RESUMO
Tissue transglutaminase is a ubiquitous and multifunctional protein that contributes to several processes such as apoptosis/survival, efferocytosis, inflammation and tissue repairing under physiological and pathological conditions. Several activities can be associated with well-established functional domains; in addition, four RNA alternative splice variants have been described, characterized by sequence divergences and residues deletion at the C-terminal domains. Tissue transglutaminase is recognized as the central player in the physiopathology of coeliac disease (CD) mainly through calcium-dependent enzymatic activities. It can be hypothesized that differential regulation of tissue transglutaminase splice variants expression in persons with CD contributes to pathology by altering the protein functionality. We characterized the expression pattern of RNA alternative splice variants by RT-PCR in peripheral cells from patients with CD under free gluten diet adhesion; we considered inflammatory parameters and specific antibodies as markers of the stage of disease. We found significant higher expression of both the full length and the shortest C-truncated splice variants in leucocytes from patients with CD in comparison with healthy individuals. As tissue transglutaminase expression and canonical enzymatic activity are linked to inflammation, we studied the RNA expression of inflammatory cytokines in peripheral leucocytes of persons with CD in relation with splice variants expression; interestingly, we found that recently diagnosed patients showed significant correlation between both the full length and the shortest alternative spliced variants with IL-1 expression. Our results points that regulation of alternative splicing of tissue transglutaminase could account for the complex physiopathology of CD.
Assuntos
Processamento Alternativo/genética , Doença Celíaca/genética , Doença Celíaca/patologia , Proteínas de Ligação ao GTP/genética , Leucócitos/imunologia , Transglutaminases/genética , Adulto , Idoso , Dieta Livre de Glúten , Feminino , Proteínas de Ligação ao GTP/biossíntese , Humanos , Interleucina-1/biossíntese , Interleucina-1/genética , Isoenzimas/biossíntese , Isoenzimas/genética , Masculino , Pessoa de Meia-Idade , Domínios Proteicos/genética , Proteína 2 Glutamina gama-Glutamiltransferase , RNA Mensageiro/genética , Transglutaminases/biossíntese , Adulto JovemRESUMO
The budding yeast Saccharomyces cerevisiae is a useful system to express recombinant proteins and analyze protein-protein interaction. Membrane-spanning proteins like plant receptor kinases find their way to the plasma membrane when expressed in yeast and seem to retain their structure and function. Here, we describe a general yeast DNA transformation procedure based on lithium acetate, salmon sperm DNA, and polyethylene glycol used to express recombinant proteins. Yeast cells expressing plant receptor kinases can be used for in vivo and in vitro studies of receptor function.
Assuntos
Clonagem Molecular/métodos , Vetores Genéticos/metabolismo , Proteína Quinase C/genética , Saccharomyces cerevisiae/genética , Solanum lycopersicum/química , Acetatos/farmacologia , Western Blotting/métodos , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Eletroforese em Gel de Poliacrilamida/métodos , Galactoquinase/genética , Galactoquinase/metabolismo , Expressão Gênica/efeitos dos fármacos , Vetores Genéticos/química , Isoenzimas/biossíntese , Isoenzimas/genética , Solanum lycopersicum/enzimologia , Solanum lycopersicum/genética , Fases de Leitura Aberta , Polietilenoglicóis/farmacologia , Regiões Promotoras Genéticas , Domínios Proteicos , Proteína Quinase C/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transformação GenéticaRESUMO
Alpha-amylases are ubiquitously distributed throughout microbials, plants and animals. It is widely accepted that omnivorous crustaceans have higher α-amylase activity and number of isoforms than carnivorous, but contradictory results have been obtained in some species, and carnivorous crustaceans have been less studied. In addition, the physiological meaning of α-amylase polymorphism in crustaceans is not well understood. In this work we studied α-amylase in a carnivorous lobster at the gene, transcript, and protein levels. It was showed that α-amylase isoenzyme composition (i.e., phenotype) in lobster determines carbohydrate digestion efficiency. Most frequent α-amylase phenotype has the lowest digestion efficiency, suggesting this is a favoured trait. We revealed that gene and intron loss have occurred in lobster α-amylase, thus lobsters express a single 1830 bp cDNA encoding a highly conserved protein with 513 amino acids. This protein gives rise to two isoenzymes in some individuals by glycosylation but not by limited proteolysis. Only the glycosylated isoenzyme could be purified by chromatography, with biochemical features similar to other animal amylases. High carbohydrate content in diet down-regulates α-amylase gene expression in lobster. However, high α-amylase activity occurs in lobster gastric juice irrespective of diet and was proposed to function as an early sensor of the carbohydrate content of diet to regulate further gene expression. We concluded that gene/isoenzyme simplicity, post-translational modifications and low Km, coupled with a tight regulation of gene expression, have arose during evolution of α-amylase in the carnivorous lobster to control excessive carbohydrate digestion in the presence of an active α-amylase.
Assuntos
Proteínas de Artrópodes , Carnivoridade/fisiologia , Regulação Enzimológica da Expressão Gênica/fisiologia , Palinuridae , alfa-Amilases , Animais , Proteínas de Artrópodes/biossíntese , Proteínas de Artrópodes/genética , DNA Complementar/genética , DNA Complementar/metabolismo , Glicosilação , Isoenzimas/biossíntese , Isoenzimas/genética , Palinuridae/genética , Palinuridae/metabolismo , Proteólise , alfa-Amilases/biossíntese , alfa-Amilases/genéticaRESUMO
BACKGROUND: Aldehyde dehydrogenase isoform 1 (ALDH1) has been shown to be a marker of cancer stem cells (CSCs). These stem cells may be responsible for tumour perpetuation as well as local and distant invasion. Several studies have shown that CSCs are more chemoradiotherapy (CRT)-resistant and may be responsible for tumour recurrence. Other studies, in contrast, have found ALDH1 expression to be indicative of a better prognosis. METHODS: We retrospectively evaluated 84 patients diagnosed and treated for laryngeal cancer between 2006 and 2011. All patients underwent curative-intent radiotherapy or CRT at our institution. 57 of the 84 tumour samples contained sufficient material for ALDH1 assessment. RESULTS: ALDH1 expression was detected in 17.5 % (10/57) of the tissue samples. None of the tumours from stage I patients tested positive for ALDH1. The relapse rate in ALDH1 + patients was 10 versus 51.2 % for ALDH1-. No differences in overall survival were observed between the groups; however, disease-free survival was 90 % for the ALDH1 + group versus 48.9 % for ALDH1- patients (p = 0.034). CONCLUSION: The patients in this study with ALDH1 + tumours had better outcomes than their counterparts with ALDH1- tumours. This finding suggests that not all CSCs are resistant to conventional cancer treatments. It may also imply that new methods of correctly identifying these cells are needed.
Assuntos
Biomarcadores Tumorais/análise , Isoenzimas/biossíntese , Neoplasias Laríngeas/patologia , Tolerância a Radiação/fisiologia , Retinal Desidrogenase/biossíntese , Adulto , Idoso , Idoso de 80 Anos ou mais , Família Aldeído Desidrogenase 1 , Intervalo Livre de Doença , Feminino , Humanos , Imuno-Histoquímica , Isoenzimas/análise , Estimativa de Kaplan-Meier , Neoplasias Laríngeas/enzimologia , Neoplasias Laríngeas/mortalidade , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/enzimologia , Recidiva Local de Neoplasia/mortalidade , Recidiva Local de Neoplasia/patologia , Células-Tronco Neoplásicas/enzimologia , Células-Tronco Neoplásicas/patologia , Prognóstico , Modelos de Riscos Proporcionais , Retinal Desidrogenase/análise , Estudos RetrospectivosRESUMO
Vinclozolin (V) is classified as a potent endocrine disruptor. The aim of the present study was to determine the effects of V on rat liver CYP regulation and on serum levels of testosterone and estradiol during pregnancy. Pregnancy decreased the liver total CYP content by 65%, enzyme activities of MROD, PROD, and PNPH, and testosterone hydroxylation activities, as well as the protein content of CYP2A and 3A. V exposure remarkably induced the protein content and enzyme activities of CYP1A, 2A, 2B and 3A subfamilies. Testosterone and estradiol were affected in an opposite manner, provoking a 3.5-fold increase in the estradiol/testosterone ratio. These results suggest that V could regulate the hepatic CYP expression through interaction with receptors and coactivators involved in its expression and may play an important role in hormonal balance during pregnancy. In addition, the results may also contribute to understanding the toxicity of V by in utero exposure.
Assuntos
Antagonistas de Androgênios/farmacologia , Sistema Enzimático do Citocromo P-450/metabolismo , Fígado/efeitos dos fármacos , Oxazóis/farmacologia , Animais , Estradiol/sangue , Feminino , Isoenzimas/biossíntese , Fígado/metabolismo , Gravidez/metabolismo , Ratos Wistar , Testosterona/sangueRESUMO
We postulate that protein kinase C α (PKCα) may contribute to the maintenance of pregnancy myometrial quiescence in humans. We studied the changes in myometrial PKCα gene products (messenger RNA [mRNA] and protein) in 4 groups of women: preterm not in labor (PT-NL), preterm in labor (PT-L), term not in labor (T-NL), and term in labor (T-L). The degree of PKCα activation was studied by comparing the levels of particulate (active) PKCα with the total PKCα protein levels and by measuring PKCα activity in the cytosolic and particulate fractions. Protein kinase Cα abundance (mRNA and protein) did not increase during myometrial quiescence (PT-NL), whereas the level of PKCα activity significantly increased during quiescence. The activity of PKCα significantly decreased in the T-NL, T-L, and PT-L groups. These findings suggest that PKCα plays a significant role in the maintenance of myometrial quiescence and that PKCα activity must decrease at the end of pregnancy allowing myometrial activation. Additionally, our data demonstrate an association between reduced PKCα activity and preterm labor, which merits further investigation.
Assuntos
Trabalho de Parto/metabolismo , Miométrio/enzimologia , Trabalho de Parto Prematuro/enzimologia , Proteína Quinase C-alfa/biossíntese , Biomarcadores/metabolismo , Ativação Enzimática/fisiologia , Feminino , Humanos , Isoenzimas/biossíntese , Isoenzimas/genética , Trabalho de Parto/genética , Trabalho de Parto Prematuro/genética , Gravidez , Proteína Quinase C-alfa/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genéticaRESUMO
The present study investigated the role of growth differentiation factor (GDF)-9 and FSH, alone or in combination, on the growth, viability and mRNA expression of FSH receptor, proliferating cell nuclear antigen (PCNA) and proteoglycan-related factors (i.e., hyaluronan synthase (HAS) 1, HAS2, versican, perlecan) in bovine secondary follicles before and after in vitro culture. After 12 days culture, sequential FSH (100 ng mL⻹) from Days 0 to 6 and 500 ng mL⻹ from Days 7 to 12) increased follicular diameter and resulted in increased antrum formation (P<0.05). Alone, 200 ng mL⻹ GDF-9 significantly reduced HAS1 mRNA levels, but increased versican and perlecan mRNA levels in whole follicles, which included the oocyte, theca and granulosa cells. Together, FSH and GDF-9 increased HAS2 and versican (VCAN) mRNA levels, but decreased PCNA mRNA expression, compared with levels in follicles cultured in α-minimum essential medium supplemented with 3.0 mg mL⻹ bovine serum albumin, 10 µg mL⻹ insulin, 5.5 µg mL⻹ transferrin, 5 ng mL⻹ selenium, 2 mM glutamine, 2mM hypoxanthine and 50 µg mL⻹ ascorbic acid (α-MEMâº). Comparisons of uncultured (0.2 mm) and α-MEM⺠cultured follicles revealed that HAS1 mRNA expression was higher, whereas VCAN expression was lower, in cultured follicles (P<0.05). Expression of HAS1, VCAN and perlecan (HSPG2) was higher in cultured than in vivo-grown (0.3 mm) follicles. In conclusion, FSH and/or GDF-9 promote follicular growth and antrum formation. Moreover, GDF-9 stimulates expression of versican and perlecan and interacts positively with FSH to increase HAS2 expression.
Assuntos
Hormônio Foliculoestimulante/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Fator 9 de Diferenciação de Crescimento/metabolismo , Técnicas de Maturação in Vitro de Oócitos/veterinária , Oogênese , Folículo Ovariano/metabolismo , RNA Mensageiro/metabolismo , Matadouros , Animais , Bovinos , Sobrevivência Celular , Feminino , Líquido Folicular/enzimologia , Líquido Folicular/metabolismo , Glucuronosiltransferase/antagonistas & inibidores , Glucuronosiltransferase/biossíntese , Glucuronosiltransferase/genética , Glucuronosiltransferase/metabolismo , Hialuronan Sintases , Isoenzimas/antagonistas & inibidores , Isoenzimas/biossíntese , Isoenzimas/genética , Isoenzimas/metabolismo , Oócitos/citologia , Oócitos/enzimologia , Oócitos/metabolismo , Folículo Ovariano/citologia , Folículo Ovariano/crescimento & desenvolvimento , Antígeno Nuclear de Célula em Proliferação/biossíntese , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteoglicanas/antagonistas & inibidores , Proteoglicanas/biossíntese , Proteoglicanas/genética , Proteoglicanas/metabolismo , Receptores do FSH/antagonistas & inibidores , Receptores do FSH/biossíntese , Receptores do FSH/genética , Receptores do FSH/metabolismo , Técnicas de Cultura de Tecidos/veterináriaRESUMO
Cysteine proteases (CPs) from the C1 family, which are similar to papain, can be found in animals and plants, as well as some viruses and prokaryotes. These enzymes have diverse physiological functions and are thus very attractive for science and industry. Jacaratia mexicana, a member of the Caricaceae plant family, contains several CPs, the principal being mexicain, found to favorably compete against papain for many industrial applications due to its high stability and specific activity. In this study, leaves of J. mexicana were used to isolate a CP-coding gene, similar to those that code for mexicain and chymomexicain. By using rapid amplification of cDNA ends (RACE) as well as oligonucleotide design from papain-like conserved amino acids (aa), a sequence of 1404 bp consisting of a 5' terminal untranslated region (UTR) of 153 bp, a 3' terminal UTR of 131 bp, with a polyadenylation (poly(A)) signal sequence and a poly(A) tail, and an open reading frame (ORF) of 1046 bp, was obtained by overlapping three partial sequences. Two full-length cDNA sequences that encode for mexicain-like proteases were cloned from mRNA (JmCP4 and JmCP5). JmCP4 is predicted to have an ORF of 1044 bp, which codifies for polypeptides that have a 26 aa signal peptide region, a 108 aa propeptide region and a mature enzyme of 214 aa. A 969 bp fragment (JmCP5) encodes for a partial sequence of a CP gene, without the signal peptide region but with a full-length propeptide region. The sequence analysis showed that this protease presented a high similarity to other plant CPs from J. mexicana, Vasconcellea cundinamarcensis, Vasconcellea stipulata, and Carica papaya, among others, mainly at the conserved catalytic site. Obtaining the sequence of this CP gene from J. mexicana provides an alternative for production in a standard system and could be an initial step towards the commercialization of this enzyme.
Assuntos
Caricaceae/genética , Cisteína Proteases/genética , Proteínas de Plantas/genética , Precursores de Proteínas/genética , RNA de Plantas/isolamento & purificação , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência de Bases , Caricaceae/enzimologia , Domínio Catalítico , Cisteína Proteases/biossíntese , Cisteína Proteases/química , DNA Complementar/biossíntese , Estabilidade Enzimática , Isoenzimas/biossíntese , Isoenzimas/química , Isoenzimas/genética , Modelos Moleculares , Dados de Sequência Molecular , Folhas de Planta/genética , Proteínas de Plantas/biossíntese , Proteínas de Plantas/química , Precursores de Proteínas/química , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificação , RNA de Plantas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Homologia Estrutural de ProteínaRESUMO
The autoinhibition/activation of the PMCA (plasma membrane Ca2+-ATPase) involves conformational changes in the membrane region of the protein that affect the amount of lipids directly associated with the transmembrane domain. The lipid-protein-dependence of PMCA isoforms 2 and 4 expressed and obtained in purified form from Saccharomyces cerevisiae was investigated using the phosphatidylcholine analogue [125I]TID-PC/16 {l-O-hexadecanoyl-2-O-[9-[[[2-[125I]iodo-4-(trifluoromemyl-3H-diazirin-3-yl)benzyl]oxy]carbonyl]nonanoyl]-sn-glycero-3-phosphocholine}, which was incorporated into mixtures of dimyristoylphosphatidylcholine and the non-ionic detergent C12E10 [deca(ethylene glycol) dodecyl ether]. We found no differences between the recombinant PMCA4 and PMCA purified from erythrocytes (ePMCA). However, titration of the half-maximal activation by Ca2+/calmodulin of PMCA2 showed 30-fold higher affinity than PMCA4. PMCA2 exhibited a lower level of labelling in the autoinhibited conformation relative to PMCA4, indicating that the lower autoinhibition was correlated with a lower exposure to lipids in the autoinhibited state. Analysis of the lipid-protein stoichiometry showed that the lipid annulus of PMCA varies: (i) in accordance to the conformational state of the enzyme; and (ii) depending on the different isoforms of PMCA. PMCA2 during Ca2+ transport changes its conformation to a lesser extent than PMCA4, an isoform more sensitive to modulation by calmodulin and acidic phospholipids. This is the first demonstration of a dynamic behaviour of annular lipids and PMCA.
Assuntos
Ativação Enzimática , Fosfolipídeos/química , ATPases Transportadoras de Cálcio da Membrana Plasmática/química , Animais , Calmodulina/química , Cromatografia de Afinidade , Eritrócitos/enzimologia , Humanos , Isoenzimas/biossíntese , Isoenzimas/química , Isoenzimas/isolamento & purificação , ATPases Transportadoras de Cálcio da Membrana Plasmática/biossíntese , ATPases Transportadoras de Cálcio da Membrana Plasmática/isolamento & purificação , Ligação Proteica , Conformação Proteica , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Saccharomyces cerevisiae , Coloração e Rotulagem , TitulometriaRESUMO
PP2A is the main serine/threonine-specific phosphatase in animal cells. The active phosphatase has been described as a holoenzyme consisting of a catalytic, a scaffolding, and a variable regulatory subunit, all encoded by multiple genes, allowing for the assembly of more than 70 different holoenzymes. The catalytic subunit can also interact with α4, TIPRL (TIP41, TOR signaling pathway regulator-like), the methyl-transferase LCMT-1, and the methyl-esterase PME-1. Here, we report that the gene encoding the catalytic subunit PP2Acα can generate two mRNA types, the standard mRNA and a shorter isoform, lacking exon 5, which we termed PP2Acα2. Higher levels of the PP2Acα2 mRNA, equivalent to the level of the longer PP2Acα mRNA, were detected in peripheral blood mononuclear cells that were left to rest for 24 h. After this time, the peripheral blood mononuclear cells are still viable and the PP2Acα2 mRNA decreases soon after they are transferred to culture medium, showing that generation of the shorter isoform depends on the incubation conditions. FLAG-tagged PP2Acα2 expressed in HEK293 is catalytically inactive. It displays a specific interaction profile with enhanced binding to the α4 regulatory subunit, but no binding to the scaffolding subunit and PME-1. Consistently, α4 out-competes PME-1 and LCMT-1 for binding to both PP2Acα isoforms in pulldown assays. Together with molecular modeling studies, this suggests that all three regulators share a common binding surface on the catalytic subunit. Our findings add important new insights into the complex mechanisms of PP2A regulation.