RESUMO
The aim of the present study was to investigate whether the acyl glucuronide of mycophenolic acid (AcMPAG) directly affects gene expression independent of guanosine (G) depletion. Human native mononuclear cells from healthy volunteers were studied. A concentration of 100 micromol/L (50 mg/L) AcMPAG, which provided effective inhibition of cell proliferation according to dose-response curves, was selected for gene expression analysis on microarray, verified by quantitative real-time polymerase chain reaction on the LightCycler. Differentially regulated genes on the microarray were 114 inosine monophosphate dehydrogenase-independent genes involved in cell proliferation, signal transduction, chemokine stimulation, endocytosis, vesicle transport, cell adhesion, and cytoskeleton. For verification, 16 genes, which were directly or indirectly related to cell proliferation, were selected for quantitative real-time polymerase chain reaction. SCNM1, ANP32E, CXCL13, CALM1, DKFZp451J0118, TPM3, CDC42, YWHAE, CXCL3, RDX, NDUFA3, and SOD1 showed no significant difference between the studied groups (P > 0.05). CCL1 gene expression was significantly regulated (P < 0.05) only in the mononuclear cell group treated with AcMPAG, whereas YWHAZ gene expression was significantly regulated only in the group treated with AcMPAG in presence of G and 8-aminoguanosine. The difference of interleukin 2 (IL2) and nucleobindin 1 (NUCB1) expression was significant between control and AcMPAG (P < 0.05) however not significant between AcMPAG in presence and absence of G and 8-aminoguanosine (P > 0.05). The expression of interleukin 2 and nucleobindin 1 revealed an effect of AcMPAG on gene expression independent of inosine monophosphate dehydrogenase inhibition.
Assuntos
Antibióticos Antineoplásicos/farmacologia , Proteínas de Ligação ao Cálcio/biossíntese , Proteínas de Ligação a DNA/biossíntese , Inibidores Enzimáticos/farmacologia , IMP Desidrogenase/antagonistas & inibidores , Interleucina-2/biossíntese , Monócitos/metabolismo , Ácido Micofenólico/farmacologia , Adulto , Proliferação de Células/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Glucuronídeos/farmacologia , Guanosina/farmacologia , Humanos , Masculino , Monócitos/efeitos dos fármacos , Proteínas do Tecido Nervoso , Nucleobindinas , Análise de Sequência com Séries de Oligonucleotídeos , RNA/biossíntese , RNA/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The aim of our study was to analyze the differential expression of miRNAs in the brains of BSE-infected cynomolgus macaques as a model for Creutzfeldt-Jakob disease (CJD). MicroRNAs (miRNAs) are small noncoding RNAs regulating gene expression by mRNA targeting. Among other functions they contribute to neuronal development and survival. Recently, the lack of miRNA processing has been shown to promote neurodegeneration and deregulation of several miRNAs has been reported to be associated with Scrapie in mice. Therefore, we hypothesized that miRNAs are also regulated in response to human prion disease. We have applied miRNA-microarrays to identify deregulated miRNA candidates in brains of BSE-infected macaques. Shock-frozen brain sections of six BSE-infected and five non-infected macaques were used to validate regulated miRNA candidates by two independent qRT-PCR-based methods. Our study revealed significant upregulation of hsa-miR-342-3p and hsa-miR-494 in the brains of BSE-infected macaques compared to non-infected animals. In a pilot study we could show that hsa-miR-342-3p was also upregulated in brain samples of human type 1 and type 2 sporadic CJD. With respect to the reported regulation of this miRNA in Scrapie-infected mice, we propose that upregulation of hsa-miR-342-3p may be a general phenomenon in late stage prion disease and might be used as a novel marker for animal and human TSEs.
RESUMO
Extracellular signal-regulated protein kinase 1 and 2 (ERK1/2) are members of the MAPK family and participate in the transduction of stimuli in cellular responses. Their long-term actions are accomplished by promoting the expression of specific genes whereas faster responses are achieved by direct phosphorylation of downstream effectors located throughout the cell. In this study we determined that hERK1 translocates to the mitochondria of HeLa cells upon a proliferative stimulus. In the mitochondrial environment, hERK1 physically associates with (i) at least 5 mitochondrial proteins with functions related to transport (i.e. VDAC1), signalling, and metabolism; (ii) histones H2A and H4; and (iii) other cytosolic proteins. This work indicates for the first time the presence of diverse ERK-complexes in mitochondria and thus provides a new perspective for assessing the functions of ERK1 in the regulation of cellular signalling and trafficking in HeLa cells.
Assuntos
Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Sequência de Aminoácidos , Proliferação de Células , Perfilação da Expressão Gênica , Glutationa Transferase/metabolismo , Células HeLa , Humanos , Mitocôndrias/metabolismo , Dados de Sequência Molecular , Proteômica/métodos , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
To identify novel genes that could be involved in oncogenesis of oral squamous cell carcinoma a microarray-based gene-expression analysis was performed using tumour samples from patients with low-stage (n=4) and high-stage (n=4) disease in a pilot study. Genes (601) were found to be significantly regulated in cancer tissue compared to adjacent intraindividual mucosa controls. Genes (25) were identified with differences in their regulation comparing samples from early-stage cancer with those from advanced disease. The gene expression pattern of 5 of 7 genes examined by real-time-PCR verified the results received from the microarray-experiment. Among these, FMO2, CPA6, TNC and SIAT1 were significantly upregulated in early disease stages. LGI1 gene expression was significantly enhanced in normal adjacent mucosa of patients with early-stage disease without showing a differential expression in carcinoma biopsies. With this pilot study several novel genes were identified, which could be related to early and late stage disease. Hypotheses from these findings are discussed and have to be confirmed in a larger study sample.
Assuntos
Carcinoma de Células Escamosas/metabolismo , Neoplasias Bucais/metabolismo , Proteínas de Neoplasias/metabolismo , Idoso , Idoso de 80 Anos ou mais , Antígenos CD/genética , Antígenos CD/metabolismo , Carboxipeptidases A/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Feminino , Secções Congeladas , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , Análise em Microsséries/métodos , Pessoa de Meia-Idade , Neoplasias Bucais/genética , Neoplasias Bucais/patologia , Proteínas de Neoplasias/genética , Estadiamento de Neoplasias , Oxigenases/genética , Oxigenases/metabolismo , Projetos Piloto , Proteínas/genética , Proteínas/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sialiltransferases/genética , Sialiltransferases/metabolismo , Tenascina/genética , Tenascina/metabolismo , Regulação para CimaRESUMO
The endoplasmic reticulum (ER) contains a highly effective protein quality control system eliminating malfolded proteins by a mechanism called ER-associated protein degradation (ERAD). Here, we unravel the topology of Der1p, a previously identified component of the ERAD system. Der1p contains four transmembrane domains, its N- and C-terminus protrude into the cytoplasm and contribute to its function. Additionally, we describe a yeast homologue of Der1p, Dfm1p, which does not seem to be involved in ERAD. In contrast, a Caenorhabditis elegans orthologue of Der1p, R151.6, is capable of complementing der1-defective phenotypes in yeast.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Sequência Conservada , DNA Fúngico/genética , Teste de Complementação Genética , Proteínas de Membrana/genética , Modelos Moleculares , Dados de Sequência Molecular , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos , SolubilidadeRESUMO
Proteins entering the endoplasmic reticulum (ER) have to acquire an export-competent structure before they are delivered to their final destination. This folding process is monitored by an ER protein quality control system. Folding-incompetent conformers are eliminated via a mechanism called ER-associated protein degradation (ERAD). Genetic studies in the yeast Saccharomyces cerevisiae have revealed that carbohydrate modification plays a crucial role in these processes. Here we show that a previously isolated der mutant (der7-1) is defective in ERAD. We identify DER7 as the gene encoding N-glycan-processing alpha-glucosidase I (EC 3.2.1.106) of the ER and demonstrate that its inactivity, due to a substitution of the conserved glycine residue at position 725 by arginine (G725R) in the der7-1 mutant, leads to ER-stress.
Assuntos
Retículo Endoplasmático/metabolismo , Glicoproteínas/metabolismo , Saccharomyces cerevisiae/enzimologia , alfa-Glucosidases/metabolismo , Saccharomyces cerevisiae/metabolismo , alfa-Glucosidases/química , alfa-Glucosidases/genéticaRESUMO
The endoplasmic reticulum (ER) harbors a protein quality control system, which monitors protein folding in the ER. Elimination of malfolded proteins is an important function of this protein quality control. Earlier studies with various soluble and transmembrane ER-associated degradation (ERAD) substrates revealed differences in the ER degradation machinery used. To unravel the nature of these differences we generated two type I membrane ERAD substrates carrying malfolded carboxypeptidase yscY (CPY*) as the ER-luminal ERAD recognition motif. Whereas the first, CT* (CPY*-TM), has no cytoplasmic domain, the second, CTG*, has the green fluorescent protein present in the cytosol. Together with CPY*, these three substrates represent topologically diverse malfolded proteins, degraded via ERAD. Our data show that degradation of all three proteins is dependent on the ubiquitin-proteasome system involving the ubiquitin-protein ligase complex Der3/Hrd1p-Hrd3p, the ubiquitin conjugating enzymes Ubc1p and Ubc7p, as well as the AAA-ATPase complex Cdc48-Ufd1-Npl4 and the 26S proteasome. In contrast to soluble CPY*, degradation of the membrane proteins CT* and CTG* does not require the ER proteins Kar2p (BiP) and Der1p. Instead, CTG* degradation requires cytosolic Hsp70, Hsp40, and Hsp104p chaperones.