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1.
Mol Cell ; 80(1): 72-86.e7, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32910895

RESUMO

Membrane protein biogenesis faces the challenge of chaperoning hydrophobic transmembrane helices for faithful membrane insertion. The guided entry of tail-anchored proteins (GET) pathway targets and inserts tail-anchored (TA) proteins into the endoplasmic reticulum (ER) membrane with an insertase (yeast Get1/Get2 or mammalian WRB/CAML) that captures the TA from a cytoplasmic chaperone (Get3 or TRC40, respectively). Here, we present cryo-electron microscopy reconstructions, native mass spectrometry, and structure-based mutagenesis of human WRB/CAML/TRC40 and yeast Get1/Get2/Get3 complexes. Get3 binding to the membrane insertase supports heterotetramer formation, and phosphatidylinositol binding at the heterotetramer interface stabilizes the insertase for efficient TA insertion in vivo. We identify a Get2/CAML cytoplasmic helix that forms a "gating" interaction with Get3/TRC40 important for TA insertion. Structural homology with YidC and the ER membrane protein complex (EMC) implicates an evolutionarily conserved insertion mechanism for divergent substrates utilizing a hydrophilic groove. Thus, we provide a detailed structural and mechanistic framework to understand TA membrane insertion.


Assuntos
Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Complexos Multiproteicos/metabolismo , Linhagem Celular , Sequência Conservada , Evolução Molecular , Humanos , Proteínas de Membrana/metabolismo , Modelos Moleculares , Fosfatidilinositóis/metabolismo , Ligação Proteica , Multimerização Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
2.
Nature ; 584(7822): 630-634, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32814900

RESUMO

Integral membrane proteins are encoded by approximately 25% of all protein-coding genes1. In eukaryotes, the majority of membrane proteins are inserted, modified and folded at the endoplasmic reticulum (ER)2. Research over the past several decades has determined how membrane proteins are targeted to the ER and how individual transmembrane domains (TMDs) are inserted into the lipid bilayer3. By contrast, very little is known about how multi-spanning membrane proteins with several TMDs are assembled within the membrane. During the assembly of TMDs, interactions between polar or charged amino acids typically stabilize the final folded configuration4-8. TMDs with hydrophilic amino acids are likely to be chaperoned during the co-translational biogenesis of membrane proteins; however, ER-resident intramembrane chaperones are poorly defined. Here we identify the PAT complex, an abundant obligate heterodimer of the widely conserved ER-resident membrane proteins CCDC47 and Asterix. The PAT complex engages nascent TMDs that contain unshielded hydrophilic side chains within the lipid bilayer, and it disengages concomitant with substrate folding. Cells that lack either subunit of the PAT complex show reduced biogenesis of numerous multi-spanning membrane proteins. Thus, the PAT complex is an intramembrane chaperone that protects TMDs during assembly to minimize misfolding of multi-spanning membrane proteins and maintain cellular protein homeostasis.


Assuntos
Proteínas de Membrana/biossíntese , Proteínas de Membrana/metabolismo , Chaperonas Moleculares/metabolismo , Complexos Multiproteicos/metabolismo , Biossíntese de Proteínas , Sequência de Aminoácidos , Asparagina/genética , Retículo Endoplasmático/metabolismo , Células HEK293 , Humanos , Bicamadas Lipídicas/metabolismo , Modelos Moleculares , Chaperonas Moleculares/química , Complexos Multiproteicos/química , Mutação , Proteínas Nucleares/metabolismo , Ligação Proteica , Dobramento de Proteína , Subunidades Proteicas/metabolismo , Especificidade por Substrato
3.
Cancer Sci ; 111(7): 2259-2274, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32391585

RESUMO

Hepatocellular carcinoma (HCC) is the most common type of liver cancer. It has a poor prognosis because it is often diagnosed at the advanced stage when treatments are limited. In addition, HCC pathogenesis is not fully understood, and this has affected early diagnosis and treatment of this disease. Human alkaline ceramidase 2 (ACER2), a key enzyme that regulates hydrolysis of cellular ceramides, affects cancer cell survival, however its role in HCC has not been well characterized. Our results showed that ACER2 is overexpressed in HCC tissues and cell lines. In addition, high ACER2 protein expression was associated with tumor growth; ACER2 knockdown resulted in decreased cell growth and migration. Sphingomyelin phosphodiesterase acid-like 3B (SMPDL3B) promoted HCC cell growth, invasion, and migration; SMPDL3B knockdown had a significant inhibitory effect on HCC tumor growth in vivo. Moreover, ACER2 positively regulated the protein level of SMPDL3B. Of note, ACER2/SMPDL3B promoted ceramide hydrolysis and S1P production. This axis induced HCC survival and could be blocked by inhibition of S1P formation. In conclusion, ACER2 promoted HCC cell survival and migration, possibly via SMPDL3B. Thus, inhibition of ACER2/SMPDL3B may be a novel therapeutic target for HCC treatment.


Assuntos
Ceramidase Alcalina/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Adulto , Idoso , Ceramidase Alcalina/metabolismo , Animais , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Xenoenxertos , Humanos , Neoplasias Hepáticas/patologia , Masculino , Proteínas de Membrana/biossíntese , Camundongos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Monoéster Fosfórico Hidrolases/biossíntese , Transdução de Sinais , Esfingomielina Fosfodiesterase/genética
4.
Oncogene ; 39(20): 4092-4102, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32231272

RESUMO

Genome-wide association studies (GWAS) have identified numerous genetic variants that are associated with lung cancer risk, but the biological mechanisms underlying these associations remain largely unknown. Here we investigated the functional relevance of a genetic region in 6q22.2 which was identified to be associated with lung cancer risk in our previous GWAS. We performed linkage disequilibrium (LD) analysis and bioinformatic prediction to screen functional SNPs linked to a tagSNP in 6q22.2 loci, followed by two case-control studies and a meta-analysis with 4403 cases and 5336 controls to identify if these functional SNPs were associated with lung cancer risk. A novel SNP rs17079281 in the DCBLD1 promoter was identified to be associated with lung cancer risk in Chinese populations. Compared with those with C allele, patients with T allele had lower risk of adenocarcinoma (adjusted OR = 0.86; 95% CI: 0.80-0.92), but not squamous cell carcinoma (adjusted OR = 0.99; 95% CI: 0.91-1.10), and patients with the C/T or T/T genotype had lower levels of DCBLD1 expression than those with C/C genotype in lung adenocarcinoma tissues. We performed functional assays to characterize its biological relevance. The results showed that the T allele of rs17079281 had higher binding affinity to transcription factor YY1 than the C allele, which suppressed DCBLD1 expression. DCBLD1 behaved like an oncogene, promoting tumor growth by influencing cell cycle progression. These findings suggest that the functional variant rs17079281C>T decreased lung adenocarcinoma risk by creating an YY1-binding site to suppress DCBLD1 expression, which may serve as a biomarker for assessing lung cancer susceptibility.


Assuntos
Adenocarcinoma de Pulmão , Regulação Neoplásica da Expressão Gênica , Neoplasias Pulmonares , Proteínas de Membrana , Proteínas de Neoplasias , Polimorfismo de Nucleotídeo Único , Elementos de Resposta , Fator de Transcrição YY1 , Células A549 , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/patologia , Animais , Cromossomos Humanos Par 6/genética , Cromossomos Humanos Par 6/metabolismo , Feminino , Loci Gênicos , Estudo de Associação Genômica Ampla , Células HEK293 , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fator de Transcrição YY1/genética , Fator de Transcrição YY1/metabolismo
5.
Curr Pharm Biotechnol ; 21(11): 1119-1128, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32297576

RESUMO

BACKGROUND: Semaphorin 3F (SEMA3F) plays a substantial role in carcinogenesis, because of its role in inducing angiogenesis, and creating a microenvironment for the developing tumor. OBJECTIVE: The purpose of this work was to assess the impact of cisplatin, depending on the concentration and exposure time on the expression pattern of SEMA3F in an endometrial cancer cell line. MATERIALS AND METHODS: Cultures of the Ishikawa endometrial cancer cells were incubated with cisplatin with the following concentrations: 2.5µM; 5µM; and 10µM and for the following periods of time: 12; 24; and 48 hours. Cells not incubated with the drug constituted the control in the experiment. To determine the effect of cisplatin on the expression of SEMA3F, the real-time quantitative reverse transcription reaction (RtqPCR; mRNA) was used, as well as the ELISA assay (protein). The statistical analysis was done with the admission of p<0.05. RESULTS: The silencing of SEMA3F expression on the transcriptome and proteome levels in a culture unexposed to the effects of cisplatin in comparison to endometrial cancer cells under the influence of cisplatin (p<0.05) were noted. Along with an increase in the concentration of the drug used, the number of copies of the gene transcript, during the shortest incubation period had a gradual increase. Only for the highest concentration of the drug, substantial statistical differences in the expression of the SEMA3F protein between 24 and 48 hour incubation periods (p<0.05) were determined. CONCLUSION: Using cisplatin in an endometrial cancer cell culture results in an increased expression of SEMA3F, which advantageously affects the normalization of the neoplastic angiogenic process and lowers the proliferation of the cells making up the mass of the tumor.


Assuntos
Antineoplásicos/farmacologia , Carcinogênese/efeitos dos fármacos , Cisplatino/farmacologia , Neoplasias do Endométrio/metabolismo , Proteínas de Membrana/biossíntese , Proteínas do Tecido Nervoso/biossíntese , RNA Mensageiro/biossíntese , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Feminino , Inativação Gênica , Humanos , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/genética , Microambiente Tumoral/efeitos dos fármacos
6.
J Cancer Res Clin Oncol ; 146(5): 1153-1167, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32189106

RESUMO

BACKGROUND: Hepatoblastoma (HB) and pediatric hepatocellular carcinoma (HCC) are the most common malignant liver tumors in childhood. Both tumor types exhibit genetic and epigenetic alterations in the WNT/ß-catenin signaling pathway, which is a key regulator of liver progenitor cells in embryonic development. The tumors demonstrate a high rate of ß-catenin mutations and gene expression changes of several WNT antagonists. However, the role of the WNT inhibitory factor secreted frizzled-related protein 1 (SFRP1) has not been addressed in pediatric liver cancer so far. RESULTS: In our study, we investigated the gene expression level, DNA methylation status and functional relevance of SFRP1 in HB cell lines and in pediatric liver tumor patient samples. SFRP1 was downregulated due to DNA promoter methylation in all tested HB cell lines. Overexpression of SFRP1 in HB cell lines diminished tumor cell proliferation, colony formation and migration potential. In addition, the SFRP1-expressing HB cell lines showed reduced WNT/ß-catenin signaling pathway activity and decreased expression of WNT target genes. To evaluate the utility of SFRP1 as a biomarker in pediatric liver cancer, we determined the gene expression level and DNA methylation status of SFRP1 in 45 pediatric liver tumor patient samples. The correlation analysis of different clinical parameters and tumor characteristics revealed a significant correlation of reduced SFRP1 expression with the presence of mutant ß-catenin. The methylation status of SFRP1 was furthermore associated to a pediatric liver tumor type with HCC-like characteristics, TERT mutations and an older age at diagnosis. CONCLUSION: Altogether, our data demonstrate that the epigenetic suppression of the WNT/ß-catenin antagonist SFRP1 has an important impact on the malignant behavior of HB cells. Although SFRP1 methylation is a common event in HCC-like pediatric liver tumors, its potential as a prognostic or diagnostic biomarker needs to be further investigated.


Assuntos
Hepatoblastoma/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Neoplasias Hepáticas/genética , Proteínas de Membrana/genética , Mutação , beta Catenina/genética , Idoso , Linhagem Celular Tumoral , Metilação de DNA , Regulação para Baixo , Feminino , Células Hep G2 , Hepatoblastoma/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/biossíntese , Neoplasias Hepáticas/metabolismo , Masculino , Proteínas de Membrana/biossíntese , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Via de Sinalização Wnt , beta Catenina/metabolismo
7.
Biochim Biophys Acta Biomembr ; 1862(6): 183272, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32169592

RESUMO

Membrane proteins exist in lipid bilayers and mediate solute transport, signal transduction, cell-cell communication and energy conversion. Their activities are fundamental for life, which make them prominent subjects of study, but access to only a limited number of high-resolution structures complicates their mechanistic understanding. The absence of such structures relates mainly to difficulties in expressing and purifying high quality membrane protein samples in large quantities. An additional layer of complexity stems from the presence of intra- and/or extra-cellular domains constituted by unstructured intrinsically disordered regions (IDR), which can be hundreds of residues long. Although IDRs form key interaction hubs that facilitate biological processes, these are regularly removed to enable structural studies. To advance mechanistic insight into intact intrinsically disordered membrane proteins, we have developed a protocol for their purification. Using engineered yeast cells for optimized expression and purification, we have purified to homogeneity two very different human membrane proteins each with >300 residues long IDRs; the sodium proton exchanger 1 and the growth hormone receptor. Subsequent to their purification we have further explored their incorporation into membrane scaffolding protein nanodiscs, which will enable future structural studies.


Assuntos
Proteínas Intrinsicamente Desordenadas/química , Proteínas de Membrana/química , Proteínas Recombinantes/química , Saccharomyces cerevisiae/genética , Humanos , Proteínas de Membrana/biossíntese , Conformação Proteica , Receptores da Somatotropina/química , Proteínas Recombinantes/biossíntese , Trocadores de Sódio-Hidrogênio/química , Leveduras/genética
8.
Metabolism ; 105: 154182, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32061660

RESUMO

The impairment of podocyte protein filtration function caused by excessive mitochondrial calcium intake is a critical feature of diabetic nephropathy (DN). Ca2+ channel transient receptor potential cation channel subfamily V member 1 (TRPV1) has been reported to protect against ischemia-reperfusion induced acute renal injury, but there is no report about its role in DN. Here, we report that dietary capsaicin potently inhibits and reverses chronic renal structural and functional damages in db/db or streptozotocin (STZ)-induced diabetic mice in a TRPV1-dependent manner. Activation of TRPV1 by capsaicin alleviated hyperglycemia-induced mitochondrial dysfunction in podocytes, accompanied by reduced mitochondria-associated membranes (MAMs) formation and fewer Ca2+ transport from endoplasmic reticulum (ER) to mitochondria. Mechanistically, TRPV1-mediated transient Ca2+ influx activated 5' AMP-activated protein kinase (AMPK) that reduced the transcription of Fundc1, a key molecule participating in MAMs formation. Inhibition of AMPK or overexpression of Fundc1 obviously blocked the inhibitory effect of capsaicin on MAMs formation and functional decline in podocytes. These findings emphasize the critical role of mitochondrial Ca2+ homeostasis in the maintenance of normal renal function and suggest an effective intervention method to counteract DN.


Assuntos
Nefropatias Diabéticas/tratamento farmacológico , Retículo Endoplasmático/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Podócitos/efeitos dos fármacos , Canais de Cátion TRPV/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Canais de Cálcio/metabolismo , Capsaicina/uso terapêutico , Dieta , Inibidores Enzimáticos/farmacologia , Hiperglicemia/tratamento farmacológico , Hiperglicemia/microbiologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/biossíntese , Camundongos , Camundongos Endogâmicos C57BL , Membranas Mitocondriais/efeitos dos fármacos , Proteínas Mitocondriais/antagonistas & inibidores , Proteínas Mitocondriais/biossíntese
9.
Neuron ; 105(6): 1007-1017.e5, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-31974009

RESUMO

LRRTM4 is a transsynaptic adhesion protein regulating glutamatergic synapse assembly on dendrites of central neurons. In the mouse retina, we find that LRRTM4 is enriched at GABAergic synapses on axon terminals of rod bipolar cells (RBCs). Knockout of LRRTM4 reduces RBC axonal GABAA and GABAC receptor clustering and disrupts presynaptic inhibition onto RBC terminals. LRRTM4 removal also perturbs the stereotyped output synapse arrangement at RBC terminals. Synaptic ribbons are normally apposed to two distinct postsynaptic "dyad" partners, but in the absence of LRRTM4, "monad" and "triad" arrangements are also formed. RBCs from retinas deficient in GABA release also demonstrate dyad mis-arrangements but maintain LRRTM4 expression, suggesting that defects in dyad organization in the LRRTM4 knockout could originate from reduced GABA receptor function. LRRTM4 is thus a key synapse organizing molecule at RBC terminals, where it regulates function of GABAergic synapses and assembly of RBC synaptic dyads.


Assuntos
Proteínas de Membrana/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Inibição Neural/fisiologia , Terminações Pré-Sinápticas/fisiologia , Células Bipolares da Retina/fisiologia , Animais , Feminino , Masculino , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/biossíntese , Proteínas do Tecido Nervoso/genética , Receptores de GABA/metabolismo , Receptores de GABA/fisiologia , Retina/metabolismo , Proteínas Vesiculares de Transporte de Aminoácidos Inibidores/genética , Ácido gama-Aminobutírico/metabolismo
10.
Protein Expr Purif ; 169: 105569, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-31945417

RESUMO

Whereas membrane proteins make up ∼23% of the human proteome, it is estimated that membrane proteins constitute more than 60% of current drug targets. With membrane proteins forming such a high percentage of drug targets relative to their abundance within the proteome, it is little wonder that drug companies need to rapidly access high quality membrane proteins for their drug discovery process. Newly devised technologies, such as rapid gene synthesis, novel detergents, and protein thermostabilisation strategies allow conventionally 'undruggable' membrane proteins to be drugged. In this review, we survey the state-of-the-art gene design, expression and purification strategies, and protein thermostabilisation methods used within a modern drug discovery programme, with a focus on G protein-coupled receptors.


Assuntos
Descoberta de Drogas/métodos , Proteínas de Membrana/biossíntese , Receptores Acoplados a Proteínas-G/biossíntese , Cromatografia de Afinidade/métodos , Clonagem Molecular/métodos , Microscopia Crioeletrônica/métodos , Cristalografia por Raios X/métodos , Engenharia Genética , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Receptores Acoplados a Proteínas-G/química , Receptores Acoplados a Proteínas-G/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/ultraestrutura
11.
Am J Physiol Endocrinol Metab ; 318(4): E441-E452, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31935111

RESUMO

During pregnancy, the uterus transitions from a quiescent state to an excitable, highly contractile state to deliver the fetus. Two important contributors essential for this transition are hormones and ion channels, both of which modulate myometrial smooth muscle cell (MSMC) excitability. Recently, the sodium (Na+) leak channel, nonselective (NALCN), was shown to contribute to a Na+ leak current in human MSMCs, and mice lacking NALCN in the uterus had dysfunctional labor. Microarray data suggested that the proquiescent hormone progesterone (P4) and the procontractile hormone estrogen (E2) regulated this channel. Here, we sought to determine whether P4 and E2 directly regulate NALCN. In human MSMCs, we found that NALCN mRNA expression decreased by 2.3-fold in the presence of E2 and increased by 5.6-fold in the presence of P4. Similarly, E2 treatment decreased, and P4 treatment restored NALCN protein expression. Additionally, E2 significantly inhibited, and P4 significantly enhanced an NALCN-dependent leak current in MSMCs. Finally, we identified estrogen response and progesterone response elements (EREs and PREs) in the NALCN promoter. With the use of luciferase assays, we showed that the PREs, but not the ERE, contributed to regulation of NALCN expression. Our findings reveal a new mechanism by which NALCN is regulated in the myometrium and suggest a novel role for NALCN in pregnancy.


Assuntos
Estradiol/farmacologia , Canais Iônicos/biossíntese , Canais Iônicos/genética , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Miócitos de Músculo Liso/metabolismo , Miométrio/metabolismo , Progesterona/farmacologia , Adulto , Linhagem Celular , Feminino , Humanos , Mutação/genética , Miócitos de Músculo Liso/efeitos dos fármacos , Miométrio/efeitos dos fármacos , Gravidez , RNA Mensageiro/biossíntese , Elementos de Resposta/efeitos dos fármacos
12.
Neuron ; 105(2): 310-321.e3, 2020 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-31761710

RESUMO

Transmembrane channel-like (TMC) 1 and 2 are required for the mechanotransduction of mouse inner ear hair cells and localize to the site of mechanotransduction in mouse hair cell stereocilia. However, it remains unclear whether TMC1 and TMC2 are indeed ion channels and whether they can sense mechanical force directly. Here we express TMC1 from the green sea turtle (CmTMC1) and TMC2 from the budgerigar (MuTMC2) in insect cells, purify and reconstitute the proteins, and show that liposome-reconstituted CmTMC1 and MuTMC2 proteins possess ion channel activity. Furthermore, by applying pressure to proteoliposomes, we demonstrate that both CmTMC1 and MuTMC2 proteins can indeed respond to mechanical stimuli. In addition, CmTMC1 mutants corresponding to human hearing loss mutants exhibit reduced or no ion channel activity. Taken together, our results show that the CmTMC1 and MuTMC2 proteins are pore-forming subunits of mechanosensitive ion channels, supporting TMC1 and TMC2 as hair cell transduction channels.


Assuntos
Canais Iônicos/metabolismo , Mecanotransdução Celular/fisiologia , Proteínas de Membrana/fisiologia , Animais , Animais Geneticamente Modificados , Linhagem Celular , Feminino , Lipossomos/metabolismo , Melopsittacus , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Mutação , Spodoptera , Tartarugas
13.
Alcohol Alcohol ; 55(1): 3-10, 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-31845992

RESUMO

AIMS: We investigated the cardiac effects of ethanol withdrawal and the possible role of AT1 receptors in such response. METHODS: Male Wistar rats were treated with increasing doses of ethanol (3 to 9%, vol./vol.) for 21 days. The cardiac effects of ethanol withdrawal were investigated 48 h after abrupt discontinuation of ethanol. Some animals were orally treated with losartan (10 mg/kg/day), a selective AT1 receptor antagonist. RESULTS: Ethanol withdrawal did not affect serum levels of creatine kinase (CK)-MB. Losartan prevented ethanol withdrawal-induced increase in superoxide anion (O2•-) production in the left ventricle (LV). However, ethanol withdrawal did no alter the levels of thiobarbituric acid reactive substances (TBARS) or the expression of Nox1, Nox2 or Nox4 were found in the LV. Ethanol withdrawal reduced the concentration of hydrogen peroxide (H2O2) in the LV and this response was prevented by losartan. Ethanol withdrawal increased catalase activity in the LV and losartan attenuated this response. No changes on superoxide dismutase (SOD) activity or expression were detected in the LV during ethanol withdrawal. The expression of AT1, AT2 or angiotensin converting enzyme (ACE) was not affected by ethanol withdrawal. Similarly, no changes on the expression of ERK1/2, SAPK/JNK, COX-1 or COX-2 were found in the LV during ethanol withdrawal. CONCLUSIONS: Ethanol withdrawal altered the cardiac oxidative state through AT1-dependent mechanisms. Our findings showed a role for angiotensin II/AT1 receptors in the initial steps of the cardiac effects induced by ethanol withdrawal.


Assuntos
Etanol/efeitos adversos , Ventrículos do Coração/metabolismo , Receptor Tipo 1 de Angiotensina/biossíntese , Síndrome de Abstinência a Substâncias/metabolismo , Superóxidos/metabolismo , Animais , Catalase/metabolismo , Creatina Quinase Forma MB/sangue , Ciclo-Oxigenase 1/biossíntese , Ciclo-Oxigenase 2/biossíntese , Peróxido de Hidrogênio/metabolismo , Losartan/farmacologia , Masculino , Proteínas de Membrana/biossíntese , Proteína Quinase 1 Ativada por Mitógeno/biossíntese , Proteína Quinase 3 Ativada por Mitógeno/biossíntese , Proteína Quinase 8 Ativada por Mitógeno/biossíntese , NADPH Oxidases/biossíntese , Peptidil Dipeptidase A/biossíntese , Ratos , Receptor Tipo 2 de Angiotensina/biossíntese , Síndrome de Abstinência a Substâncias/sangue , Síndrome de Abstinência a Substâncias/prevenção & controle , Superóxido Dismutase/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismo
14.
J Med Chem ; 63(2): 847-879, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31860309

RESUMO

Trypanosoma protists are pathogens leading to a spectrum of devastating infectious diseases. The range of available chemotherapeutics against Trypanosoma is limited, and the existing therapies are partially ineffective and cause serious adverse effects. Formation of the PEX14-PEX5 complex is essential for protein import into the parasites' glycosomes. This transport is critical for parasite metabolism and failure leads to mislocalization of glycosomal enzymes, with fatal consequences for the parasite. Hence, inhibiting the PEX14-PEX5 protein-protein interaction (PPI) is an attractive way to affect multiple metabolic pathways. Herein, we have used structure-guided computational screening and optimization to develop the first line of compounds that inhibit PEX14-PEX5 PPI. The optimization was driven by several X-ray structures, NMR binding data, and molecular dynamics simulations. Importantly, the developed compounds show significant cellular activity against Trypanosoma, including the human pathogen Trypanosoma brucei gambiense and Trypanosoma cruzi parasites.


Assuntos
Proteínas de Membrana/antagonistas & inibidores , Proteínas de Protozoários/antagonistas & inibidores , Piridinas/síntese química , Piridinas/farmacologia , Tripanossomicidas/síntese química , Tripanossomicidas/farmacologia , Animais , Cristalografia por Raios X , Desenho de Fármacos , Humanos , Espectroscopia de Ressonância Magnética , Proteínas de Membrana/biossíntese , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Mioblastos/efeitos dos fármacos , Mioblastos/parasitologia , Proteínas de Protozoários/biossíntese , Ratos , Relação Estrutura-Atividade , Trypanosoma brucei gambiense/efeitos dos fármacos , Trypanosoma brucei gambiense/metabolismo , Trypanosoma brucei rhodesiense/efeitos dos fármacos
15.
Am J Surg Pathol ; 44(1): 61-67, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31498176

RESUMO

Prostate cancer is well known to metastasize to the testis and is not an uncommon finding on castration performed for advanced disease. Although germ cell tumors make up the majority of testis neoplasms, there are more rare tumors, such as rete testis adenocarcinoma, that can mimic metastatic disease. NKX3.1 and prostein (P501S) are antibodies highly specific for prostate origin. Relatively little is known of the expression of these markers in testicular tissue. We investigated the expression of NKX3.1 and P501S in testicular tissues, sex cord-stromal tumors, germ cell tumors, and rete testis adenocarcinoma. We found strong diffuse nuclear staining for NKX3.1 in Sertoli cells of the testis. Expression of NKX3.1 was seen in 0/3 ovarian Sertoli cell tumors, 1/4 testicular Sertoli cell tumors, and in the Sertoli cell component of 1/12 ovarian Sertoli-Leydig cell tumors. We found moderate, diffuse cytoplasmic positivity for P501S in rete testis epithelium and in testicular Leydig cells. P501S also highlighted Leydig cells in 9/12 Sertoli-Leydig cell tumors of the ovary. Two of 3 Leydig cell tumors of the testis showed weak to moderate, diffuse cytoplasmic staining for P501S. All cases of embryonal carcinoma and pure seminoma were negative for both NKX3.1 and P501S. One case of rete testis adenocarcinoma showed patchy positivity for both NKX3.1 and P501S. In conclusion, NKX3.1 shows routine expression in Sertoli cells and P501S shows routine expression in Leydig cells and rete testis epithelium. In addition, these markers can be positive in sex cord-stromal tumors and rete testis adenocarcinoma.


Assuntos
Adenocarcinoma/metabolismo , Biomarcadores Tumorais/biossíntese , Proteínas de Homeodomínio/biossíntese , Proteínas de Membrana/biossíntese , Neoplasias Ovarianas/metabolismo , Tumores do Estroma Gonadal e dos Cordões Sexuais/metabolismo , Neoplasias Testiculares/metabolismo , Fatores de Transcrição/biossíntese , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino
16.
Eur Rev Med Pharmacol Sci ; 23(23): 10324-10331, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31841187

RESUMO

OBJECTIVE: Oral squamous cell carcinoma (OSCC), the most frequent head and neck cancer, has a high potential for metastasis. MiR-126 plays an important role in the tumorigenesis of many tumors; however, there were little studies in OSCC. The purpose of our study was to explore how miR-126 and ADAM9 worked on migration and invasion in OSCC. PATIENTS AND METHODS: The quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) was applied to detect the mRNA level of miR-126 and ADAM9. The transwell assay was utilized to calculate the migratory and invasive capacities in the OSCC cells. The luciferase report assay was utilized to verify that ADAM9 was a direct target of miR-126. RESULTS: MicroR-126 was downregulated in OSCC tissues and cell lines SCC25 and HSC3. ADAM9 was predicted to be a direct target of miR-126 and was upregulated in the OSCC cells. In addition, miR-126 suppressed the migratory and invasive ability via mediating the expression of ADAM9 by directly targeting its mRNA 3'-noncoding region (UTR), whose partial functions was reversed by ADAM9. CONCLUSIONS: MiR-126 inhibited the migratory and invasive capacities of OSCC by directly targeting the 3'-UTR of ADAM9 mRNA. It is suggested that miR-126/ADAM9 axis may play an essential role in inhibiting the abilities of migration and invasion in oral squamous cell carcinoma cells.


Assuntos
Proteínas ADAM/fisiologia , Carcinoma de Células Escamosas/fisiopatologia , Movimento Celular/fisiologia , Proteínas de Membrana/fisiologia , MicroRNAs/fisiologia , Neoplasias Bucais/fisiopatologia , Invasividade Neoplásica/fisiopatologia , Regiões 3' não Traduzidas/fisiologia , Proteínas ADAM/biossíntese , Apoptose/fisiologia , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Proteínas de Membrana/biossíntese , MicroRNAs/biossíntese , Neoplasias Bucais/metabolismo , Regulação para Cima
17.
Asian Pac J Cancer Prev ; 20(12): 3679-3687, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31870109

RESUMO

BACKGROUND: Radioresistance remains a challenge for cancer radiotherapy. The present study aims to investigate the role of TMPRSS4 in triple negative breast cancer (TNBC) cell radiosensitivity. MATERIALS AND METHODS: After transfection of MDA-MD-468 triple negative breast cancer cells line by using the lentivirus vector, the effect of TMPRSS4 down-regulation on TNBC radiosensitivity was evaluated by using cloning assay and CCK-8 assay. The CCK-8 assay was also used for performing cell proliferation analysis. Western blot was carried out to detect the expression of certain proteins related to cell cycle pathways (cyclin D1), cell apoptosis pathways (Bax, Bcl2, and Caspase3), DNA damage and DNA damage repair (TRF2, Ku80 , Ë H2AX) . The cell cycle and cell apoptosis were also investigated using flow cytometer analysis. RESULTS: TMPRSS4 expression was down-regulated in MDA-MB-468 cells which enhanced MDA-MB-468 cells radiosensitivity. TMPRSS4 silencing also improved IR induced cell proliferation ability reduction and promoted cell arrested at G2/M phase mediated by 6 Gy IR associated with cyclin D1 expression inhibition. Moreover, TMPRSS4 inhibition enhanced TNBC apoptosis induced by 6 Gy IR following by over-expression of (Bax, Caspase3) and down-regulation of Bcl2 as the pro-apoptotic and anti-apoptotic proteins, respectively. Otherwise, TMPRSS4 down-regulation increases  DNA damage induced by 6 Gy IR and delays DNA damage repair respectively illustrated by downregulation of TRF2 and permanent increase of Ku80 and Ë H2AX expression at 1 h and 10 h post-IR. CONCLUSION: Down-regulation of TMPRSS4 increases triple negative breast cancer cell radiosensitivity and the use of TMPRSS4 inhibitor can be encouraged for improving radiotherapy effectiveness in TNBC radioresistant patients.


Assuntos
Apoptose/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/genética , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Tolerância a Radiação/genética , Serina Endopeptidases/biossíntese , Serina Endopeptidases/genética , Neoplasias de Mama Triplo Negativas/radioterapia , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/efeitos da radiação , Regulação para Baixo/genética , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos da radiação , Regulação Neoplásica da Expressão Gênica/genética , Humanos
18.
J Cereb Blood Flow Metab ; 39(12): 2355-2367, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31566073

RESUMO

RNA binding motif 3 (RBM3) is a powerful neuroprotectant that inhibits neurodegenerative cell death in vivo and is a promising therapeutic target in brain ischemia. RBM3 is increased by the hormone fibroblast growth factor 21 (FGF21) in an age- and temperature-dependent manner in rat cortical neurons. FGF21 receptor binding is controlled by the transmembrane protein ß-klotho, which is mostly absent in the adult brain. We discovered that RBM3/ß-klotho is unexpectedly high in the human infant vs. adult brain (hippocampus/prefrontal cortex). The use of tissue homogenates in that study precluded a comparison of RBM3/ß-klotho expression among different CNS cell-types, thus, omitted key evidence (i.e. confirmation of neuronal expression) that would otherwise provide a critical link to support their possible direct neuroprotective effects in humans. This report addresses that knowledge gap. High-quality fixed human hippocampus, cortex, and hypothalamic tissues were acquired from the NIH Neurobiobank (<1 yr (premature born) infants, 1 yr, 4 yr, and 34 yr). Dual labeling of cell-type markers vs. RBM3/ß-klotho revealed enriched staining of targets in neurons in the developing brain. Identifying that RBM3/ß-klotho is abundant in neurons in the immature brain is fundamentally important to guide protocol design and conceptual frameworks germane to future testing of these neuroprotective pathways in humans.


Assuntos
Encéfalo/crescimento & desenvolvimento , Regulação da Expressão Gênica , Proteínas de Membrana/biossíntese , Neurônios/metabolismo , Proteínas de Ligação a RNA/biossíntese , Adulto , Animais , Encéfalo/citologia , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Neurônios/citologia , Ratos
19.
Am J Physiol Renal Physiol ; 317(6): F1605-F1611, 2019 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-31566428

RESUMO

The transient receptor potential canonical 6 (TRPC6) channel and podocin are colocalized in the glomerular slit diaphragm as an important complex to maintain podocyte function. Gain of TRPC6 function and loss of podocin function induce podocyte injury. We have previously shown that high glucose induces apoptosis of podocytes by activating TRPC6; however, whether the activated TRPC6 can alter podocin expression remains unknown. Western blot analysis and confocal microscopy were used to examine both expression levels of TRPC6, podocin, and nephrin and morphological changes of podocytes in response to high glucose. High glucose increased the expression of TRPC6 but reduced the expression of podocin and nephrin, in both cultured human podocytes and type 1 diabetic rat kidneys. The decreased podocin was diminished in TRPC6 knockdown podocytes. High glucose elevated intracellular Ca2+ in control podocytes but not in TRPC6 knockdown podocytes. High glucose also elevated the expression of a tight junction protein, zonula occludens-1, and induced the redistribution of zonula occludens-1 and loss of podocyte processes. These data together suggest that high glucose reduces protein levels of podocin by activating TRPC6 and induces morphological changes of cultured podocytes.


Assuntos
Glucose/farmacologia , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/biossíntese , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/biossíntese , Podócitos/metabolismo , Canal de Cátion TRPC6/biossíntese , Animais , Cálcio/metabolismo , Linhagem Celular , Células Cultivadas , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Técnicas de Silenciamento de Genes , Humanos , Podócitos/efeitos dos fármacos , Ratos , Canal de Cátion TRPC6/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/biossíntese
20.
mBio ; 10(5)2019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31615956

RESUMO

Tail-anchored membrane proteins (TAMPs) are a distinct subset of inner membrane proteins (IMPs) characterized by a single C-terminal transmembrane domain (TMD) that is responsible for both targeting and anchoring. Little is known about the routing of TAMPs in bacteria. Here, we have investigated the role of TMD hydrophobicity in tail-anchor function in Escherichia coli and its influence on the choice of targeting/insertion pathway. We created a set of synthetic, fluorescent TAMPs that vary in the hydrophobicity of their TMDs and corresponding control polypeptides that are extended at their C terminus to create regular type II IMPs. Surprisingly, we observed that TAMPs have a much lower TMD hydrophobicity threshold for efficient targeting and membrane insertion than their type II counterparts. Using strains conditional for the expression of known membrane-targeting and insertion factors, we show that TAMPs with strongly hydrophobic TMDs require the signal recognition particle (SRP) for targeting. Neither the SecYEG translocon nor YidC appears to be essential for the membrane insertion of any of the TAMPs studied. In contrast, corresponding type II IMPs with a TMD of sufficient hydrophobicity to promote membrane insertion followed an SRP- and SecYEG translocon-dependent pathway. Together, these data indicate that the capacity of a TMD to promote the biogenesis of E. coli IMPs is strongly dependent upon the polypeptide context in which it is presented.IMPORTANCE A subset of membrane proteins is targeted to and inserted into the membrane via a hydrophobic transmembrane domain (TMD) that is positioned at the very C terminus of the protein. The biogenesis of these so-called tail-anchored proteins (TAMPs) has been studied in detail in eukaryotic cells. Various partly redundant pathways were identified, the choice for which depends in part on the hydrophobicity of the TMD. Much less is known about bacterial TAMPs. The significance of our research is in identifying the role of TMD hydrophobicity in the routing of E. coli TAMPs. Our data suggest that both the nature of the TMD and its role in routing can be very different for TAMPs versus "regular" membrane proteins. Elucidating these position-specific effects of TMDs will increase our understanding of how prokaryotic cells face the challenge of producing a wide variety of membrane proteins.


Assuntos
Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Membrana/biossíntese , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/química , Ligação Proteica
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