESUMO

A disintegrin and metalloproteinase 3 (ADAM3) is a sperm membrane protein critical for sperm migration from the uterus into the oviduct and sperm-egg binding in mice. Disruption of PRSS37 results in male infertility concurrent with the absence of mature ADAM3 from cauda epididymal sperm. However, how PRSS37 modulates ADAM3 maturation remains largely unclear. Here, we determine the PRSS37 interactome by GFP immunoprecipitation coupled with mass spectrometry in PRSS37-EGFP knock-in mice. Three molecular chaperones (CLGN, CALR3 and PDILT) and three ADAM proteins (ADAM2, ADAM6B and ADAM4) were identified to be interacting with PRSS37. Coincidently, five of them (except ADAM4) have been reported to interact with ADAM3 precursor and regulate its maturation. We further demonstrated that PRSS37 also interacts directly with ADAM3 precursor and its deficiency impedes the association between PDILT and ADAM3. This could contribute to improper translocation of ADAM3 to the germ cell surface, leading to ADAM3 loss in PRSS37-null mature sperm. The understanding of the maturation mechanisms of pivotal sperm plasma membrane proteins will pave the way toward novel strategies for contraception and the treatment of unexplained male infertility.


Assuntos
Infertilidade Masculina , Glicoproteínas de Membrana , Proteínas ADAM/genética , Animais , Epididimo , Feminino , Masculino , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Isomerases de Dissulfetos de Proteínas , Serina Proteases , Espermatozoides
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