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Environmental stresses disturb the endoplasmic reticulum (ER) protein folding. However, primary metabolic responses induced by ER stress remain unclear. Thus, we investigated the morphophysiological and metabolomic changes under ER stress, induced by dithiothreitol (DTT) and tunicamycin (TM) treatments in sorghum seedlings from 24 to 96 h. The ER stress caused lipid peroxidation and increased the expression of SbBiP1, SbPDI, and SbIRE1. The development impairment was more pronounced in roots than in shoots as distinct metabolomic profiles were observed. DTT decreased root length, lateral roots, and root hair, while TM decreased mainly the root length. At 24 h, under ER stresses, the glutamic acid and o-acetyl-serine were biomarkers in the shoots. While homoserine, pyroglutamic acid, and phosphoric acid were candidates for roots. At the latest time (96 h), kestose and galactinol were key metabolites for shoots under DTT and TM, respectively. In roots, palatinose, trehalose, and alanine were common markers for DTT and TM late exposure. The accumulation of sugars such as arabinose and kestose occurred mainly in roots in the presence of DTT at a later time, which also inhibited glycolysis and the tricarboxylic acid cycle (TCA). Amino acid metabolism was induced, which also contributed TCA components decreasing, such as succinate in shoots and citrate in roots. Thus, our study may provide new insights into primary metabolism modulated by ER stress and seedling development.

Assuntos

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Transforming growth factor-beta 1 (TGF-ß1) is a cytokine with marked pro-fibrotic action on cardiac fibroblasts (CF). TGF-ß1 induces CF-to-cardiac myofibroblast (CMF) differentiation, defined by an increase in α-smooth muscle cells (α-SMA), collagen secretion and it has a cytoprotective effect against stimuli that induce apoptosis. In the Endoplasmic Reticulum (ER) lumen, misfolded protein accumulation triggers ER stress and induces apoptosis, and this process plays a critical role in cell death mediated by Ischemia/Reperfusion (I/R) injury and by ER stress inducers, such as Tunicamycin (Tn). Here, we studied the regulation of CHOP, a proapoptotic ER-stress-related transcription factor in CF under simulated I/R (sI/R) or exposed to Tn. Even though TGF-ß1 has been shown to participate in ER stress, its regulatory effect on CF apoptosis and ER stress-induced by sI/R or TN has not been evaluated yet. CF from neonatal rats were exposed to sI/R, and cell death was evaluated by cell count and apoptosis by flow cytometry. ER stress was assessed by western blot against CHOP. Our results evidenced that sI/R (8/24) h or Tn triggers CF apoptosis and an increase in CHOP protein levels. TGF-ß1 pre-treatment partially prevented apoptosis induced by sI/R or Tn. Furthermore, TGF-ß1 pre-treatment completely prevented CHOP increase by sI/R or Tn. Additionally, we found a decrease in α-SMA expression induced by sI/R and in collagen secretion induced by Tn, which were not prevented by TGF-ß1 treatment. In conclusion, TGF-ß1 partially protects CF apoptosis induced by sI/R or Tn, through a mechanism that would involve ER stress.

Assuntos

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lysA gene encoding meso-diaminopimelic acid (DAP) decarboxylase enzyme that catalyzes L-lysine biosynthesis in the aspartate pathway in Streptomyces clavuligerus was overexpressed, and its effects on cephamycin C (CephC), clavulanic acid (CA), and tunicamycin productions were investigated. Multicopy expression of lysA gene under the control of glpF promoter (glpFp) in S. clavuligerus pCOlysA led to higher expression levels ranging from 2- to 6-fold increase at both lysA gene and CephC biosynthetic gene cluster at T36 and T48 of TSBG fermentation. These results accorded well with CephC production. Thus, 1.86- and 3.14-fold higher volumetric as well as 1.26- and 1.71-fold increased specific CephC yields were recorded in S. clavuligerus pCOlysA in comparison with the wild-type and its control strain, respectively, at 48th h. Increasing the expression of lysA provided 4.3 times more tunicamycin yields in the recombinant strain. These findings suggested that lysA overexpression in S. clavuligerus made the strain more productive for CephC and tunicamycin. The results also supported the presence of complex interactions among antibiotic biosynthesis pathways in S. clavuligerus.

Assuntos

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Melanoma is responsible for most deaths among skin cancers and conventional and palliative care chemotherapy are limited due to the development of chemoresistance. We used proteomic analysis to identify cellular responses that lead to chemoresistance of human melanoma cell lines to cisplatin. A systems approach to the proteomic data indicated the participation of specific cellular processes such as oxidative phosphorylation, mitochondrial organization and homeostasis, as well as the unfolded protein response (UPR) to be required for the survival of cells treated with cisplatin. Prohibitin (PHB) was among the proteins consistently accumulated, interacting with the functional clusters associated with resistance to cisplatin. We showed PHB accumulated at different levels in melanoma cell lines under stressing stimuli, such as (i) treatment with temozolomide (TMZ), dacarbazine (DTIC) and cisplatin; (ii) serum deprivation; (iii) tunicamycin, an UPR inducer. Prohibitin accumulated in the mitochondria of melanoma cells after cisplatin and tunicamycin treatment and its de novo accumulation led to chemoresistance melanoma cell lines. In contrast, PHB knock-down sensitized melanoma cells to cisplatin and tunicamycin treatment. We conclude that PHB participates in the survival of cells exposed to different stress stimuli, and can therefore serve as a target for the sensitization of melanoma cells to chemotherapy.

Assuntos

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Parkinson's disease (PD) is a neurodegenerative disorder characterized by progressive death of dopaminergic neurons of the substantia nigra pars compacta (SNpc), leading to the major clinical abnormalities that characterize this disease. Although PD's etiology is unknown, α-synuclein aggregation plays a pivotal role in PD pathogenesis, which could be associated to some pathological processes such as oxidative stress, endoplasmic reticulum (ER) stress, impaired protein degradation, and mitochondrial dysfunction. Increasing experimental evidence indicates that ER stress is involved in PD, however most of the described results employed cultured cell lines and genetically modified animal models. In this study, we developed a new ER stress rat model employing the well-known ER stressor tunicamycin (Tm). To evaluate if ER stress was able to induce PD features, we performed an intranigral injection of Tm (0.1 µg/cerebral hemisphere) and animals (male Wistar rats) were analyzed 7 days post injection. The classical 6-OHDA neurotoxin model (1 µg/cerebral hemisphere) was used as an established positive control for PD. We show that Tm injection induced locomotor impairment, dopaminergic neurons death, and activation of astroglia. In addition, we observed an extensive α-synuclein oligomerization in SNpc of Tm-injected animals when compared with DMSO-injected controls. Finally, both Tm and 6-OHDA treated animals presented increased levels of ER stress markers. Taken together, these findings show for the first time that the ER stressor Tm recapitulates some of the phenotypic characteristics observed in rodent models of PD, reinforcing the concept that ER stress could be an important contributor to the pathophysiology of PD. Therefore, we propose the intranigral Tm injection as a new ER stress-based model for the study of PD in vivo.

Assuntos

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The sustainability of global crop production is critically dependent on improving tolerance of crop plants to various types of environmental stress. Thus, identification of genes that confer stress tolerance in crops has become a top priority especially in view of expected changes in global climatic patterns. Drought stress is one of the abiotic stresses that can result in dramatic loss of crop productivity. In this work, we show that transgenic expression of a highly conserved cell death suppressor, Bax Inhibitor-1 from Arabidopsis thaliana (AtBI-1), can confer increased tolerance of sugarcane plants to long-term (>20 days) water stress conditions. This robust trait is correlated with an increased tolerance of the transgenic sugarcane plants, especially in the roots, to induction of endoplasmic reticulum (ER) stress by the protein glycosylation inhibitor tunicamycin. Our findings suggest that suppression of ER stress in C4 grasses, which include important crops such as sorghum and maize, can be an effective means of conferring improved tolerance to long-term water deficit. This result could potentially lead to improved resilience and yield of major crops in the world.

Assuntos

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Bufo arenarum eggs at late blastula and gastrula were treated with tunicamycin, an inhibitor of glycoprotein glycosylation, to investigate its effects on morphogenesis and neural induction. Because of the low permeability of the amphibian egg to a number of drugs, the blastocoel was opened surgically prior to treatment. Almost all of the eggs treated with the antimetabolite, at a concentration of 10 µg/ml, from late blastula stage for 24h exhibited exogastrulation. The effect is dose- and stage-dependent as shown by the lower proportion of exogastrulae obtained when eggs are treated at a lower concentration (5 µg/ml) or after the onset of gastrulation. Treatment with the antimetabolite did not interfere with neural induction, as partial exogastrulae developed a small neural tube. The most striking biochemical effect was an enhanced uptake of glucose, mannose and leucine. The incorporation of mannose into acid-insoluble material was severely inhibited by tunicamycin, with a concomitant decrease of leucine incorporation into the acid-soluble pool.