scholarly journals Mitochondria maintain maturation and secretion of lipoprotein lipase in the endoplasmic reticulum

2006 ◽  
Vol 396 (1) ◽  
pp. 173-182 ◽  
Author(s):  
Karin Osibow ◽  
Sasa Frank ◽  
Roland Malli ◽  
Rudolf Zechner ◽  
Wolfgang F. Graier
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Protein Folding ◽  
Lipoprotein Lipase ◽  
Chinese Hamster ◽  
Protein Levels ◽  
Dependent Activity ◽  
Dependent Protein ◽  
The Impact ◽  
Reversible Reduction

Considering the physiological Ca2+ dynamics within the ER (endoplasmic reticulum), it remains unclear how efficient protein folding is maintained in living cells. Thus, utilizing the strictly folding-dependent activity and secretion of LPL (lipoprotein lipase), we evaluated the impact of ER Ca2+ content and mitochondrial contribution to Ca2+-dependent protein folding. Exhaustive ER Ca2+ depletion by inhibition of sarcoplasmic/endoplasmic reticulum Ca2+-ATPases caused strong, but reversible, reduction of cell-associated and released activity of constitutive and adenovirus-encoded human LPL in CHO-K1 (Chinese-hamster ovary K1) and endothelial cells respectively, which was not due to decline of mRNA or intracellular protein levels. In contrast, stimulation with the IP3 (inositol 1,4,5-trisphosphate)-generating agonist histamine only moderately and transiently affected LPL maturation in endothelial cells that paralleled a basically preserved ER Ca2+ content. However, in the absence of extracellular Ca2+ or upon prevention of transmitochondrial Ca2+ flux, LPL maturation discontinued upon histamine stimulation. Collectively, these data indicate that Ca2+-dependent protein folding in the ER is predominantly controlled by intraluminal Ca2+ and is largely maintained during physiological cell stimulation owing to efficient ER Ca2+ refilling. Since Ca2+ entry and mitochondrial Ca2+ homoeostasis are crucial for continuous Ca2+-dependent protein maturation in the ER, their pathological alterations may result in dysfunctional protein folding.

scholarly journals p32 protein levels are integral to mitochondrial and endoplasmic reticulum morphology, cell metabolism and survival

2013 ◽  
Vol 453 (3) ◽  
pp. 381-391 ◽  
Author(s):  
MengJie Hu ◽  
Simon A. Crawford ◽  
Darren C. Henstridge ◽  
Ivan H. W. Ng ◽  
Esther J. H. Boey ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Death ◽  
Stress Responses ◽  
Binding Protein ◽  
Cell Metabolism ◽  
Mitochondrial Protein ◽  
Basal Respiration ◽  
Protein Levels ◽  
Stress Dependent ◽  
The Impact

p32 [also known as HABP1 (hyaluronan-binding protein 1), gC1qR (receptor for globular head domains complement 1q) or C1qbp (complement 1q-binding protein)] has been shown previously to have both mitochondrial and non-mitochondrial localization and functions. In the present study, we show for the first time that endogenous p32 protein is a mitochondrial protein in HeLa cells under control and stress conditions. In defining the impact of altering p32 levels in these cells, we demonstrate that the overexpression of p32 increased mitochondrial fibrils. Conversely, siRNA-mediated p32 knockdown enhanced mitochondrial fragmentation accompanied by a loss of detectable levels of the mitochondrial fusion mediator proteins Mfn (mitofusin) 1 and Mfn2. More detailed ultrastructure analysis by transmission electron microscopy revealed aberrant mitochondrial structures with less and/or fragmented cristae and reduced mitochondrial matrix density as well as more punctate ER (endoplasmic reticulum) with noticeable dissociation of their ribosomes. The analysis of mitochondrial bioenergetics showed significantly reduced capacities in basal respiration and oxidative ATP turnover following p32 depletion. Furthermore, siRNA-mediated p32 knockdown resulted in differential stress-dependent effects on cell death, with enhanced cell death observed in the presence of hyperosmotic stress or cisplatin treatment, but decreased cell death in the presence of arsenite. Taken together, our studies highlight the critical contributions of the p32 protein to the morphology of mitochondria and ER under normal cellular conditions, as well as important roles of the p32 protein in cellular metabolism and various stress responses.

scholarly journals ET-1 mediated vasoconstriction leads to memory impairment and synaptic dysfunction reminiscent of Alzheimer’s disease phenotype

2020 ◽  
Author(s):  
Latha Diwakar ◽  
Ruturaj Gowaikar ◽  
Keerthana Chithanathan ◽  
Gnanabharathi ◽  
Deepika Singh Tomar ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Blood Flow ◽  
Synaptic Plasticity ◽  
Spatial Memory ◽  
Memory Impairment ◽  
Protein Translation ◽  
Mtor Signaling ◽  
Dependent Protein ◽  
The Impact ◽  
Activity Dependent

Abstract Background Cerebrovascular lesions seen as white matter hyperintensity in MRI of elderly population provides evidence for micro-infracts and micro-bleeds, which contribute to vascular dementia. Such vascular insult may be caused by impairment in blood flow in specific area in brain involving small vessels and these could gradually worsen the pathology leading to cognitive deficits. In the present study we developed a transient model of vaso-constriction to study the impact of such pathology on cognition specifically, memory impairment. The molecular underpinnings were also studied to comprehend the commonalities between vascular dementia and Alzheimer’s disease. Methods Vascular constriction was achieved by bilateral injection of ET-1 (Endothelin − 1; a 21 amino acid vasoconstricting peptide) into lateral ventricles of C57 mice. The impact on endothelial cells lining of blood vessels was examined by staining for CD31, a marker of endothelial cells. Contextual fear conditioning, Novel object recognition and Morris water maze task was performed to ascertain associative learning and spatial memory deficits. Activity dependent protein translation, which is dependent on Akt1-mTOR signaling was also examined since this is critical for synaptic plasticity. Results There was considerable decrease in CD31 expression in endothelial cells lining the blood vessels around the hippocampal region. The impediment in cerebral blood flow following ET-1 injection lead to deficits in associative learning and spatial memory after 7 days. Activity dependent protein translation, which is critical for synaptic plasticity was absent in synaptoneurosomes prepared from hippocampal tissue of endothelin injected mice. Further, Akt1- mTOR signaling cascade was downregulated due to decreased Akt1 phosphorylation indicating that this could be the cause for loss of activity dependent protein translation. However, these effects were reversed after 30 days indicating the ephemeral nature of deficits following a single vascular insult. Conclusions Vasoconstriction caused by bilateral injection of ET-1 leads to prominent associative and spatial memory deficit. Decline in activity dependent protein translation in hippocampus and loss of Akt1-mTOR signaling demonstrates potential molecular mechanism impacting synaptic plasticity during vasoconstriction akin to early deficits in AD mice.

PTEN expression in endothelial cells is down-regulated by uPAR to promote angiogenesis

2015 ◽  
Vol 114 (08) ◽  
pp. 379-389 ◽  
Author(s):  
Matthias Unseld ◽  
Anastasia Chilla ◽  
Clemens Pausz ◽  
Rula Mawas ◽  
Johannes Breuss ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
In Vitro Assays ◽  
Soluble Form ◽  
Dependent Manner ◽  
Drug Interference ◽  
Protein Levels ◽  
Novel Target ◽  
The Impact

SummaryThe tumour suppressor phosphatase and tensin homologue (PTEN), mutated or lost in many human cancers, is a major regulator of angiogenesis. However, the cellular mechanism of PTEN regulation in endothelial cells so far remains elusive. Here, we characterise the urokinase receptor (uPAR, CD87) and its tumour-derived soluble form, suPAR, as a key molecule of regulating PTEN in endothelial cells. We observed uPAR-deficient endothelial cells to express enhanced PTEN mRNA- and protein levels. Consistently, uPAR expression in endogenous negative uPAR cells, down-regulated PTEN and activated the PI3K/Akt pathway. Additionally, we found that integrin adhesion receptors act as trans-membrane signaling partners for uPAR to repress PTEN transcription in a NF-κB-dependent manner. Functional in vitro assays with endothelial cells, derived from uPAR-deficient and PTEN heterozygous crossbred mice, demonstrated the impact of uPAR- dependent PTEN regulation on cell motility and survival. In an in vivo murine angiogenesis model uPAR-deficient PTEN heterozygous animals increased the impaired angiogenic phenotype of uPAR knockout mice and were able to reverse the high invasive potential of PTEN heterozygots. Our data provide first evidence that endogenous as well as exogenous soluble uPAR down-regulated PTEN in endothelial cells to support angiogenesis. The uPAR-induced PTEN regulation might represent a novel target for drug interference, and may lead to the development of new therapeutic strategies in anti-angiogenic treatment.

scholarly journals Endoplasmic Reticulum (ER) Stress-Generated Extracellular Vesicles (Microparticles) Self-Perpetuate ER Stress and Mediate Endothelial Cell Dysfunction Independently of Cell Survival

2020 ◽  
Vol 7 ◽  
Author(s):  
Aisha Osman ◽  
Heba El-Gamal ◽  
Mazhar Pasha ◽  
Asad Zeidan ◽  
Hesham M. Korashy ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Endothelial Cell ◽  
Endothelial Dysfunction ◽  
Endothelial Function ◽  
Er Stress ◽  
Extracellular Vesicles ◽  
Chemical Chaperone ◽  
No Release ◽  
The Impact

Circulating extracellular vesicles (EVs) are recognized as biomarkers and effectors of endothelial dysfunction, the initiating step of cardiovascular abnormalities. Among these EVs, microparticles (MPs) are vesicles directly released from the cytoplasmic membrane of activated cells. MPs were shown to induce endothelial dysfunction through the activation of endoplasmic reticulum (ER) stress. However, it is not known whether ER stress can lead to MPs release from endothelial cells and what biological messages are carried by these MPs. Therefore, we aimed to assess the impact of ER stress on MPs shedding from endothelial cells, and to investigate their effects on endothelial cell function. EA.hy926 endothelial cells or human umbilical vein endothelial cells (HUVECs) were treated for 24 h with ER stress inducers, thapsigargin or dithiothreitol (DTT), in the presence or absence of 4-Phenylbutyric acid (PBA), a chemical chaperone to inhibit ER stress. Then, MPs were isolated and used to treat cells (10–20 μg/mL) for 24–48 h before assessing ER stress response, angiogenic capacity, nitric oxide (NO) release, autophagy and apoptosis. ER stress (thapsigargin or DDT)-generated MPs did not differ quantitatively from controls; however, they carried deleterious messages for endothelial function. Exposure of endothelial cells to ER stress-generated MPs increased mRNA and protein expression of key ER stress markers, indicating a vicious circle activation of ER stress. ER stress (thapsigargin)-generated MPs impaired the angiogenic capacity of HUVECs and reduced NO release, indicating an impaired endothelial function. While ER stress (thapsigargin)-generated MPs altered the release of inflammatory cytokines, they did not, however, affect autophagy or apoptosis in HUVECs. This work enhances the general understanding of the deleterious effects carried out by MPs in medical conditions where ER stress is sustainably activated such as diabetes and metabolic syndrome.

scholarly journals N-Glycan–dependent protein folding and endoplasmic reticulum retention regulate GPI-anchor processing

2017 ◽  
Vol 217 (2) ◽  
pp. 585-599 ◽  
Author(s):  
Yi-Shi Liu ◽  
Xin-Yu Guo ◽  
Tetsuya Hirata ◽  
Yao Rong ◽  
Daisuke Motooka ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Folding ◽  
Cell Surface ◽  
Er Stress ◽  
Posttranslational Modification ◽  
Acyl Chain ◽  
Genetic Screens ◽  
Gpi Anchor ◽  
Er Retention ◽  
Dependent Protein

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a conserved posttranslational modification in the endoplasmic reticulum (ER). Soon after GPI is attached, an acyl chain on the GPI inositol is removed by post-GPI attachment to proteins 1 (PGAP1), a GPI-inositol deacylase. This is crucial for switching GPI-anchored proteins (GPI-APs) from protein folding to transport states. We performed haploid genetic screens to identify factors regulating GPI-inositol deacylation, identifying seven genes. In particular, calnexin cycle impairment caused inefficient GPI-inositol deacylation. Calnexin was specifically associated with GPI-APs, dependent on N-glycan and GPI moieties, and assisted efficient GPI-inositol deacylation by PGAP1. Under chronic ER stress caused by misfolded GPI-APs, inositol-acylated GPI-APs were exposed on the cell surface. These results indicated that N-glycans participate in quality control and temporal ER retention of GPI-APs, ensuring their correct folding and GPI processing before exiting from the ER. Once the system is disrupted by ER stress, unprocessed GPI-APs become exposed on the cell surface.

scholarly journals Calcium Release from Endoplasmic Reticulum Involves Calmodulin-Mediated NADPH Oxidase-Derived Reactive Oxygen Species Production in Endothelial Cells

2019 ◽  
Vol 20 (7) ◽  
pp. 1644 ◽  
Author(s):  
Ryugo Sakurada ◽  
Keiichi Odagiri ◽  
Akio Hakamata ◽  
Chiaki Kamiya ◽  
Jiazhang Wei ◽  
...  
Keyword(s):  
Reactive Oxygen Species ◽  
Endothelial Cells ◽  
Endoplasmic Reticulum ◽  
Calcium Release ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Reactive Oxygen ◽  
Ros Production ◽  
Oxygen Species ◽  
Dependent Protein ◽  
Dependent Pathway

Background: Previous studies demonstrated that calcium/calmodulin (Ca2+/CaM) activates nicotinamide adenine dinucleotide phosphate oxidases (NOX). In endothelial cells, the elevation of intracellular Ca2+ level consists of two components: Ca2+ mobilization from the endoplasmic reticulum (ER) and the subsequent store-operated Ca2+ entry. However, little is known about which component of Ca2+ increase is required to activate NOX in endothelial cells. Here, we investigated the mechanism that regulates NOX-derived reactive oxygen species (ROS) production via a Ca2+/CaM-dependent pathway. Methods: We measured ROS production using a fluorescent indicator in endothelial cells and performed phosphorylation assays. Results: Bradykinin (BK) increased NOX-derived cytosolic ROS. When cells were exposed to BK with either a nominal Ca2+-free or 1 mM of extracellular Ca2+ concentration modified Tyrode’s solution, no difference in BK-induced ROS production was observed; however, chelating of cytosolic Ca2+ by BAPTA/AM or the depletion of ER Ca2+ contents by thapsigargin eliminated BK-induced ROS production. BK-induced ROS production was inhibited by a CaM inhibitor; however, a Ca2+/CaM-dependent protein kinase II (CaMKII) inhibitor did not affect BK-induced ROS production. Furthermore, BK stimulation did not increase phosphorylation of NOX2, NOX4, and NOX5. Conclusions: BK-induced NOX-derived ROS production was mediated via a Ca2+/CaM-dependent pathway; however, it was independent from NOX phosphorylation. This was strictly regulated by ER Ca2+ contents.

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