1,720,986 research outputs found
Overexpression of hsa-miR-663a reduces expression of PLOD3 encoded LH3 protein and secretion of Collagen IV
No full textIn the past decade, microRNAs have been uncovered as key regulators of gene expression at post-transcriptional level by the mean of their ability to interact with complementary regions of transcripts and gain repression of translation or RNA degradation. In the attempt to discover novel miRNA induced by ER stress and identify their target genes we performed a combined analysis by the use of miRNA array and 2D-DIGE/mass-spectrometry approach. Comparative analysis of the microRNA profiles revealed that, in the cells exposed to the ER stress inducer Thapsigargin (TG), twenty-four miRNAs are differentially expressed. Among them, hsa-miR-663a turned out to be the most significant TG induced miR. Interestingly, ontological analysis showed that about 26% of predicted miR-663a target genes are related to secretory pathway functions. To identify, miR-663a target genes we performed proteomic analysis by the combined use of 2D-DIGE and mass spectrometry of cells over-expressing miR-663a revealing that miR-663a could potentially target a number of genes having a function within the secretory pathway. In particular, we show that miR-663a, directly targets PLOD3 mRNA encoding Lysyl hydroxylase 3 (LH3) protein, a multifunctional collagen-modifying enzyme. In miR-663a over expressing cells, miR-663a reduces LH3 expression. Reduction of the galactosylhydroxylysine-glucosyltransferase (GGT) activity of LH3 disrupts the localization of type IV collagen, which showed retention in the Endoplasmic Reticulum. Our results suggest that miR-663a could modulate collagen secretion in pathophysiological conditions
Structural and Functional Significance of the Endoplasmic Reticulum Unfolded Protein Response Transducers and Chaperones at the Mitochondria–ER Contacts: A Cancer Perspective
No full textIn the last decades, the endoplasmic reticulum (ER) has emerged as a key coordinator of cellular homeostasis, thanks to its physical interconnection to almost all intracellular organelles. In particular, an intense and mutual crosstalk between the ER and mitochondria occurs at the mitochondria–ER contacts (MERCs). MERCs ensure a fine-tuned regulation of fundamental cellular processes, involving cell fate decision, mitochondria dynamics, metabolism, and proteostasis, which plays a pivotal role in the tumorigenesis and therapeutic response of cancer cells. Intriguingly, recent studies have shown that different components of the unfolded protein response (UPR) machinery, including PERK, IRE1α, and ER chaperones, localize at MERCs. These proteins appear to exhibit multifaceted roles that expand beyond protein folding and UPR transduction and are often related to the control of calcium fluxes to the mitochondria, thus acquiring relevance to cell survival and death. In this review, we highlight the novel functions played by PERK, IRE1α, and ER chaperones at MERCs focusing on their impact on tumor development
Endoplasmic reticulum stress reduces COPII vesicles formation and modifies Sec23a cycling at ERESs G. Amodio1, O. Moltedo1, S. Franceschelli1, P. Remondelli2 1Dipartimento di Farmacia, Univ. of Salerno, Italy 2Dipartimento di Medicina e Chirurgia, Univ. of Salerno, Italy
No full textCOPII vesicles bud from the ER at ER Exit Sites (ERESs) to mediate the exit from the Endoplasmic Reticulum (ER) of newly synthesized proteins. Previously, we demonstrated that ER Stress rapidly impairs the anterograde transport to the Golgi complex and the formation of COPII vesicles (Amodio et al., 2009). In a recent work (Amodio et al., 2013) we found that the reduced permanence of Sec23a at the ERES could be the mean through which ER Stress modulates COPII assembling and vesicular trafficking. Sec23a is one of the component of the COPII vesicles coat and its GTPase activating function on Sar1 is one of the key mechanisms of COPII assembly. Interestingly, we found that during ER Stress the association to the ER membrane of Sec23a is reduced. Concomitantly, FRAP and FLIP analysis of Sec23a revealed that ER stress accelerates its recycling kinetics on ER membrane. These results prompted us to analyze the role of post-translational modifications of Sec23a in the regulation of its function during ER Stress. Surprisingly, we found that Sec23a is mono-ubiquitinated in mammalian cells on two different cysteines and that the induction of ER stress reduces the amount of mono-ubiquitinated Sec23a. The biological scope of Sec23a cysteine mono-ubiquitination has yet to be elucidated but recent evidences demonstrating that ubiquitination on cysteines regulates signal transduction and membrane translocation (Grou et al., 2008; Shannon and Weerapana, 2013) open new fields of investigation about Sec23a ubiquitination and modulation of COPII function
Sec23a as the key molecular target of ER Stress dependent modulation of COPII assembling G. Amodio1, O. Moltedo1, V. Pecoraro1, P. Remondelli2 1Dipartimento di Farmacia, Univ. of Salerno, Italy 2Dipartimento di Medicina e Chirurgia , Univ. of Salerno, Italy
No full textExit from the Endoplasmic Reticulum (ER) of newly synthesized proteins is mediated by COPII vesicles that bud from the ER at the ER Exit Sites (ERESs). Disruption of ER homeostasis causes accumulation of unfolded and misfolded proteins in the ER. This condition is referred as ER stress. Several evidences suggest a link between the ER stress and the vesicular trafficking within of the early secretory pathway. Previously, we demonstrated that ER Stress rapidly impairs the anterograde transport to the Golgi complex and the formation of COPII vesicles. Sec23a is one of the component of the COPII vesicles coat and its GTPase activating function on Sar1 is one of the key mechanisms of COPII assembly. Interestingly, we found that ER Stress reduces the association to the ER membrane of Sec23a. Concomitantly, FRAP and FLIP analysis of Sec23a revealed that ER stress accelerates its recycling kinetics on ER membrane. The reduced permanence of Sec23a at the ERES could be the mean through which ER Stress modulates COPII assembling and vesicular trafficking. Moreover, we found that Sec23a is mono-ubiquitinated in mammalian cells in two different sites of its second β-barrel domainand that the induction of ER stress reduces the amount of mono-ubiquitinated Sec23a. Noteworthy, this modification is a mono-ubiquitination on two different cysteines of the β-Barrel domain that is implicated in the binding of Sec23a to the ER membrane. Emerging evidences have demonstrated that ubiquitination on cysteines is implicated in many processes including signal transduction and membrane translocation. The regulatory function of cysteine ubiquitination, its localization in the β-Barrel domain and its impairment during ER stress strictly support the idea that Sec23a is the moleculartarget of ER Stress-dependent modulation of vesicular trafficking and that the regulation of Sec23a ubiquitination is the molecular mechanism involved in this phenomena
Targeting the Endoplasmic Reticulum Unfolded Protein Response to Counteract the Oxidative Stress-Induced Endothelial Dysfunction
Full text linkIn endothelial cells, the tight control of the redox environment is essential for the maintenance of vascular homeostasis. The imbalance between ROS production and antioxidant response can induce endothelial dysfunction, the initial event of many cardiovascular diseases. Recent studies have revealed that the endoplasmic reticulum could be a new player in the promotion of the pro- or antioxidative pathways and that in such a modulation, the unfolded protein response (UPR) pathways play an essential role. The UPR consists of a set of conserved signalling pathways evolved to restore the proteostasis during protein misfolding within the endoplasmic reticulum. Although the first outcome of the UPR pathways is the promotion of an adaptive response, the persistent activation of UPR leads to increased oxidative stress and cell death. This molecular switch has been correlated to the onset or to the exacerbation of the endothelial dysfunction in cardiovascular diseases. In this review, we highlight the multiple chances of the UPR to induce or ameliorate oxidative disturbances and propose the UPR pathways as a new therapeutic target for the clinical management of endothelial dysfunction
In the Huh7 Hepatoma Cells Diclofenac and Indonethacin Activate Differently the Unfolded Protein Response and Induce ER Stress Apoptosis
No full textNon-steroidal anti-inflammatory drugs (NSAIDs) are cyclooxygenases (COXs) inhibitors frequently used in the treatment of acute and chronic inflammation. Side effects of NSAIDs are often due to their ability to induce apoptosis. Located at the Endoplasmic Reticulum membranes a tripartite signalling pathway, collectively known as the Unfolded Protein Response (UPR), decides survival or death of cells exposed to cytotoxic agents. To shed light on the molecular events responsible for the cytotoxicity of NSAIDs, we analysed the ability of diclofenac and indomethacin to activate the UPR in the human hepatoma cell line Huh7. We report that both NSAIDs can induce differently the single arms of the UPR. We show that indomethacin turns on the PERK and, only in part, the ATF6 and IRE1 pathways. Instead, diclofenac reduces the expression of ATF6 and does not stimulate the IRE1 endonuclease, which drives the expression of the prosurvival factor XBP1. Diclofenac, as well as indomethacin, is able to activate efficiently only the PERK pathway of the UPR, which induces the expression of the proapoptotic GADD153/CHOP protein. Our results highlight the importance of the UPR in evaluating the potential of drugs to induce apoptosis
Identification of Cysteine Ubiquitylation Sites on the Sec23A Protein of the COPII Complex Required for Vesicle Formation from the ER
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The urokinase receptor takes control of cell migration through a mechanism involving integrins and fMLF receptors.
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