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1

Analysis of Unfolded Protein Response in Arabidopsis  

PubMed Central

The unfolded protein response (UPR) is fundamental for development and adaption in eukaryotic cells. Arabidopsis has become one of the best model systems to uncover conserved mechanisms of the UPR in multicellular eukaryotes as well as organism-specific regulation of the UPR in plants. Monitoring the UPR in planta is an elemental approach to identifying regulatory components and to revealing molecular mechanisms of the plant UPR. In this chapter, we provide protocols for the induction and analyses of plant UPR at a molecular level in Arabidopsis. Three kinds of ER stress treatment methods and quantitation of the plant UPR activation are described here.

Chen, Yani; Brandizzi, Federica

2014-01-01

2

Autophagy Counterbalances Endoplasmic Reticulum Expansion during the Unfolded Protein Response  

Microsoft Academic Search

The protein folding capacity of the endoplasmic reticulum (ER) is regulated by the unfolded protein response (UPR). The UPR senses unfolded proteins in the ER lumen and transmits that information to the cell nucleus, where it drives a transcriptional program that is tailored to re-establish homeostasis. Using thin section electron microscopy, we found that yeast cells expand their ER volume

Sebastián Bernales; Kent L McDonald; Peter Walter

2006-01-01

3

The Unfolded Protein Response and Gastrointestinal Disease  

PubMed Central

As the inner lining of the gastrointestinal tract, the intestinal epithelium serves an essential role in innate immune function at the interface between the host and microbiota. Given the unique environmental challenges and thus physiologic secretory functions of this surface, it is exquisitely sensitive to perturbations that affect its capacity to resolve endoplasmic reticulum (ER) stress. Genetic deletion of factors involved in the unfolded protein response (UPR), which functions in the resolution of ER stress that arises from misfolded proteins, result in spontaneous intestinal inflammation closely mimicking human inflammatory bowel disease (IBD). This is demonstrated by observations wherein deletion of genes such as Xbp1 and Agr2 profoundly affects the intestinal epithelium and results in spontaneous intestinal inflammation. Moreover, both genes, along with others (e.g. ORDML3) represent genetic risk factors for human IBD, both Crohn’s disease and ulcerative colitis. Here we review the current mechanistic understanding for how unresolved ER stress can lead to intestinal inflammation, and highlight the findings that implicate ER stress as a genetically affected biological pathway in IBD. We further discuss environmental and microbial factors that might impact on the epithelium’s capacity to resolve ER stress, and which may constitute exogenous factors that may precipitate disease in genetically susceptible individuals.

Kaser, Arthur; Adolph, Timon Erik; Blumberg, Richard S

2013-01-01

4

The impact of the unfolded protein response on human disease  

PubMed Central

A central function of the endoplasmic reticulum (ER) is to coordinate protein biosynthetic and secretory activities in the cell. Alterations in ER homeostasis cause accumulation of misfolded/unfolded proteins in the ER. To maintain ER homeostasis, eukaryotic cells have evolved the unfolded protein response (UPR), an essential adaptive intracellular signaling pathway that responds to metabolic, oxidative stress, and inflammatory response pathways. The UPR has been implicated in a variety of diseases including metabolic disease, neurodegenerative disease, inflammatory disease, and cancer. Signaling components of the UPR are emerging as potential targets for intervention and treatment of human disease.

Wang, Shiyu

2012-01-01

5

Engineering of chaperone systems and of the unfolded protein response  

PubMed Central

Production of recombinant proteins in mammalian cells is a successful technology that delivers protein pharmaceuticals for therapies and for diagnosis of human disorders. Cost effective production of protein biopharmaceuticals requires extensive optimization through cell and fermentation process engineering at the upstream and chemical engineering of purification processes at the downstream side of the production process. The majority of protein pharmaceuticals are secreted proteins. Accumulating evidence suggests that the folding and processing of these proteins in the endoplasmic reticulum (ER) is a general rate- and yield limiting step for their production. We will summarize our knowledge of protein folding in the ER and of signal transduction pathways activated by accumulation of unfolded proteins in the ER, collectively called the unfolded protein response (UPR). On the basis of this knowledge we will evaluate engineering approaches to increase cell specific productivities through engineering of the ER-resident protein folding machinery and of the UPR.

Khan, Saeed U.

2008-01-01

6

The unfolded protein response during prostate cancer development  

Microsoft Academic Search

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) induces the unfolded protein response (UPR). The UPR\\u000a promotes cell survival by adjusting ER protein folding capacity but if homeostasis cannot be re-established, apoptosis is\\u000a induced. The execution of life\\/death decisions is regulated by the three UPR branches (IRE1, PERK, ATF6) and their downstream\\u000a effectors. Events that offset the balance of

Alex Yick-Lun So; Erwin de la Fuente; Peter Walter; Marc Shuman; Sebastián Bernales

2009-01-01

7

The Endoplasmic Reticulum and the Unfolded Protein Response  

PubMed Central

The endoplasmic reticulum (ER) is the site where proteins enter the secretory pathway. Proteins are translocated into the ER lumen in an unfolded state and require protein chaperones and catalysts of protein folding to attain their final appropriate conformation. A sensitive surveillance mechanism exists to prevent misfolded proteins from transiting the secretory pathway and ensures that persistently misfolded proteins are directed towards a degradative pathway. In addition, those processes that prevent accumulation of unfolded proteins in the ER lumen are highly regulated by an intracellular signaling pathway known as the unfolded protein response (UPR). The UPR provides a mechanism by which cells can rapidly adapt to alterations in client protein-folding load in the ER lumen by expanding the capacity for protein folding. In addition, a variety of insults that disrupt protein folding in the ER lumen also activate the UPR. These include changes in intralumenal calcium, altered glycosylation, nutrient deprivation, pathogen infection, expression of folding-defective proteins, and changes in redox status. Persistent protein misfolding initiates apoptotic cascades that are now known to play fundamental roles in the pathogenesis of multiple human diseases including diabetes, atherosclerosis and neurodegenerative diseases.

Malhotra, Jyoti D.; Kaufman, Randal J.

2009-01-01

8

The Cell Biology of the Unfolded Protein Response  

PubMed Central

The Unfolded Protein Response (UPR) is an ensemble of signal transduction pathways that respond to perturbations in the oxidative, pro-folding environment of the endoplasmic reticulum. During the past decade, ongoing research implicated these pathways in maintaining homeostasis of cells and organisms exposed to various stresses. Herein, we highlight recent findings regarding the functional role of the UPR in both normal and pathophysiological processes.

Diehl, J. Alan; Fuchs, Serge Y.; Koumenis, Costantinos

2011-01-01

9

Cellular unfolded protein response against viruses used in gene therapy.  

PubMed

Viruses are excellent vehicles for gene therapy due to their natural ability to infect and deliver the cargo to specific tissues with high efficiency. Although such vectors are usually "gutted" and are replication defective, they are subjected to clearance by the host cells by immune recognition and destruction. Unfolded protein response (UPR) is a naturally evolved cyto-protective signaling pathway which is triggered due to endoplasmic reticulum (ER) stress caused by accumulation of unfolded/misfolded proteins in its lumen. The UPR signaling consists of three signaling pathways, namely PKR-like ER kinase, activating transcription factor 6, and inositol-requiring protein-1. Once activated, UPR triggers the production of ER molecular chaperones and stress response proteins to help reduce the protein load within the ER. This occurs by degradation of the misfolded proteins and ensues in the arrest of protein translation machinery. If the burden of protein load in ER is beyond its processing capacity, UPR can activate pro-apoptotic pathways or autophagy leading to cell death. Viruses are naturally evolved in hijacking the host cellular translation machinery to generate a large amount of proteins. This phenomenon disrupts ER homeostasis and leads to ER stress. Alternatively, in the case of gutted vectors used in gene therapy, the excess load of recombinant vectors administered and encountered by the cell can trigger UPR. Thus, in the context of gene therapy, UPR becomes a major roadblock that can potentially trigger inflammatory responses against the vectors and reduce the efficiency of gene transfer. PMID:24904562

Sen, Dwaipayan; Balakrishnan, Balaji; Jayandharan, Giridhara R

2014-01-01

10

Cellular unfolded protein response against viruses used in gene therapy  

PubMed Central

Viruses are excellent vehicles for gene therapy due to their natural ability to infect and deliver the cargo to specific tissues with high efficiency. Although such vectors are usually “gutted” and are replication defective, they are subjected to clearance by the host cells by immune recognition and destruction. Unfolded protein response (UPR) is a naturally evolved cyto-protective signaling pathway which is triggered due to endoplasmic reticulum (ER) stress caused by accumulation of unfolded/misfolded proteins in its lumen. The UPR signaling consists of three signaling pathways, namely PKR-like ER kinase, activating transcription factor 6, and inositol-requiring protein-1. Once activated, UPR triggers the production of ER molecular chaperones and stress response proteins to help reduce the protein load within the ER. This occurs by degradation of the misfolded proteins and ensues in the arrest of protein translation machinery. If the burden of protein load in ER is beyond its processing capacity, UPR can activate pro-apoptotic pathways or autophagy leading to cell death. Viruses are naturally evolved in hijacking the host cellular translation machinery to generate a large amount of proteins. This phenomenon disrupts ER homeostasis and leads to ER stress. Alternatively, in the case of gutted vectors used in gene therapy, the excess load of recombinant vectors administered and encountered by the cell can trigger UPR. Thus, in the context of gene therapy, UPR becomes a major roadblock that can potentially trigger inflammatory responses against the vectors and reduce the efficiency of gene transfer.

Sen, Dwaipayan; Balakrishnan, Balaji; Jayandharan, Giridhara R.

2014-01-01

11

Autophagy Counterbalances Endoplasmic Reticulum Expansion during the Unfolded Protein Response  

PubMed Central

The protein folding capacity of the endoplasmic reticulum (ER) is regulated by the unfolded protein response (UPR). The UPR senses unfolded proteins in the ER lumen and transmits that information to the cell nucleus, where it drives a transcriptional program that is tailored to re-establish homeostasis. Using thin section electron microscopy, we found that yeast cells expand their ER volume at least 5-fold under UPR-inducing conditions. Surprisingly, we discovered that ER proliferation is accompanied by the formation of autophagosome-like structures that are densely and selectively packed with membrane stacks derived from the UPR-expanded ER. In analogy to pexophagy and mitophagy, which are autophagic processes that selectively sequester and degrade peroxisomes and mitochondria, the ER-specific autophagic process described utilizes several autophagy genes: they are induced by the UPR and are essential for the survival of cells subjected to severe ER stress. Intriguingly, cell survival does not require vacuolar proteases, indicating that ER sequestration into autophagosome-like structures, rather than their degradation, is the important step. Selective ER sequestration may help cells to maintain a new steady-state level of ER abundance even in the face of continuously accumulating unfolded proteins.

Bernales, Sebastian; McDonald, Kent L; Walter, Peter

2006-01-01

12

Endoplasmic Reticulum Stress: Signaling the Unfolded Protein Response  

NSDL National Science Digital Library

The endoplasmic reticulum (ER) is the cellular site of newly synthesized secretory and membrane proteins. Such proteins must be properly folded and posttranslationally modified before exit from the organelle. Proper protein folding and modification requires molecular chaperone proteins as well as an ER environment conducive for these reactions. When ER lumenal conditions are altered or chaperone capacity is overwhelmed, the cell activates signaling cascades that attempt to deal with the altered conditions and restore a favorable folding environment. Such alterations are referred to as ER stress, and the response activated is the unfolded protein response (UPR). When the UPR is perturbed or not sufficient to deal with the stress conditions, apoptotic cell death is initiated. This review will examine UPR signaling that results in cell protective responses, as well as the mechanisms leading to apoptosis induction, which can lead to pathological states due to chronic ER stress.

2007-06-01

13

Targeting the unfolded protein response in heart diseases.  

PubMed

In neurological disease and diabetes, the unfolded protein response (UPR) has been investigated for years, while its function in heart disease is less well understood. All three branches of the UPR are involved in ischaemia/reperfusion and can either protect or impair heart function. Recently, UPR has been found to play a role in arrhythmogenesis during human heart failure, and blocking UPR has an antiarrhythmic effect. This review will discuss the rationale for and challenges to targeting UPR in heart disease. PMID:24865516

Liu, Man; Dudley, Samuel C

2014-07-01

14

REGULATION OF THE UNFOLDED PROTEIN RESPONSE BY MICRORNAS  

PubMed Central

The unfolded protein response (UPR) is an adaptive response to stress that is caused by an accumulation of misfolded proteins in the lumen of endoplasmic reticulum (ER) and is therefore an important component of cellular homeostasis. During ER stress, the UPR increases the protein folding capacity of the endoplasmic reticulum to relieve the stress, and failure to recover leads to apoptosis. Specific cellular mechanisms, therefore, are required for the cellular recovery phase after UPR activation. In this review, we discuss the potential role of microRNAs as key regulators of this pathway. Using bioinformatics, we identified a number of microRNAs that are predicted to decrease the mRNA expression levels for a number of critical components of the UPR and discuss how microRNAs may play an essential role in turning off the UPR after the stress has subsided.

Madanecki, Piotr; Piotrowski, Arkadiusz; Ochocka, Renata; Collawn, James F.; Bartoszewski, Rafal

2013-01-01

15

Estrogen receptor mediates a distinct mitochondrial unfolded protein response.  

PubMed

Unfolded protein responses (UPRs) of the endoplasmic reticulum and mitochondrial matrix have been described. Here, we show that the accumulation of proteins in the inter-membrane space (IMS) of mitochondria in the breast cancer cell line MCF-7 activates a distinct UPR. Upon IMS stress, overproduction of reactive oxygen species (ROS) and phosphorylation of AKT triggers estrogen receptor (ER) activity, which further upregulates the transcription of the mitochondrial regulator NRF1 and the IMS protease OMI (officially known as HTRA2). Moreover, we demonstrate that the IMS stress-induced UPR culminates in increased proteasome activity. Given our previous report on a proteasome- and OMI-dependent checkpoint that limits the import of IMS proteins, the findings presented in this study suggest that this newly discovered UPR acts as a cytoprotective response to overcome IMS stress. PMID:21486948

Papa, Luena; Germain, Doris

2011-05-01

16

Unfolded protein response in hepatitis C virus infection  

PubMed Central

Hepatitis C virus (HCV) is a single-stranded, positive-sense RNA virus of clinical importance. The virus establishes a chronic infection and can progress from chronic hepatitis, steatosis to fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). The mechanisms of viral persistence and pathogenesis are poorly understood. Recently the unfolded protein response (UPR), a cellular homeostatic response to endoplasmic reticulum (ER) stress, has emerged to be a major contributing factor in many human diseases. It is also evident that viruses interact with the host UPR in many different ways and the outcome could be pro-viral, anti-viral or pathogenic, depending on the particular type of infection. Here we present evidence for the elicitation of chronic ER stress in HCV infection. We analyze the UPR signaling pathways involved in HCV infection, the various levels of UPR regulation by different viral proteins and finally, we propose several mechanisms by which the virus provokes the UPR.

Chan, Shiu-Wan

2014-01-01

17

Unfolded protein response, treatment and CMT1B  

PubMed Central

CMT1B is the second most frequent autosomal dominant inherited neuropathy and is caused by assorted mutations of the myelin protein zero (MPZ) gene. MPZ mutations cause neuropathy gain of function mechanisms that are largely independent MPZs normal role of mediating myelin compaction. Whether there are only a few or multiple pathogenic mechanisms that cause CMT1B is unknown. Arg98Cys and Ser63Del MPZ are two CMT1B causing mutations that have been shown to cause neuropathy in mice at least in part by activating the unfolded protein response (UPR). We have recently treated Arg98Cys mice with derivatives of curcumin that improved the neuropathy and reduced UPR activation.1 Future studies will address whether manipulating the UPR will be a common or rare strategy for treating CMT1B or other forms of inherited neuropathies.

Bai, Yunhong; Patzko, Agnes; Shy, Michael E.

2013-01-01

18

The Unfolded Protein Response in Fatty Liver Disease  

PubMed Central

The Unfolded Protein Response (UPR) is a protective cellular response activated under conditions of endoplasmic reticulum (ER) stress. The hepatic UPR is activated in several forms of liver disease including nonalcoholic fatty liver disease (NAFLD). Recent data defining the role of the UPR in hepatic lipid metabolism have identified molecular mechanisms that may underlie the association between UPR activation and NAFLD. It has become increasingly evident that the IRE?/Xbp1 pathway of the UPR is critical for hepatic lipid homeostasis, and dysregulation of this evolutionarily conserved pathway is associated with human non-alcoholic steatohepatitis (NASH). Although increasing evidence has delineated the importance of UPR pathway signaling in fatty liver disorders, the regulation of the hepatic UPR in normal physiology and fatty liver disorders remains incompletely understood. Understanding the role of the UPR in hepatic lipid metabolism may lead to the identification of novel therapeutic targets for the treatment of NAFLD.

Green, Richard M.

2014-01-01

19

Intersection of the unfolded protein response and hepatic lipid metabolism  

PubMed Central

The liver plays a central role in whole-body lipid metabolism by governing the synthesis, oxidization, transport and excretion of lipids. The unfolded protein response (UPR) was identified as a signal transduction system that is activated by ER stress. Recent studies revealed a critical role of the UPR in hepatic lipid metabolism. The IRE1/XBP1 branch of the UPR is activated by high dietary carbohydrates and controls the expression of genes involved in fatty acid and cholesterol biosynthesis. PERK mediated eIF2? phosphorylation is also required for the expression of lipogenic genes and the development of hepatic steatosis, likely by activating C/EBP and PPAR? transcription factors. Further studies to define the molecular pathways that lead to the activation of the UPR by nutritional cues in the liver, and their contribution to human metabolic disorders such as hepatic steatosis, atherosclerosis and type 2 diabetes that are associated with dysregulation of lipid homeostasis, are warranted.

2011-01-01

20

The unfolded protein response affects readthrough of premature termination codons.  

PubMed

One-third of monogenic inherited diseases result from premature termination codons (PTCs). Readthrough of in-frame PTCs enables synthesis of full-length functional proteins. However, extended variability in the response to readthrough treatment is found among patients, which correlates with the level of nonsense transcripts. Here, we aimed to reveal cellular pathways affecting this inter-patient variability. We show that activation of the unfolded protein response (UPR) governs the response to readthrough treatment by regulating the levels of transcripts carrying PTCs. Quantitative proteomic analyses showed substantial differences in UPR activation between patients carrying PTCs, correlating with their response. We further found a significant inverse correlation between the UPR and nonsense-mediated mRNA decay (NMD), suggesting a feedback loop between these homeostatic pathways. We uncovered and characterized the mechanism underlying this NMD-UPR feedback loop, which augments both UPR activation and NMD attenuation. Importantly, this feedback loop enhances the response to readthrough treatment, highlighting its clinical importance. Altogether, our study demonstrates the importance of the UPR and its regulatory network for genetic diseases caused by PTCs and for cell homeostasis under normal conditions. PMID:24705877

Oren, Yifat S; McClure, Michelle L; Rowe, Steven M; Sorscher, Eric J; Bester, Assaf C; Manor, Miriam; Kerem, Eitan; Rivlin, Joseph; Zahdeh, Fouad; Mann, Matthias; Geiger, Tamar; Kerem, Batsheva

2014-05-01

21

The unfolded protein response affects readthrough of premature termination codons  

PubMed Central

One-third of monogenic inherited diseases result from premature termination codons (PTCs). Readthrough of in-frame PTCs enables synthesis of full-length functional proteins. However, extended variability in the response to readthrough treatment is found among patients, which correlates with the level of nonsense transcripts. Here, we aimed to reveal cellular pathways affecting this inter-patient variability. We show that activation of the unfolded protein response (UPR) governs the response to readthrough treatment by regulating the levels of transcripts carrying PTCs. Quantitative proteomic analyses showed substantial differences in UPR activation between patients carrying PTCs, correlating with their response. We further found a significant inverse correlation between the UPR and nonsense-mediated mRNA decay (NMD), suggesting a feedback loop between these homeostatic pathways. We uncovered and characterized the mechanism underlying this NMD-UPR feedback loop, which augments both UPR activation and NMD attenuation. Importantly, this feedback loop enhances the response to readthrough treatment, highlighting its clinical importance. Altogether, our study demonstrates the importance of the UPR and its regulatory network for genetic diseases caused by PTCs and for cell homeostasis under normal conditions.

Oren, Yifat S; McClure, Michelle L; Rowe, Steven M; Sorscher, Eric J; Bester, Assaf C; Manor, Miriam; Kerem, Eitan; Rivlin, Joseph; Zahdeh, Fouad; Mann, Matthias; Geiger, Tamar; Kerem, Batsheva

2014-01-01

22

Insulin regulates the unfolded protein response in human adipose tissue.  

PubMed

Endoplasmic reticulum (ER) stress is increased in obesity and is postulated to be a major contributor to many obesity-related pathologies. Little is known about what causes ER stress in obese people. Here, we show that insulin upregulated the unfolded protein response (UPR), an adaptive reaction to ER stress, in vitro in 3T3-L1 adipocytes and in vivo, in subcutaneous (sc) adipose tissue of nondiabetic subjects, where it increased the UPR dose dependently over the entire physiologic insulin range (from ? 35 to ? 1,450 pmol/L). The insulin-induced UPR was not due to increased glucose uptake/metabolism and oxidative stress. It was associated, however, with increased protein synthesis, with accumulation of ubiquitination associated proteins, and with multiple posttranslational protein modifications (acetylations, methylations, nitrosylations, succinylation, and ubiquitinations), some of which are potential causes for ER stress. These results reveal a new physiologic role of insulin and provide a putative mechanism for the development of ER stress in obesity. They may also have clinical and therapeutic implications, e.g., in diabetic patients treated with high doses of insulin. PMID:24130338

Boden, Guenther; Cheung, Peter; Salehi, Sajad; Homko, Carol; Loveland-Jones, Catherine; Jayarajan, Senthil; Stein, T Peter; Williams, Kevin Jon; Liu, Ming-Lin; Barrero, Carlos A; Merali, Salim

2014-03-01

23

The unfolded protein response has a protective role in yeast models of classic galactosemia.  

PubMed

Classic galactosemia is a human autosomal recessive disorder caused by mutations in the GALT gene (GAL7 in yeast), which encodes the enzyme galactose-1-phosphate uridyltransferase. Here we show that the unfolded protein response pathway is triggered by galactose in two yeast models of galactosemia: lithium-treated cells and the gal7? mutant. The synthesis of galactose-1-phosphate is essential to trigger the unfolded protein response under these conditions because the deletion of the galactokinase-encoding gene GAL1 completely abolishes unfolded protein response activation and galactose toxicity. Impairment of the unfolded protein response in both yeast models makes cells even more sensitive to galactose, unmasking its cytotoxic effect. These results indicate that endoplasmic reticulum stress is induced under galactosemic conditions and underscores the importance of the unfolded protein response pathway to cellular adaptation in these models of classic galactosemia. PMID:24077966

De-Souza, Evandro A; Pimentel, Felipe S A; Machado, Caio M; Martins, Larissa S; da-Silva, Wagner S; Montero-Lomelí, Mónica; Masuda, Claudio A

2014-01-01

24

The unfolded protein response has a protective role in yeast models of classic galactosemia  

PubMed Central

Classic galactosemia is a human autosomal recessive disorder caused by mutations in the GALT gene (GAL7 in yeast), which encodes the enzyme galactose-1-phosphate uridyltransferase. Here we show that the unfolded protein response pathway is triggered by galactose in two yeast models of galactosemia: lithium-treated cells and the gal7? mutant. The synthesis of galactose-1-phosphate is essential to trigger the unfolded protein response under these conditions because the deletion of the galactokinase-encoding gene GAL1 completely abolishes unfolded protein response activation and galactose toxicity. Impairment of the unfolded protein response in both yeast models makes cells even more sensitive to galactose, unmasking its cytotoxic effect. These results indicate that endoplasmic reticulum stress is induced under galactosemic conditions and underscores the importance of the unfolded protein response pathway to cellular adaptation in these models of classic galactosemia.

De-Souza, Evandro A.; Pimentel, Felipe S. A.; Machado, Caio M.; Martins, Larissa S.; da-Silva, Wagner S.; Montero-Lomeli, Monica; Masuda, Claudio A.

2014-01-01

25

ER stress response, peroxisome proliferation, mitochondrial unfolded protein response and Golgi stress response.  

PubMed

The endoplasmic reticulum (ER) response has been thought a cytoprotective mechanism to cope with accumulation of unfolded proteins in the ER. Recent progress has made a quantum leap revealing that ER stress response can be regarded as an autoregulatory system adjusting the ER capacity to cellular demand. This Copernican change raised a novel fundamental question in cell biology: how do cells regulate the capacity of each organelle in accordance with cellular needs? Although this fundamental question has not been fully addressed yet, research about each organelle has been advancing. The proliferation of the peroxisome is regulated by the PPAR alpha pathway, whereas the abundance of mitochondria appears to be regulated by mitochondrial retrograde signaling and the mitochondrial unfolded protein response. The functional capacity of the Golgi apparatus may be regulated by the mechanism of the Golgi stress response. These observations strongly suggest the existence of an elaborate network of organelle autoregulation in eukaryotic cells. PMID:19504573

Yoshida, Hiderou

2009-09-01

26

Hydrogen Bonding between Sugar and Protein Is Responsible for Inhibition of Dehydration-Induced Protein Unfolding  

Microsoft Academic Search

The nature of the interaction responsible for the inhibition of protein unfolding and subsequent damage by sugars during dehydration is unclear. The relationship between sample moisture content measured by coulometric Karl Fischer titration and the apparent moisture content predicted by the area of the protein side chain carboxylate band at approximately 1580 cm?1in infrared spectra of dried protein–sugar samples was

S. Dean Allison; Byeong Chang; Theodore W. Randolph; John F. Carpenter

1999-01-01

27

Development of Antiproliferative Phenylmaleimides that Activate the Unfolded Protein Response  

PubMed Central

The current paper presents the synthesis and evaluation of a series of maleimides that were designed to inhibit the Cdc25 phosphatase by alkylation of catalytically essential cysteine residues. Although in HepB3 cell culture assays the analogues did exhibit antiproliferative IC50 values ranging from sub-micromolar to greater than 100 µM, inhibition of Cdc25 through cysteine alkylation could not be demonstrated. It was also found that analysis using fluorescence activated cell sorting (FACS) following treatment with the most potent analogue (1t) did not provide data consistent with inhibition at one specific point in the cell cycle, as would be expected if Cdc25A were inhibited. Further studies with a subset of analogues resulted in a correlation of antiproliferative potencies with activation of the unfolded protein response (UPR). The UPR is a regulatory pathway that temporarily suspends protein production when misfolding of proteins occurs within the endoplastic reticulum (ER). In addition, ER chaperones that promote proper refolding become upregulated. If cellular damage cannot be resolved by these mechanisms, then the UPR can initiate apoptosis. The current study indicates that these maleimide analogues lead to UPR activation, which is predictive of the selective antiproliferative activity of the series.

Muus, Ulrike; Hose, Curtis; Yao, Wei; Kosakowska-Cholody, Teresa; Farnsworth, David; Dyba, Marzena; Lountos, George T.; Waugh, David S.; Monks, Anne; Burke, Terrence R.; Michejda, Christopher J.

2010-01-01

28

Arsenic trioxide induces unfolded protein response in vascular endothelial cells.  

PubMed

Chronic arsenic exposure has been linked to endothelial dysfunction and apoptosis. We investigate the involvement of unfolded protein response (UPR) signaling in the arsenic-mediated cytotoxicity of the SVEC4-10 mouse endothelial cells. The SVEC4-10 cells underwent apoptosis in response to As2O3 dose- and time-dependently, accompanied by increased accumulation of calcium, and activation of caspase-3. These phenomena were completely inhibited by ?-lipoic acid (LA), which did not scavenge ROS over-production, but were only partially or not ameliorated by tiron, a potent superoxide scavenger. Moreover, arsenic activated UPR, leading to phosphorylation of eukaryotic translation initiation factor 2 subunit ? (eIF2?), induction of ATF4, and processing of ATF6. Treatment with arsenic also triggered the expression of endoplasmic reticulum (ER) stress markers, GRP78 (glucose-regulated protein), and CHOP (C/EBP homologous protein). The activation of eIF2?, ATF4 and ATF6 and expression of GRP78 and CHOP are repressed by both LA and tiron, indicating arsenic-induced UPR is mediated through ROS-dependent and ROS-independent pathways. Arsenic also induced ER stress-inducible genes, BAX, PUMA (p53 upregulated modulator of apoptosis), TRB3 (tribbles-related protein 3), and SNAT2 (sodium-dependent neutral amino acid transporter 2). Consistent with intracellular calcium and cell viability data, ROS may not be important in arsenic-induced death, because tiron did not affect the expression of these pro-apoptotic genes. In addition, pretreatment with salubrinal, a selective inhibitor of eIF2? dephosphorylation, enhanced arsenic-induced GRP78 and CHOP expression and partially prevented arsenic cytotoxicity in SVEC4-10 cells. Taken together, these results suggest that arsenic-induced endothelial cytotoxicity is associated with ER stress, which is mediated by ROS-dependent and ROS-independent signaling. PMID:23892647

Weng, Ching-Yi; Chiou, Shu-Yuan; Wang, Lisu; Kou, Mei-Chun; Wang, Ying-Jan; Wu, Ming-Jiuan

2014-02-01

29

Adaptive Unfolded Protein Response Attenuates Alcohol-induced Pancreatic Damage  

PubMed Central

BACKGROUND & AIMS Endoplasmic reticulum (ER) stress responses (collectively known the unfolded protein response, UPR) have important roles in several human disorders, but their contribution to alcoholic pancreatitis is not known. We investigated the role of X box-binding protein 1 (XBP1), an UPR regulator, in prevention of alcohol-induced ER stress in the exocrine pancreas. METHODS Wild-type and Xbp1+/? mice were fed control or ethanol diets for 4 weeks. Pancreatic tissue samples were then examined by light and electron microscopy to determine pancreatic alterations; UPR regulators were analyzed biochemically. RESULTS In wild-type mice, ethanol activated a UPR, increasing pancreatic levels of XBP1 and XBP1 targets such as protein disulfide isomerase (PDI). In these mice, pancreatic damage was minor. In ethanol-fed Xbp1+/? mice, XBP1 and PDI levels were significantly lower than in ethanol-fed, wild-type mice. The combination of XBP1 deficiency and ethanol feeding reduced expression of regulators of ER function and the upregulation of pro-apoptotic signals. Moreover, ethanol feeding induced oxidation of PDI, which might compromise PDI-mediated disulfide bond formation during ER protein folding. In ethanol-fed Xbp1+/? mice, ER stress was associated with disorganized and dilated ER, loss of zymogen granules, accumulation of autophagic vacuoles, and increased acinar cell death. CONCLUSIONS Chronic ethanol feeding causes oxidative ER stress, which activates a UPR and increases XBP1 levels and activity. A defective UPR, due to XBP1 deficiency, results in ER dysfunction and acinar cell pathology.

Lugea, Aurelia; Tischler, David; Nguyen, Janie; Gong, Jun; Gukovsky, Ilya; French, Samuel W.; Gorelick, Fred S.; Pandol, Stephen J.

2010-01-01

30

Unfolded protein response in Gaucher disease: from human to Drosophila  

PubMed Central

Background In Gaucher disease (GD), resulting from mutations in the GBA gene, mutant ?-glucocerebrosidase (GCase) molecules are recognized as misfolded in the endoplasmic reticulum (ER). They are retrotranslocated to the cytoplasm, where they are ubiquitinated and undergo proteasomal degradation in a process known as the ER Associated Degradation (ERAD). We have shown in the past that the degree of ERAD of mutant GCase correlates with GD severity. Persistent presence of mutant, misfolded protein molecules in the ER leads to ER stress and evokes the unfolded protein response (UPR). Methods We investigated the presence of UPR in several GD models, using molecular and behavioral assays. Results Our results show the existence of UPR in skin fibroblasts from GD patients and carriers of GD mutations. We could recapitulate UPR in two different Drosophila models for carriers of GD mutations: flies heterozygous for the endogenous mutant GBA orthologs and flies expressing the human N370S or L444P mutant GCase variants. We encountered early death in both fly models, indicating the deleterious effect of mutant GCase during development. The double heterozygous flies, and the transgenic flies, expressing mutant GCase in dopaminergic/serotonergic cells developed locomotion deficit. Conclusion Our results strongly suggest that mutant GCase induces the UPR in GD patients as well as in carriers of GD mutations and leads to development of locomotion deficit in flies heterozygous for GD mutations.

2013-01-01

31

Aberrant islet unfolded protein response in type 2 diabetes  

PubMed Central

The endoplasmic reticulum adapts to fluctuations in demand and copes with stress through an adaptive signaling cascade called the unfolded protein response (UPR). Accumulating evidence indicates that the canonical UPR is critical to the survival and function of insulin-producing pancreatic ?-cells, and alterations in the UPR may contribute to the pathogenesis of type 2 diabetes. However, the dynamic regulation of UPR molecules in the islets of animal models and humans with type 2 diabetes remains to be elucidated. Here, we analyzed the expression of activating factor 6 (ATF6?) and spliced X-box binding protein 1 (sXBP1), and phosphorylation of eukaryotic initiation factor 2 (eIF2?), to evaluate the three distinct branches of the UPR in the pancreatic islets of mice with diet- or genetic-induced obesity and insulin resistance. ATF6 and sXBP1 expression was predominantly found in the ?-cells, where hyperglycemia coincided with a decline in expression in both experimental models and in humans with type 2 diabetes. These data suggest alterations in the expression of UPR mediators may contribute to the decline in islet function in type 2 diabetes in mice and humans.

Engin, Feyza; Nguyen, Truc; Yermalovich, Alena; Hotamisligil, Gokhan S.

2014-01-01

32

The unfolded protein response selectively targets active smoothened mutants.  

PubMed

The Hedgehog signaling pathway, an essential regulator of developmental patterning, has been implicated in playing causative and survival roles in a range of human cancers. The signal-transducing component of the pathway, Smoothened, has revealed itself to be an efficacious therapeutic target in combating oncogenic signaling. However, therapeutic challenges remain in cases where tumors acquire resistance to Smoothened antagonists, and also in cases where signaling is driven by active Smoothened mutants that exhibit reduced sensitivity to these compounds. We previously demonstrated that active Smoothened mutants are subjected to prolonged endoplasmic reticulum (ER) retention, likely due to their mutations triggering conformation shifts that are detected by ER quality control. We attempted to exploit this biology and demonstrate that deregulated Hedgehog signaling driven by active Smoothened mutants is specifically attenuated by ER stressors that induce the unfolded protein response (UPR). Upon UPR induction, active Smoothened mutants are targeted by ER-associated degradation, resulting in attenuation of inappropriate pathway activity. Accordingly, we found that the UPR agonist thapsigargin attenuated mutant Smoothened-induced phenotypes in vivo in Drosophila melanogaster. Wild-type Smoothened and physiological Hedgehog patterning were not affected, suggesting that UPR modulation may provide a novel therapeutic window to be evaluated for targeting active Smoothened mutants in disease. PMID:23572559

Marada, Suresh; Stewart, Daniel P; Bodeen, William J; Han, Young-Goo; Ogden, Stacey K

2013-06-01

33

The mitochondrial unfolded protein response and increased longevity: Cause, consequence, or correlation?  

PubMed

The mitochondrial unfolded protein response is a conserved pathway that allows mitochondrial chaperones and other factors to be induced in response to mitochondrial dysfunction. Activation of this pathway has been proposed to underlie lifespan extension from knockdown or mutation of several nuclear encoded mitochondrial genes in Caenorhabditis elegans. In some cases, however, induction of the mitochondrial unfolded protein response is associated with a reduction of lifespan in both yeast and C. elegans. It also has yet to be demonstrated that induction of the mitochondrial unfolded protein response is sufficient to increase lifespan in the absence of overt mitochondrial dysfunction. In this perspective, we briefly review the evidence for and against a direct pro-longevity role of the mitochondrial unfolded protein response and suggest important areas of investigation for experimentally addressing this question. PMID:24518875

Bennett, Christopher F; Kaeberlein, Matt

2014-08-01

34

Endurance training in mice increases the unfolded protein response induced by a high-fat diet.  

PubMed

Certain conditions, such as several weeks of high-fat diet, disrupt endoplasmic reticulum (ER) homeostasis and activate an adaptive pathway referred as the unfolded protein response. When the unfolded protein response fails, the result is the development of inflammation and insulin resistance. These two pathological states are known to be improved by regular exercise training but the mechanisms remain largely undetermined. As it has recently been shown that the unfolded protein response is regulated by exercise, we hypothesised that concomitant treadmill exercise training (HFD+ex) prevents ER homeostasis disruption and its downstream consequences induced by a 6-week high-fat diet (HFD) in mice by activating the protective unfolded protein response. Several well-documented markers of the unfolded protein response were measured in the soleus and tibialis anterior muscles as well as in the liver and pancreas. In HFD mice, an increase in these markers was observed (from 2- to 15-fold, P < 0.05) in all tissues studied. The combination of HFD+ex increased the expression of several markers further, up to 100 % compared to HFD alone (P < 0.05). HFD increased inflammatory markers both in the plasma (IL-6 protein, 2.5 ± 0.52-fold; MIP-1? protein, 1.3 ± 0.13-fold; P < 0.05) and in the tissues studied, and treadmill exercise attenuated the inflammatory state induced by HFD (P < 0.05). However, treadmill exercise could not reverse HFD-induced whole body glucose intolerance, assessed by OGTT (AUC, 1.8 ± 0.29-fold, P < 0.05). In conclusion, our results show that a HFD activated the unfolded protein response in mouse tissues in vivo, and that endurance training promoted this response. We speculate that the potentiation of the unfolded protein response by endurance training may represent a positive adaptation protecting against further cellular stress. PMID:23011781

Deldicque, Louise; Cani, Patrice D; Delzenne, Nathalie M; Baar, Keith; Francaux, Marc

2013-06-01

35

Unfolded protein response in Fuchs Endothelial Corneal Dystrophy: a Unifying Pathogenic Pathway?  

PubMed Central

Purpose To assess for activation of the unfolded protein response in corneal endothelium of Fuchs endothelial corneal dystrophy patients. Design Retrospective comparative case series of laboratory specimens Methods Corneal specimens of patients with Fuchs dystrophy and controls with corneal pathologies other than Fuchs dystrophy were evaluated by transmission electron microscopy (TEM) to evaluate for structural changes of the rough endoplasmic reticulum in corneal endothelium. TEM images were evaluated for alterations of rough endoplasmic reticulum as sign of unfolded protein response. Normal autopsy eyes, Fuchs dystrophy, and keratoconus corneas were used for immunohistochemistry. Immunohistochemistry was performed on formalin-fixed, paraffin-embedded sections of patient corneas for three unfolded protein response markers (GRP78, phospho-eIF2?, CHOP) and two apoptosis markers (Caspase 3 and 9). Immunohistochemistry signal quantitation of corneal endothelium for evaluation of marker expression was performed using automated software. Corneal sections were assessed quantitatively for levels of immunohistochemistry marker expression. Results TEM showed enlargement of rough endoplasmic reticulum in corneal endothelium of all Fuchs dystrophy specimens. Immunohistochemistry quantitation demonstrated a significant increase in mean signal in corneal endothelium from Fuchs dystrophy patients for markers GRP78, phospho-eIF2?, CHOP and caspase 9, compared with non- Fuchs dystrophy corneas (p < 0.05). Conclusions Results of both TEM and immunohistochemistry indicate activation of unfolded protein response in Fuchs dystrophy. Unfolded protein response activation leads to endothelial cell apoptosis in Fuchs dystrophy and may play a central pathogenic role in this disease.

Engler, Christoph; Kelliher, Clare; Spitze, Arielle R.; Speck, Caroline L.; Eberhart, Charles G.; Jun, Albert S.

2009-01-01

36

Membrane lipid saturation activates endoplasmic reticulum unfolded protein response transducers through their transmembrane domains  

PubMed Central

Endoplasmic reticulum (ER) stress sensors use a related luminal domain to monitor the unfolded protein load and convey the signal to downstream effectors, signaling an unfolded protein response (UPR) that maintains compartment-specific protein folding homeostasis. Surprisingly, perturbation of cellular lipid composition also activates the UPR, with important consequences in obesity and diabetes. However, it is unclear if direct sensing of the lipid perturbation contributes to UPR activation. We found that mutant mammalian ER stress sensors, IRE1? and PERK, lacking their luminal unfolded protein stress-sensing domain, nonetheless retained responsiveness to increased lipid saturation. Lipid saturation-mediated activation in cells required an ER-spanning transmembrane domain and was positively regulated in vitro by acyl-chain saturation in reconstituted liposomes. These observations suggest that direct sensing of the lipid composition of the ER membrane contributes to the UPR.

Volmer, Romain; van der Ploeg, Kattria; Ron, David

2013-01-01

37

IRE1 Signaling Affects Cell Fate During the Unfolded Protein Response  

Microsoft Academic Search

Endoplasmic reticulum (ER) stress activates a set of signaling pathways, collectively termed the unfolded protein response (UPR). The three UPR branches (IRE1, PERK, and ATF6) promote cell survival by reducing misfolded protein levels. UPR signaling also promotes apoptotic cell death if ER stress is not alleviated. How the UPR integrates its cytoprotective and proapoptotic outputs to select between life or

Jonathan H. Lin; Han Li; Douglas Yasumura; Hannah R. Cohen; Chao Zhang; Barbara Panning; Kevan M. Shokat; Matthew M. LaVail; Peter Walter

2007-01-01

38

The unfolded protein response: controlling cell fate decisions under ER stress and beyond  

Microsoft Academic Search

Protein-folding stress at the endoplasmic reticulum (ER) is a salient feature of specialized secretory cells and is also involved in the pathogenesis of many human diseases. ER stress is buffered by the activation of the unfolded protein response (UPR), a homeostatic signalling network that orchestrates the recovery of ER function, and failure to adapt to ER stress results in apoptosis.

Claudio Hetz

2012-01-01

39

Cigarette Smoke Induces an Unfolded Protein Response in the Human Lung A Proteomic Approach  

Microsoft Academic Search

Cigarette smoking, which exposes the lung to high concentrations of reactive oxidant species (ROS) is the major risk factor for chronic obstructive pulmonary disease (COPD). Recent studies indicate that ROS interfere with protein folding in the endoplasmic reticulum and elicit a compensatory response termed the ''unfolded protein re- sponse'' (UPR). The importance of the UPR lies in its ability to

Steven G. Kelsen; Xunbao Duan; Rong Ji; Oscar Perez; Chunli Liu; Salim Merali

2007-01-01

40

Japanese Encephalitis Virus Infection Initiates Endoplasmic Reticulum Stress and an Unfolded Protein Response  

Microsoft Academic Search

The malfunctioning of the endoplasmic reticulum (ER) of cells in hosts ranging from yeast to mammals can trigger an unfolded protein response (UPR). Such malfunctioning can result from a variety of ER stresses, including the inhibition of protein glycosylation and calcium imbalance. To cope with ER stresses, cells may rely on the UPR to send a signal(s) from the ER

Hong-Lin Su; Ching-Len Liao; Yi-Ling Lin

2002-01-01

41

The endoplasmic reticulum and unfolded protein response in the control of mammalian recombinant protein production.  

PubMed

The endoplasmic reticulum (ER) of eukaryotic cells is involved in the synthesis and processing of proteins and lipids in the secretory pathway. These processing events that proteins undergo in the ER may present major limiting steps for recombinant protein production. Increased protein synthesis, accumulation of improperly processed or mis-folded protein can induce ER stress. To cope with ER stress, the ER has quality control mechanisms, such as the unfolded protein response (UPR) and ER-associated degradation to restore homeostasis. ER stress and UPR activation trigger multiple physiological cellular changes. Here we review cellular mechanisms that cope with ER stress and illustrate how this knowledge can be applied to increase the efficiency of recombinant protein expression. PMID:24752815

Hussain, Hirra; Maldonado-Agurto, Rodrigo; Dickson, Alan J

2014-08-01

42

The Yeast Rab GTPase Ypt1 Modulates Unfolded Protein Response Dynamics by Regulating the Stability of HAC1 RNA  

Microsoft Academic Search

The unfolded protein response (UPR) is a conserved mechanism that mitigates accumulation of unfolded proteins in the ER. The yeast UPR is subject to intricate post-transcriptional regulation, involving recruitment of the RNA encoding the Hac1 transcription factor to the ER and its unconventional splicing. To investigate the mechanisms underlying regulation of the UPR, we screened the yeast proteome for proteins

Nikoleta G. Tsvetanova; Daniel P. Riordan; Patrick O. Brown

2012-01-01

43

Metabolic stress promotes renal tubular inflammation by triggering the unfolded protein response  

Microsoft Academic Search

The renal epithelium contributes to the development of inflammation during ischemic injury. Ischemia induces endoplasmic reticulum (ER) stress and activates the unfolded protein response (UPR). Ischemic tissues generate distress signals and inflammation that activates fibrogenesis and may promote adaptive immunity. Interestingly, the UPR may activate inflammation pathways. Our aim was to test whether the UPR is activated during metabolic stress

S Fougeray; N Bouvier; P Beaune; C Legendre; D Anglicheau; E Thervet; N Pallet

2011-01-01

44

Overexpression of Myocilin in the Drosophila Eye Activates the Unfolded Protein Response: Implications for Glaucoma  

Microsoft Academic Search

BackgroundGlaucoma is the world's second leading cause of bilateral blindness with progressive loss of vision due to retinal ganglion cell death. Myocilin has been associated with congenital glaucoma and 2–4% of primary open angle glaucoma (POAG) cases, but the pathogenic mechanisms remain largely unknown. Among several hypotheses, activation of the unfolded protein response (UPR) has emerged as a possible disease

Mary Anna Carbone; Julien F. Ayroles; Akihiko Yamamoto; Tatiana V. Morozova; Steven A. West; Michael M. Magwire; Trudy F. C. Mackay; Robert R. H. Anholt; Olaf Sporns

2009-01-01

45

Unfolded protein response to autophagy as a promising druggable target for anticancer therapy  

PubMed Central

The endoplasmic reticulum (ER) is responsible for protein processing. In rapidly proliferating tumor cells, the ER tends to be overloaded with unfolded and misfolded proteins due to high metabolic demand. With the limited protein-folding capacity of the ER, tumor cells often suffer from more ER stress than do normal cells. Thus, cellular stress responses to cope with ER stress, such as the unfolded protein response (UPR) and autophagy, might be more activated in cancer cells than in normal cells. The complex signaling pathways from the UPR to autophagy provide promising druggable targets; a number of UPR/autophagy-targeted anticancer agents are currently in development in preclinical and clinical studies. In this short review we will discuss the potential anticancer efficacy of modulators of cellular stress responses, especially UPR and autophagy, on the basis of their signaling pathways. In addition, the current developmental status of the UPR/autophagy-targeted agents will be discussed.

Suh, Dong Hoon; Kim, Mi-Kyung; Kim, Hee Seung; Chung, Hyun Hoon; Song, Yong Sang

2012-01-01

46

Endoplasmic Reticulum Stress and Unfolded Protein Response Pathways: Potential for Treating Age-related Retinal Degeneration  

PubMed Central

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) and their aggregation impair normal cellular function and can be toxic, leading to cell death. Prolonged expression of misfolded proteins triggers ER stress, which initiates a cascade of reactions called the unfolded protein response (UPR). Protein misfolding is the basis for a variety of disorders known as ER storage or conformational diseases. There are an increasing number of eye disorders associated with misfolded proteins and pathologic ER responses, including retinitis pigmentosa (RP). Herein we review the basic cellular and molecular biology of UPR with focus on pathways that could be potential targets for treating retinal degenerative diseases.

Haeri, Mohammad; Knox, Barry E

2012-01-01

47

PI 3-kinase regulatory subunits as regulators of the unfolded protein response.  

PubMed

The endoplasmic reticulum (ER) consists of an interconnected, membranous network that is the major site for the synthesis and folding of integral membrane and secretory proteins. Within the ER lumen, protein folding is facilitated by molecular chaperones and a variety of enzymes that ensure that polypeptides obtain their appropriate, tertiary conformation (Dobson, C. M. (2004). Principles of protein folding, misfolding and aggregation. Semin. Cell Dev. Biol. 15, 3-16; Ni, M., and Lee, A. S. (2007). ER chaperones in mammalian development and human diseases. FEBS Lett. 581, 3641-3651.). Physiological conditions that increase protein synthesis or stimuli that disturb the processes by which proteins obtain their native conformation, create an imbalance between the protein-folding demand and capacity of the ER. This results in the accumulation of unfolded or improperly folded proteins in the ER lumen and a state of ER stress. The cellular response, referred to as the unfolded protein response (UPR), results in activation of three linked signal transduction pathways: PKR-like kinase (PERK), inositol requiring 1 ? (IRE1?), and activating transcription factor 6? (ATF6?) (Ron, D., and Walter, P. (2007). Signal integration in the endoplasmic reticulum unfolded protein response. Nat. Rev. Mol. Cell. Biol. 8, 519-529; Schroder, M., and Kaufman, R. (2005). ER stress and the unfolded protein response. Mutat. Res./Fundam. Mol. Mech. Mutagen. 569, 29-63.). Collectively, the combined actions of these signaling cascades serve to reduce ER stress through attenuation of translation to reduce protein synthesis and through activation of transcriptional programs that ultimately serve to increase ER protein-folding capacity. Recently, we and Park et al. have characterized a novel function for the p85? and p85? subunits as modulators of the UPR by virtue of their ability to facilitate the nuclear entry of XBP-1s following induction of ER stress (Park, S. W., Zhou, Y., Lee, J., Lu, A., Sun, C., Chung, J., Ueki, K., and Ozcan, U. (2010). Regulatory subunits of PI3K, p85alpha and p85 beta, interact with XBP1 and increase its nuclear translocation. Nat. Med. 16, 429-437; Winnay, J. N., Boucher, J., Mori, M. A., Ueki, K., and Kahn, C. R. (2010). A regulatory subunit of phosphoinositide 3-kinase increases the nuclear accumulation of X-box-binding protein-1 to modulate the unfolded protein response. Nat. Med. 16, 438-445.). This chapter describes the recently elucidated role for the regulatory subunits of PI 3-kinase as modulators of the UPR and provides methods to measure UPR pathway activation. PMID:21266249

Winnay, Jonathon N; Kahn, C Ronald

2011-01-01

48

Inhibition of protein translocation at the endoplasmic reticulum promotes activation of the unfolded protein response  

PubMed Central

Selective small-molecule inhibitors represent powerful tools for the dissection of complex biological processes. ESI (eeyarestatin I) is a novel modulator of ER (endoplasmic reticulum) function. In the present study, we show that in addition to acutely inhibiting ERAD (ER-associated degradation), ESI causes production of mislocalized polypeptides that are ubiquitinated and degraded. Unexpectedly, our results suggest that these non-translocated polypeptides promote activation of the UPR (unfolded protein response), and indeed we can recapitulate UPR activation with an alternative and quite distinct inhibitor of ER translocation. These results suggest that the accumulation of non-translocated proteins in the cytosol may represent a novel mechanism that contributes to UPR activation.

McKibbin, Craig; Mares, Alina; Piacenti, Michela; Williams, Helen; Roboti, Peristera; Puumalainen, Marjo; Callan, Anna C.; Lesiak-Mieczkowska, Karolina; Linder, Stig; Harant, Hanna; High, Stephen; Flitsch, Sabine L.; Whitehead, Roger C.; Swanton, Eileithyia

2011-01-01

49

Hypoxia signalling through mTOR and the unfolded protein response in cancer  

Microsoft Academic Search

Hypoxia occurs in the majority of tumours, promoting angiogenesis, metastasis and resistance to therapy. Responses to hypoxia are orchestrated in part through activation of the hypoxia-inducible factor family of transcription factors (HIFs). Recently, two additional O2-sensitive signalling pathways have also been implicated: signalling through the mammalian target of rapamycin (mTOR) kinase and signalling through activation of the unfolded protein response

Marianne Koritzinsky

2008-01-01

50

Stress responses in flavivirus-infected cells: activation of unfolded protein response and autophagy  

PubMed Central

The Flavivirus is a genus of RNA viruses that includes multiple long known human, animal, and zoonotic pathogens such as Dengue virus, yellow fever virus, West Nile virus, or Japanese encephalitis virus, as well as other less known viruses that represent potential threats for human and animal health such as Usutu or Zika viruses. Flavivirus replication is based on endoplasmic reticulum-derived structures. Membrane remodeling and accumulation of viral factors induce endoplasmic reticulum stress that results in activation of a cellular signaling response termed unfolded protein response (UPR), which can be modulated by the viruses for their own benefit. Concomitant with the activation of the UPR, an upregulation of the autophagic pathway in cells infected with different flaviviruses has also been described. This review addresses the current knowledge of the relationship between endoplasmic reticulum stress, UPR, and autophagy in flavivirus-infected cells and the growing evidences for an involvement of these cellular pathways in the replication and pathogenesis of these viruses.

Blazquez, Ana-Belen; Escribano-Romero, Estela; Merino-Ramos, Teresa; Saiz, Juan-Carlos; Martin-Acebes, Miguel A.

2014-01-01

51

Engineering of chaperone systems and of the unfolded protein response  

Microsoft Academic Search

Production of recombinant proteins in mammalian cells is a successful technology that delivers protein pharmaceuticals for\\u000a therapies and for diagnosis of human disorders. Cost effective production of protein biopharmaceuticals requires extensive\\u000a optimization through cell and fermentation process engineering at the upstream and chemical engineering of purification processes\\u000a at the downstream side of the production process. The majority of protein pharmaceuticals

Saeed U. Khan; Martin Schröder

2008-01-01

52

Role of the Unfolded Protein Response in ? Cell Compensation and Failure during Diabetes  

PubMed Central

Pancreatic ? cell failure leads to diabetes development. During disease progression, ? cells adapt their secretory capacity to compensate the elevated glycaemia and the peripheral insulin resistance. This compensatory mechanism involves a fine-tuned regulation to modulate the endoplasmic reticulum (ER) capacity and quality control to prevent unfolded proinsulin accumulation, a major protein synthetized within the ? cell. These signalling pathways are collectively termed unfolded protein response (UPR). The UPR machinery is required to preserve ER homeostasis and ? cell integrity. Moreover, UPR actors play a key role by regulating ER folding capacity, increasing the degradation of misfolded proteins, and limiting the mRNA translation rate. Recent genetic and biochemical studies on mouse models and human UPR sensor mutations demonstrate a clear requirement of the UPR machinery to prevent ? cell failure and increase ? cell mass and adaptation throughout the progression of diabetes. In this review we will highlight the specific role of UPR actors in ? cell compensation and failure during diabetes.

Rabhi, Nabil; Salas, Elisabet; Froguel, Philippe; Annicotte, Jean-Sebastien

2014-01-01

53

Protein Unfolding: Rigidity Lost  

NASA Astrophysics Data System (ADS)

We present a study of protein unfolding using the FIRST software (which determines the rigid and flexible regions within a protein) on a set of structurally diverse proteins[1,2]. The proteins are modeled as networks of covalent bonds and non-covalent interactions including hydrogen bonds, salt bridges, and hydrophobic tethers. These flexible hydrophobic tethers between non-polar atoms model the hydrophobic effect, which drives protein folding. Breaking the hydrogen bonds and salt bridges according to their relative strength reduces the mean atomic coordination, < r >, and simulates the process of unfolding by thermal denaturation. As the mean coordination decreases, we observe the emergence of flexible regions from the rigid core. The transition state is determined from the peak in the fluctuations in the number of independent bond-rotational degrees of freedom (floppy modes) with respect to the mean coordination. As the protein denatures, it loses rigidity at the transition state: going from structurally stable, with the majority of the atoms in the rigid core, to completely flexible and unfolded. This transition occurs at a critical mean coordination of 2.405 for most proteins, the same mean coordination as the transition point for network glasses. Such universal behavior identifies the mean coordination as the relevant structural parameter, or reaction coordinate, for protein unfolding. [1] D. J. Jacobs, A.J. Rader, Leslie A. Kuhn, and M.F. Thorpe Protein Flexibility Prediction Using Graph Theory. Proteins, 44, 150-165, 2001. [2] A.J. Rader, B.M. Hespenheide, L.K. Kuhn, and M.F. Thorpe Protein Unfolding: Rigidity Lost. Proc. Natl. Acad. Sci., 97, 3540-3545, 2002.

Rader, Andrew; Hespenheide, Brandon M.; Kuhn, Leslie A.; Thorpe, M. F.

2003-03-01

54

Gcn4p and Novel Upstream Activating Sequences Regulate Targets of the Unfolded Protein Response  

PubMed Central

Eukaryotic cells respond to accumulation of unfolded proteins in the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR), a signal transduction pathway that communicates between the ER and the nucleus. In yeast, a large set of UPR target genes has been experimentally determined, but the previously characterized unfolded protein response element (UPRE), an upstream activating sequence (UAS) found in the promoter of the UPR target gene KAR2, cannot account for the transcriptional regulation of most genes in this set. To address this puzzle, we analyzed the promoters of UPR target genes computationally, identifying as candidate UASs short sequences that are statistically overrepresented. We tested the most promising of these candidate UASs for biological activity, and identified two novel UPREs, which are necessary and sufficient for UPR activation of promoters. A genetic screen for activators of the novel motifs revealed that the transcription factor Gcn4p plays an essential and previously unrecognized role in the UPR: Gcn4p and its activator Gcn2p are required for induction of a majority of UPR target genes during ER stress. Both Hac1p and Gcn4p bind target gene promoters to stimulate transcriptional induction. Regulation of Gcn4p levels in response to changing physiological conditions may function as an additional means to modulate the UPR. The discovery of a role for Gcn4p in the yeast UPR reveals an additional level of complexity and demonstrates a surprising conservation of the signaling circuit between yeast and metazoan cells.

Patil, Christopher K; Li, Hao

2004-01-01

55

Unfolding of Proteins: Thermal and Mechanical Unfolding  

NASA Technical Reports Server (NTRS)

We have employed a Hamiltonian model based on a self-consistent Gaussian appoximation to examine the unfolding process of proteins in external - both mechanical and thermal - force elds. The motivation was to investigate the unfolding pathways of proteins by including only the essence of the important interactions of the native-state topology. Furthermore, if such a model can indeed correctly predict the physics of protein unfolding, it can complement more computationally expensive simulations and theoretical work. The self-consistent Gaussian approximation by Micheletti et al. has been incorporated in our model to make the model mathematically tractable by signi cantly reducing the computational cost. All thermodynamic properties and pair contact probabilities are calculated by simply evaluating the values of a series of Incomplete Gamma functions in an iterative manner. We have compared our results to previous molecular dynamics simulation and experimental data for the mechanical unfolding of the giant muscle protein Titin (1TIT). Our model, especially in light of its simplicity and excellent agreement with experiment and simulation, demonstrates the basic physical elements necessary to capture the mechanism of protein unfolding in an external force field.

Hur, Joe S.; Darve, Eric

2004-01-01

56

Severe Injury Is Associated With Insulin Resistance, Endoplasmic Reticulum Stress Response, and Unfolded Protein Response  

PubMed Central

Objective We determined whether postburn hyperglycemia and insulin resistance are associated with endoplasmic reticulum (ER) stress/unfolded protein response (UPR) activation leading to impaired insulin receptor signaling. Background Inflammation and cellular stress, hallmarks of severely burned and critically ill patients, have been causally linked to insulin resistance in type 2 diabetes via induction of ER stress and the UPR. Methods Twenty severely burned pediatric patients were compared with 36 nonburned children. Clinical markers, protein, and GeneChip analysis were used to identify transcriptional changes in ER stress and UPR and insulin resistance–related signaling cascades in peripheral blood leukocytes, fat, and muscle at admission and up to 466 days postburn. Results Burn-induced inflammatory and stress responses are accompanied by profound insulin resistance and hyperglycemia. Genomic and protein analysis revealed that burn injury was associated with alterations in the signaling pathways that affect insulin resistance, ER/sarcoplasmic reticulum stress, inflammation, and cell growth/apoptosis up to 466 days postburn. Conclusion Burn-induced insulin resistance is associated with persistent ER/sarcoplasmic reticulum stress/UPR and subsequent suppressed insulin receptor signaling over a prolonged period of time.

Jeschke, Marc G.; Finnerty, Celeste C.; Herndon, David N.; Song, Juquan; Boehning, Darren; Tompkins, Ronald G.; Baker, Henry V.; Gauglitz, Gerd G.

2012-01-01

57

Physiological unfolded protein response regulated by OASIS family members, transmembrane bZIP transcription factors.  

PubMed

The endoplasmic reticulum (ER) plays role in the maintenance of numerous aspects of cellular and organismal homeostasis by folding, modifying, and exporting nascent secretory and transmembrane proteins. Failure of the ER's adaptive capacity results in accumulation of unfolded or malfolded proteins in the ER lumen (ER stress). To avoid cellular damage, mammalian cells activate the specific signals from the ER to the cytosol or nucleus to enhance the capacity for protein folding, attenuate the synthesis of proteins, and degrade unfolded proteins. These signaling pathways are collectively known as the unfolded protein response (UPR). The canonical branches of the UPR are mediated by three ER membrane-bound proteins, PERK, IRE1, and ATF6. These ER stress transducers basically play important roles in cell survival after ER stress. Recently, novel types of ER stress transducers, OASIS family members that share a region of high sequence similarity with ATF6 have been identified. They have a transmembrane domain, which allows them to associate with the ER, and possess a transcription-activation domain and a bZIP domain. OASIS family proteins include OASIS, BBF2H7, CREBH, AIbZIP, and Luman. Despite the structural similarities among OASIS family proteins and ATF6, differences in activating stimuli, tissue distribution, and response element binding indicate specialized functions of each member on regulating the UPR in the specific organs and tissues. Here, we summarize our current understanding of biochemical characteristics and in vivo functions of OASIS family proteins, particularly focusing on OASIS and BBF2H7. A growing body of new works suggests that the UPR branches regulated by OASIS family members play essential roles in cell differentiation and maturation or maintenance of basal cellular homeostasis in mammals. PMID:21438114

Kondo, Shinichi; Saito, Atsushi; Asada, Rie; Kanemoto, Soshi; Imaizumi, Kazunori

2011-04-01

58

Organismal regulation of XBP-1-mediated unfolded protein response during development and immune activation  

PubMed Central

The increased demand on protein folding in the endoplasmic reticulum (ER) during bacterial infection activates the unfolded protein response (UPR). OCTR-1—a G protein-coupled catecholamine receptor expressed in neurons—suppresses innate immunity by downregulating a non-canonical UPR pathway and the p38 MAPK pathway. Here, we show that OCTR-1 also regulates the canonical UPR pathway, which is controlled by XBP-1, at the organismal level. Importantly, XBP-1 is not under OCTR-1 control during development, only at the adult stage. Our results indicate that the nervous system temporally controls the UPR pathway to maintain ER homeostasis during development and immune activation.

Sun, Jingru; Liu, Yiyong; Aballay, Alejandro

2012-01-01

59

Stressing Out the ER: A Role of the Unfolded Protein Response in Prion-Related Disorders  

PubMed Central

Transmissible Spongiform Encephalopathies are fatal and infectious neurodegenerative diseases characterized by extensive neuronal apoptosis and the accumulation of an abnormally folded form of the cellular prion protein (PrP), denoted PrPSC. Compelling evidence suggests the involvement of several signaling pathways in prion pathogenesis, including proteasome dysfunction, alterations in the protein maturation pathways and the unfolded protein response. Recent reports indicate that endoplasmic reticulum stress due to the PrP misfolding may be a critical factor mediating neuronal dysfunction in prion diseases. These findings have applications for developing novel strategies for treatment and early diagnosis of transmissible spongiform encephalopathies and other neurodegenerative diseases.

Hetz, Claudio A.; Soto, Claudio

2009-01-01

60

Effects of Inactivation and Constitutive Expression of the Unfolded- Protein Response Pathway on Protein Production in the Yeast Saccharomyces cerevisiae  

Microsoft Academic Search

One strategy to obtain better yields of secreted proteins has been overexpression of single endoplasmic reticulum-resident foldases or chaperones. We report here that manipulation of the unfolded-protein response (UPR) pathway regulator, HAC1, affects production of both native and foreign proteins in the yeast Saccha- romyces cerevisiae. The effects of HAC1 deletion and overexpression on the production of a native protein,

Mari Valkonen; Merja Penttila; Markku Saloheimo

2003-01-01

61

Inducible membranes in yeast: relation to the unfolded-protein-response pathway.  

PubMed

Overproduction of an endoplasmic reticulum (ER)-resident membrane protein (cytochrome P450 52A3) and of a secretory protein (invertase) was used to study the regulation of the luminal ER protein Kar2p under conditions that lead to ER proliferation and secretory overload, respectively. In both cases we found (i) a significant increase of Kar2 protein and mRNA levels, (ii) a transcriptional regulation based on the the function of the 22 bp unfolded-protein-response element of the KAR2 promoter and (iii) an essential role of the transmembrane kinase Ire1p for upregulation of KAR2 gene expression. These results show that the same mechanism operates when KAR2 induction is triggered by overproduction of cytochrome P450 or invertase and that this mechanism shares the known features of the unfolded-protein-response pathway. Disruption of the IRE1 gene resulted in a marked decrease of the invertase protein levels produced. In contrast, a functional IRE1 gene was not required to reach high-level production of the integral membrane protein cytochrome P450 52A3, Moreover, IRE1 gene disruption did not prevent P450-induced ER proliferation. We suggest that Ire1p-mediated KAR2 induction is, in the case of cytochrome P450 52A3 overproduction, a process which follows on ER proliferation, thereby monitoring the increase of ER size and adjusting the level of Kar2p accordingly. PMID:9364746

Menzel, R; Vogel, F; Kärgel, E; Schunck, W H

1997-10-01

62

The unfolded protein response regulates an angiogenic response by the kidney epithelium during ischemic stress.  

PubMed

Ischemic injuries permanently affect kidney tissue and challenge cell viability, promoting inflammation and fibrogenesis. Ischemia results in nutrient deprivation, which triggers endoplasmic reticulum stress, ultimately resulting in the unfolded protein response (UPR). The aim of this study was to test whether the UPR could promote an angiogenic response independently of the HIF-1? pathway during ischemic stress in the human kidney epithelium. Glucose deprivation induced the secretion of vascular endothelial growth factor A (VEGFA), basic fibroblast growth factor (bFGF) and angiogenin (ANG) in human kidney epithelial cells independently of HIF-1?. Glucose deprivation, but not hypoxia, triggered endoplasmic reticulum stress and activated the UPR. RNA interference-mediated inhibition of the gene encoding the kinase PERK decreased VEGFA and bFGF expression, but neither gene was affected by the inhibition of IRE1? or ATF6. Furthermore, we show that the expression of angiogenin, which inhibits protein synthesis, is regulated by both IRE1? and PERK, which could constitute a complementary function of the UPR in the repression of translation. In a rat model of acute ischemic stress, we show that the UPR is activated in parallel with VEGFA, bFGF, and ANG expression and independently of HIF-1?. PMID:22403402

Bouvier, Nicolas; Fougeray, Sophie; Beaune, Philippe; Thervet, Eric; Pallet, Nicolas

2012-04-27

63

Spliced X-box binding protein 1 couples the unfolded protein response to hexosamine biosynthetic pathway.  

PubMed

The hexosamine biosynthetic pathway (HBP) generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress. PMID:24630721

Wang, Zhao V; Deng, Yingfeng; Gao, Ningguo; Pedrozo, Zully; Li, Dan L; Morales, Cyndi R; Criollo, Alfredo; Luo, Xiang; Tan, Wei; Jiang, Nan; Lehrman, Mark A; Rothermel, Beverly A; Lee, Ann-Hwee; Lavandero, Sergio; Mammen, Pradeep P A; Ferdous, Anwarul; Gillette, Thomas G; Scherer, Philipp E; Hill, Joseph A

2014-03-13

64

Unfolded Protein Response and Activated Degradative Pathways Regulation in GNE Myopathy  

PubMed Central

Although intracellular beta amyloid (A?) accumulation is known as an early upstream event in the degenerative course of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) myopathy, the process by which A?deposits initiate various degradative pathways, and their relationship have not been fully clarified. We studied the possible secondary responses after amyloid beta precursor protein (A?PP) deposition including unfolded protein response (UPR), ubiquitin proteasome system (UPS) activation and its correlation with autophagy system. Eight GNE myopathy patients and five individuals with normal muscle morphology were included in this study. We performed immunofluorescence and immunoblotting to investigate the expression of A?PP, phosphorylated tau (p-tau) and endoplasmic reticulum molecular chaperones. Proteasome activities were measured by cleavage of fluorogenic substrates. The expression of proteasome subunits and linkers between proteasomal and autophagy systems were also evaluated by immunoblotting and relative quantitative real-time RT-PCR. Four molecular chaperones, glucose-regulated protein 94 (GRP94), glucose-regulated protein 78 (GRP78), calreticulin and calnexin and valosin containing protein (VCP) were highly expressed in GNE myopathy. 20S proteasome subunits, three main proteasome proteolytic activities, and the factors linking UPS and autophagy system were also increased. Our study suggests that A?PP deposition results in endoplasmic reticulum stress (ERS) and highly expressed VCP deliver unfolded proteins from endoplasmic reticulum to proteosomal system which is activated in endoplasmic reticulum associated degradation (ERAD) in GNE myopathy. Excessive ubiquitinated unfolded proteins are exported by proteins that connect UPS and autophagy to autophagy system, which is activated as an alternative pathway for degradation.

Li, Honghao; Chen, Qi; Liu, Fuchen; Zhang, Xuemei; Li, Wei; Liu, Shuping; Zhao, Yuying; Gong, Yaoqin; Yan, Chuanzhu

2013-01-01

65

Acute nutrient regulation of the unfolded protein response and integrated stress response in cultured rat pancreatic islets  

Microsoft Academic Search

Aims\\/hypothesis  Inadequate chaperone function relative to client protein load in the endoplasmic reticulum triggers an adaptive unfolded protein\\u000a response (UPR), including the integrated stress response (ISR), the latter being also activated by other types of stresses.\\u000a It is well established that pancreatic beta cells, which synthesise and secrete insulin upon nutrient stimulation, are markedly\\u000a affected by pathological disruption or excessive activation

H. Elouil; M. Bensellam; Y. Guiot; D. Vander Mierde; S. M. A. Pascal; F. C. Schuit; J. C. Jonas

2007-01-01

66

The Unfolded Protein Response and Chemical Chaperones Reduce Protein Misfolding and Colitis in Mice  

PubMed Central

BACKGROUND & AIMS Endoplasmic reticulum (ER) stress has been associated with development of inflammatory bowel disease. We examined the effects of ER stress–induced chaperone response and the orally active chemical chaperones tauroursodeoxycholate (TUDCA) and 4-phenylbutyrate (PBA), which facilitate protein folding and reduce ER stress, in mice with colitis. METHODS We used dextran sulfate sodium (DSS) to induce colitis in mice that do not express the transcription factor ATF6? or the protein chaperone P58IPK. We examined the effects of TUDCA and PBA in cultured intestinal epithelial cells (IECs); in wild-type, P58IPK?/?, and Atf6??/? mice with colitis; and in Il10?/? mice. RESULTS P58IPK?/? and Atf6??/? mice developed more severe colitis following administration of DSS than wild-type mice. IECs from P58IPK?/? mice had excessive ER stress, and apoptotic signaling was activated in IECs from Atf6??/? mice. Inflammatory stimuli induced ER stress signals in cultured IECs, which were reduced by incubation with TUDCA or PBA. Oral administration of either PBA or TUDCA reduced features of DSS-induced acute and chronic colitis in wild-type mice, the colitis that develops in Il10?/? mice, and DSS-induced colitis in P58IPK?/? and Atf6??/? mice. Reduced signs of colonic inflammation in these mice were associated with significantly decreased ER stress in colonic epithelial cells. CONCLUSIONS The unfolded protein response induces expression of genes that encode chaperones involved in ER protein folding; these factors prevent induction of colitis in mice. Chemical chaperones such as TUDCA and PBA alleviate different forms of colitis in mice and might be developed for treatment of inflammatory bowel diseases.

CAO, STEWART SIYAN; ZIMMERMANN, ELLEN M.; CHUANG, BRANDY-MENGCHIEH; SONG, BENBO; NWOKOYE, ANOSIKE; WILKINSON, J. ERBY; EATON, KATHRYN A.; KAUFMAN, RANDAL J.

2013-01-01

67

Surveillance-Activated Defenses Block the ROS-Induced Mitochondrial Unfolded Protein Response  

PubMed Central

Disturbance of cellular functions results in the activation of stress-signaling pathways that aim at restoring homeostasis. We performed a genome-wide screen to identify components of the signal transduction of the mitochondrial unfolded protein response (UPRmt) to a nuclear chaperone promoter. We used the ROS generating complex I inhibitor paraquat to induce the UPRmt, and we employed RNAi exposure post-embryonically to allow testing genes whose knockdown results in embryonic lethality. We identified 54 novel regulators of the ROS–induced UPRmt. Activation of the UPRmt, but not of other stress-signaling pathways, failed when homeostasis of basic cellular mechanisms such as translation and protein transport were impaired. These mechanisms are monitored by a recently discovered surveillance system that interprets interruption of these processes as pathogen attack and depends on signaling through the JNK-like MAP-kinase KGB-1. Mutation of kgb-1 abrogated the inhibition of ROS–induced UPRmt, suggesting that surveillance-activated defenses specifically inhibit the UPRmt but do not compromise activation of the heat shock response, the UPR of the endoplasmic reticulum, or the SKN-1/Nrf2 mediated response to cytosolic stress. In addition, we identified PIFK-1, the orthologue of the Drosophila PI 4-kinase four wheel drive (FWD), and found that it is the only known factor so far that is essential for the unfolded protein responses of both mitochondria and endoplasmic reticulum. This suggests that both UPRs may share a common membrane associated mechanism.

Runkel, Eva D.; Liu, Shu; Baumeister, Ralf; Schulze, Ekkehard

2013-01-01

68

New Insights into Translational Regulation in the Endoplasmic Reticulum Unfolded Protein Response  

PubMed Central

Homeostasis of the protein-folding environment in the endoplasmic reticulum (ER) is maintained by signal transduction pathways that collectively constitute an unfolded protein response (UPR). These affect bulk protein synthesis and thereby the levels of ER stress, but also culminate in regulated expression of specific mRNAs, such as that encoding the transcription factor ATF4. Mechanisms linking eukaryotic initiation factor 2 (eIF2) phosphorylation to control of unfolded protein load in the ER were elucidated more than 10 years ago, but recent work has highlighted the diversity of processes that impinge on eIF2 activity and revealed that there are multiple mechanisms by which changes in eIF2 activity can modulate the translation of individual mRNAs. In addition, the potential for affecting this step of translation initiation pharmacologically is becoming clearer. Furthermore, it is now clear that another strand of the UPR, controlled by the endoribonuclease inositol-requiring enzyme 1 (IRE1), also affects rates of protein synthesis in stressed cells and that its effector function, mediated by the transcription factor X-box-binding protein 1 (XBP1), is subject to important mRNA-specific translational regulation. These new insights into the convergence of translational control and the UPR will be reviewed here.

Pavitt, Graham D.; Ron, David

2012-01-01

69

The Unfolded Protein Response, Degradation from the Endoplasmic Reticulum, and Cancer  

PubMed Central

The endoplasmic reticulum (ER) is an essential organelle involved in many cellular functions including protein folding and secretion, lipid biosynthesis, and calcium homeostasis. Proteins destined for the cell surface or for secretion are made in the ER, where they are folded and assembled into multi-subunit complexes. The ER plays a vital role in cellular protein quality control by extracting and degrading proteins that are not correctly folded or assembled into native complexes. This process, known as ER-associated degradation (ERAD), ensures that only properly folded and assembled proteins are transported to their final destinations. Besides its role in protein folding and transport in the secretory pathway, the ER regulates the biosynthesis of cholesterol and other membrane lipids. ERAD is an important means to ensure that levels of the responsible enzymes are appropriately maintained. The ER is also a major organelle for oxygen and nutrient sensing as cells adapt to their microenvironment. Stresses that disrupt ER function lead to accumulation of unfolded proteins in the ER, a condition known as ER stress. Cells adapt to ER stress by activating an integrated signal transduction pathway called the unfolded protein response (UPR). The UPR represents a survival response by the cells to restore ER homeostasis. If ER stress persists, cells activate mechanisms that result in cell death. Chronic ER stress is increasingly being recognized as a factor in many human diseases such as diabetes, neurodegenerative disorders, and cancer. In this review, we discuss the roles of the UPR and ERAD in cancer and suggest directions for future research.

Tsai, Yien Che; Weissman, Allan M.

2010-01-01

70

Regulation of basal cellular physiology by the homeostatic unfolded protein response  

PubMed Central

The extensive membrane network of the endoplasmic reticulum (ER) is physically juxtaposed to and functionally entwined with essentially all other cellular compartments. Therefore, the ER must sense diverse and constantly changing physiological inputs so it can adjust its numerous functions to maintain cellular homeostasis. A growing body of new work suggests that the unfolded protein response (UPR), traditionally charged with signaling protein misfolding stress from the ER, has been co-opted for the maintenance of basal cellular homeostasis. Thus, the UPR can be activated, and its output modulated, by signals far outside the realm of protein misfolding. These findings are revealing that the UPR causally contributes to disease not just by its role in protein folding but also through its broad influence on cellular physiology.

2010-01-01

71

Improvement of Foreign-Protein Production in Aspergillus niger var. awamori by Constitutive Induction of the Unfolded-Protein Response  

PubMed Central

Unfolded-protein response (UPR) denotes the upregulation of endoplasmic reticulum (ER)-resident chaperone and foldase genes and numerous other genes involved in secretory functions during the accumulation of unfolded proteins into the ER. Overexpression of individual foldases and chaperones has been used in attempts to improve protein production in different production systems. We describe here a novel strategy to improve foreign-protein production. We show that the constitutive induction of the UPR pathway in Aspergillus niger var. awamori can be achieved by expressing the activated form of the transcription factor hacA. This induction enhances the production of Trametes versicolor laccase by up to sevenfold and of bovine preprochymosin by up to 2.8-fold in this biotechnically important fungus. The regulatory range of UPR was studied by analyzing the mRNA levels of novel A. niger var. awamori genes involved in different secretory functions. This revealed both similarities and differences to corresponding studies in Saccharomyces cerevisiae.

Valkonen, Mari; Ward, Michael; Wang, Huaming; Penttila, Merja; Saloheimo, Markku

2003-01-01

72

Functional and Genomic Analyses Reveal an Essential Coordination between the Unfolded Protein Response and ER-Associated Degradation  

Microsoft Academic Search

The unfolded protein response (UPR) regulates gene expression in response to stress in the endoplasmic reticulum (ER). We determined the transcriptional scope of the UPR using DNA microarrays. Rather than regulating only ER-resident chaperones and phospholipid biosynthesis, as anticipated from earlier work, the UPR affects multiple ER and secretory pathway functions. Studies of UPR targets engaged in ER-associated protein degradation

Kevin J. Travers; Christopher K. Patil; Lisa Wodicka; David J. Lockhart; Jonathan S. Weissman; Peter Walter

2000-01-01

73

A new paradigm: innate immune sensing of viruses via the unfolded protein response  

PubMed Central

The immune system depends upon combinations of signals to mount appropriate responses: pathogen specific signals in the context of co-stimulatory “danger” signals drive immune strength and accuracy. Viral infections trigger anti-viral type I interferon (IFN) responses by stimulating endosomal and cytosolic pattern recognition receptors (PRRs). However, viruses have also evolved many strategies to counteract IFN responses. Are there intracellular danger signals that enhance immune responses to viruses? During infection, viruses place a heavy demand on the protein folding machinery of the host endoplasmic reticulum (ER). To survive ER stress, host cells mount an unfolded protein response (UPR) to decrease ER protein load and enhance protein-folding capacity. Viruses also directly elicit the UPR to enhance their replication. Increasing evidence supports an intersection between the host UPR and inflammation, in particular the production of pro-inflammatory cytokines and type I IFN. The UPR directly activates pro-inflammatory cytokine transcription factors and dramatically enhances cytokine production in response to viral PRR engagement. Additionally, viral PRR engagement may stimulate specific pathways within the UPR to enhance cytokine production. Through these mechanisms, viral detection via the UPR and inflammatory cytokine production are intertwined. Consequently, the UPR response is perfectly poised to act as an infection-triggered “danger” signal. The UPR may serve as an internal “co-stimulatory” signal that (1) provides specificity and (2) critically augments responses to overcome viral subterfuge. Further work is needed to test this hypothesis during viral infections.

Smith, Judith A.

2014-01-01

74

Cell death. Opposing unfolded-protein-response signals converge on death receptor 5 to control apoptosis.  

PubMed

Protein folding by the endoplasmic reticulum (ER) is physiologically critical; its disruption causes ER stress and augments disease. ER stress activates the unfolded protein response (UPR) to restore homeostasis. If stress persists, the UPR induces apoptotic cell death, but the mechanisms remain elusive. Here, we report that unmitigated ER stress promoted apoptosis through cell-autonomous, UPR-controlled activation of death receptor 5 (DR5). ER stressors induced DR5 transcription via the UPR mediator CHOP; however, the UPR sensor IRE1? transiently catalyzed DR5 mRNA decay, which allowed time for adaptation. Persistent ER stress built up intracellular DR5 protein, driving ligand-independent DR5 activation and apoptosis engagement via caspase-8. Thus, DR5 integrates opposing UPR signals to couple ER stress and apoptotic cell fate. PMID:24994655

Lu, Min; Lawrence, David A; Marsters, Scot; Acosta-Alvear, Diego; Kimmig, Philipp; Mendez, Aaron S; Paton, Adrienne W; Paton, James C; Walter, Peter; Ashkenazi, Avi

2014-07-01

75

Protein Unfolding and Alzheimer's  

NASA Astrophysics Data System (ADS)

Early interaction events of beta-amyloid (A?) proteins with neurons have been associated with the pathogenesis of Alzheimer's disease. Knowledge pertaining to the role of lipid molecules, particularly cholesterol, in modulating the single A? interactions with neurons at the atomic length and picosecond time resolutions, remains unclear. In our research, we have used atomistic molecular dynamics simulations to explore early molecular events including protein insertion kinetics, protein unfolding, and protein-induced membrane disruption of A? in lipid domains that mimic the nanoscopic raft and non-raft regions of the neural membrane. In this talk, I will summarize our current work on investigating the role of cholesterol in regulating the A? interaction events with membranes at the molecular level. I will also explain how our results will provide new insights into understanding the pathogenesis of Alzheimer's disease associated with the A? proteins.

Cheng, Kelvin

2012-10-01

76

Mif1: a missing link between the unfolded protein response pathway and ER-associated protein degradation?  

PubMed

Eukaryotic cells have three different mechanisms to deal with the accumulation of unfolded proteins in the endoplasmic reticulum: (1) In cells in which unfolded polypeptides accumulate, translation initiation is inhibited to prevent further accumulation of unfolded proteins. (2) Expression of proteins involved in polypeptide folding is strongly enhanced by a process called the Unfolded Protein Response (UPR). (3) Proteins missing the proper tertiary structure are degraded by the ER-Associated protein Degradation (ERAD) mechanism. Recent studies in S. cerevisiae have shown that the processes of UPR and ERAD are functionally linked to each other. Cells lacking a functional ERAD show a constitutive activation of UPR. In addition, many of the components of ERAD are under the direct transcriptional control of UPR. Finally, while neither UPR nor ERAD are essential for cell viability, deletion of both pathways results in severe growth impairment. UPR and ERAD are conserved between yeast and mammalian cells. One of the components of mammalian UPR is the protease presenilin-1. Mutations in the gene for presenilin-1 cause early-onset familial Alzheimer disease. Interestingly, inhibition of proteolysis by the ubiquitin-26S proteasome system has also been described for Alzheimer s disease. This suggests a link between UPR and ERAD in mammalian cells. The recently identified gene Mif1 is a possible candidate to form a direct link between UPR and ERAD in mammalian cells. The Mif1 gene is under the direct control of UPR. Mif1 is a trans-ER-membrane protein, with both the N- and the C-termini facing the cytoplasmic side of the ER membrane. It contains an N-terminal ubiquitin-like domain. It is anticipated that Mif1 may associate through its ubiquitin-like domain with the 26S proteasome, in this way connecting the protein degradation machinery to the ER membrane and resulting in an efficient ERAD. PMID:12370023

van Laar, T; van der Eb, A J; Terleth, C

2001-06-01

77

Endoplasmic Reticulum and the Unfolded Protein Response: Dynamics and Metabolic Integration  

PubMed Central

The endoplasmic reticulum (ER) is a dynamic intracellular organelle with multiple functions essential for cellular homeostasis, development, and stress responsiveness. In response to cellular stress, a well-established signaling cascade, the unfolded protein response (UPR), is activated. This intricate mechanism is an important means of reestablishing cellular homeostasis and alleviating the inciting stress. Now, emerging evidence has demonstrated that the UPR influences cellular metabolism through diverse mechanisms, including calcium and lipid transfer, raising the prospect of involvement of these processes in the pathogenesis of disease, including neurodegeneration, cancer, diabetes mellitus and cardiovascular disease. Here, we review the distinct functions of the ER and UPR from a metabolic point of view, highlighting their association with prevalent pathologies.

Bravo, Roberto; Parra, Valentina; Gatica, Damian; Rodriguez, Andrea E.; Torrealba, Natalia; Paredes, Felipe; Wang, Zhao V.; Zorzano, Antonio; Hill, Joseph A.; Jaimovich, Enrique; Quest, Andrew F.G.; Lavandero, Sergio

2013-01-01

78

Phenyl Acyl Acids Attenuate the Unfolded Protein Response in Tunicamycin-Treated Neuroblastoma Cells  

PubMed Central

Understanding how neural cells handle proteostasis stress in the endoplasmic reticulum (ER) is important to decipher the mechanisms that underlie the cell death associated with neurodegenerative diseases and to design appropriate therapeutic tools. Here we have compared the sensitivity of a human neuroblastoma cell line (SH-SY5H) to the ER stress caused by an inhibitor of protein glycosylation with that observed in human embryonic kidney (HEK-293T) cells. In response to stress, SH-SY5H cells increase the expression of mRNA encoding downstream effectors of ER stress sensors and transcription factors related to the unfolded protein response (the spliced X-box binding protein 1, CCAAT-enhancer-binding protein homologous protein, endoplasmic reticulum-localized DnaJ homologue 4 and asparagine synthetase). Tunicamycin-induced death of SH-SY5H cells was prevented by terminal aromatic substituted butyric or valeric acids, in association with a decrease in the mRNA expression of stress-related factors, and in the accumulation of the ATF4 protein. Interestingly, this decrease in ATF4 protein occurs without modifying the phosphorylation of the translation initiation factor eIF2?. Together, these results show that when short chain phenyl acyl acids alleviate ER stress in SH-SY5H cells their survival is enhanced.

Zamarbide, Marta; Martinez-Pinilla, Eva; Ricobaraza, Ana

2013-01-01

79

IRE1 signaling affects cell fate during the unfolded protein response.  

PubMed

Endoplasmic reticulum (ER) stress activates a set of signaling pathways, collectively termed the unfolded protein response (UPR). The three UPR branches (IRE1, PERK, and ATF6) promote cell survival by reducing misfolded protein levels. UPR signaling also promotes apoptotic cell death if ER stress is not alleviated. How the UPR integrates its cytoprotective and proapoptotic outputs to select between life or death cell fates is unknown. We found that IRE1 and ATF6 activities were attenuated by persistent ER stress in human cells. By contrast, PERK signaling, including translational inhibition and proapoptotic transcription regulator Chop induction, was maintained. When IRE1 activity was sustained artificially, cell survival was enhanced, suggesting a causal link between the duration of UPR branch signaling and life or death cell fate after ER stress. Key findings from our studies in cell culture were recapitulated in photoreceptors expressing mutant rhodopsin in animal models of retinitis pigmentosa. PMID:17991856

Lin, Jonathan H; Li, Han; Yasumura, Douglas; Cohen, Hannah R; Zhang, Chao; Panning, Barbara; Shokat, Kevan M; Lavail, Matthew M; Walter, Peter

2007-11-01

80

Isoprenoid biosynthetic pathway inhibition disrupts monoclonal protein secretion and induces the unfolded protein response pathway in multiple myeloma cells  

PubMed Central

Myeloma is characterized by the overproduction and secretion of monoclonal protein. Inhibitors of the isoprenoid biosynthetic pathway (IBP) have pleiotropic effects in myeloma cells. To investigate whether IBP inhibition interferes with monoclonal protein secretion, human myeloma cells were treated with specific inhibitors of the IBP or prenyltransferases. These studies demonstrate that agents that inhibit Rab geranylgeranylation disrupt light chain trafficking, lead to accumulation of light chain in the endoplasmic reticulum, activate the unfolded protein response pathway and induce apoptosis. These studies provide a novel mechanism of action for IBP inhibitors and suggest that further exploration of Rab-targeted agents in myeloma is warranted.

Holstein, Sarah A.; Hohl, Raymond J.

2010-01-01

81

The unfolded protein response transducer IRE1? prevents ER stress-induced hepatic steatosis  

PubMed Central

The endoplasmic reticulum (ER) is the cellular organelle responsible for protein folding and assembly, lipid and sterol biosynthesis, and calcium storage. The unfolded protein response (UPR) is an adaptive intracellular stress response to accumulation of unfolded or misfolded proteins in the ER. In this study, we show that the most conserved UPR sensor inositol-requiring enzyme 1 ? (IRE1?), an ER transmembrane protein kinase/endoribonuclease, is required to maintain hepatic lipid homeostasis under ER stress conditions through repressing hepatic lipid accumulation and maintaining lipoprotein secretion. To elucidate physiological roles of IRE1?-mediated signalling in the liver, we generated hepatocyte-specific Ire1?-null mice by utilizing an albumin promoter-controlled Cre recombinase-mediated deletion. Deletion of Ire1? caused defective induction of genes encoding functions in ER-to-Golgi protein transport, oxidative protein folding, and ER-associated degradation (ERAD) of misfolded proteins, and led to selective induction of pro-apoptotic UPR trans-activators. We show that IRE1? is required to maintain the secretion efficiency of selective proteins. In the absence of ER stress, mice with hepatocyte-specific Ire1? deletion displayed modest hepatosteatosis that became profound after induction of ER stress. Further investigation revealed that IRE1? represses expression of key metabolic transcriptional regulators, including CCAAT/enhancer-binding protein (C/EBP) ?, C/EBP?, peroxisome proliferator-activated receptor ? (PPAR?), and enzymes involved in triglyceride biosynthesis. IRE1? was also found to be required for efficient secretion of apolipoproteins upon disruption of ER homeostasis. Consistent with a role for IRE1? in preventing intracellular lipid accumulation, mice with hepatocyte-specific deletion of Ire1? developed severe hepatic steatosis after treatment with an ER stress-inducing anti-cancer drug Bortezomib, upon expression of a misfolding-prone human blood clotting factor VIII, or after partial hepatectomy. The identification of IRE1? as a key regulator to prevent hepatic steatosis provides novel insights into ER stress mechanisms in fatty liver diseases associated with toxic liver injuries.

Zhang, Kezhong; Wang, Shiyu; Malhotra, Jyoti; Hassler, Justin R; Back, Sung Hoon; Wang, Guohui; Chang, Lin; Xu, Wenbo; Miao, Hongzhi; Leonardi, Roberta; Chen, Y Eugene; Jackowski, Suzanne; Kaufman, Randal J

2011-01-01

82

Activation of the Unfolded Protein Response Is Required for Defenses against Bacterial PoreForming Toxin In Vivo  

Microsoft Academic Search

Pore-forming toxins (PFTs) constitute the single largest class of proteinaceous bacterial virulence factors and are made by many of the most important bacterial pathogens. Host responses to these toxins are complex and poorly understood. We find that the endoplasmic reticulum unfolded protein response (UPR) is activated upon exposure to PFTs both in Caenorhabditis elegans and in mammalian cells. Activation of

Larry J. Bischof; Cheng-Yuan Kao; Ferdinand C. O. Los; Manuel R. Gonzalez; Zhouxin Shen; Steven P. Briggs; F. Gisou van der Goot; Raffi V. Aroian

2008-01-01

83

Activation of the endoplasmic reticulum unfolded protein response by lipid disequilibrium without disturbed proteostasis in vivo  

PubMed Central

The Mediator is a conserved transcriptional coregulator complex required for eukaryotic gene expression. In Caenorhabditis elegans, the Mediator subunit mdt-15 is essential for the expression of genes involved in fatty acid metabolism and ingestion-associated stress responses. mdt-15 loss of function causes defects in reproduction and mobility and shortens lifespan. In the present study, we find that worms with mutated or depleted mdt-15 (mdt-15 worms) exhibit decreased membrane phospholipid desaturation, especially in phosphatidylcholine. Accordingly, mdt-15 worms exhibit disturbed endoplasmic reticulum (ER) homeostasis, as indicated by a constitutively activated ER unfolded protein response (UPRER). Activation of this stress response is only partially the consequence of reduced membrane lipid desaturation, implicating other mdt-15–regulated processes in maintaining ER homeostasis. Interestingly, mdt-15 inactivation or depletion of the lipid metabolism enzymes stearoyl-CoA-desaturases (SCD) and S-adenosyl methionine synthetase (sams-1) activates the UPRER without promoting misfolded protein aggregates. Moreover, these worms exhibit wild-type sensitivity to chemically induced protein misfolding, and they do not display synthetic lethality with mutations in UPRER genes, which cause protein misfolding. Therefore, the constitutively activated UPRER in mdt-15, SCD, and sams-1 worms is not the consequence of proteotoxic stress but likely is the direct result of changes in ER membrane fluidity and composition. Together, our data suggest that the UPRER is induced directly upon membrane disequilibrium and thus monitors altered ER homeostasis.

Hou, Nicole S.; Gutschmidt, Aljona; Choi, Daniel Y.; Pather, Keouna; Shi, Xun; Watts, Jennifer L.; Hoppe, Thorsten; Taubert, Stefan

2014-01-01

84

Amyloidogenesis of Natively Unfolded Proteins  

PubMed Central

Aggregation and subsequent development of protein deposition diseases originate from conformational changes in corresponding amyloidogenic proteins. The accumulated data support the model where protein fibrillogenesis proceeds via the formation of a relatively unfolded amyloidogenic conformation, which shares many structural properties with the pre-molten globule state, a partially folded intermediate first found during the equilibrium and kinetic (un)folding studies of several globular proteins and later described as one of the structural forms of natively unfolded proteins. The flexibility of this structural form is essential for the conformational rearrangements driving the formation of the core cross-beta structure of the amyloid fibril. Obviously, molecular mechanisms describing amyloidogenesis of ordered and natively unfolded proteins are different. For ordered protein to fibrillate, its unique and rigid structure has to be destabilized and partially unfolded. On the other hand, fibrillogenesis of a natively unfolded protein involves the formation of partially folded conformation; i.e., partial folding rather than unfolding. In this review recent findings are surveyed to illustrate some unique features of the natively unfolded proteins amyloidogenesis.

Uversky, Vladimir N.

2009-01-01

85

Luminal Ca2+ depletion during the unfolded protein response in Xenopus oocytes: cause and consequence.  

PubMed

The endoplasmic reticulum (ER) is a Ca(2+) storing organelle that plays a critical role in the synthesis, folding and post-translational modifications of many proteins. The ER enters into a condition of stress when the load of newly synthesized proteins exceeds its folding and processing capacity. This activates a signal transduction pathway called the unfolded protein response (UPR) that attempts to restore homeostasis. The precise role of ER Ca(2+) in the initiation of the UPR has not been defined. Specifically, it has not been established whether ER Ca(2+) dysregulation is a cause or consequence of ER stress. Here, we report that partial depletion of ER Ca(2+) stores induces a significant induction of the UPR, and leads to the retention of a normally secreted protein Carboxypeptidase Y. Moreover, inhibition of protein glycosylation by tunicamycin rapidly induced an ER Ca(2+) leak into the cytosol. However, blockade of the translocon with emetine inhibited the tunicamycin-induced Ca(2+) release. Furthermore, emetine treatment blocked elF2? phosphorylation and reduced expression of the chaperone BiP. These findings suggest that Ca(2+) may be both a cause and a consequence of ER protein misfolding. Thus, it appears that ER Ca(2+) leak is a significant co-factor for the initiation of the UPR. PMID:23415071

Paredes, R Madelaine; Bollo, Mariana; Holstein, Deborah; Lechleiter, James D

2013-04-01

86

"Cellular Stress/The Unfolded Protein Response: Relevance to Sleep and Sleep Disorders"  

PubMed Central

Summary Recent transcript profiling and microarray studies are beginning to unveil some of the mysteries of sleep. One of the most important clues has been the identification of the ER resident chaperone, BiP that increases with sleep deprivation in all species studied. BiP, an ER resident chaperone is the key cellular marker and master regulator of a signaling pathway called the ER stress response or unfolded protein response. The ER stress response occurs in 3 phases. It is healthy, protective and adaptive when the ER stress is moderate. Failure of the adaptive response leads to the activation of an inflammatory response. When the ER stress burden is great and prolonged, executioner pathways are activated. Collectively this work provides new evidence that modest sleep deprivation induces cellular stress that activates an adaptive response. Aging tilts the response to sleep deprivation from one that is adaptive and protective to one that is maladaptive. Understanding the pathways activated by sleep loss and the mechanisms by which they occur will allow the development of therapies to protect the brain during prolonged wakefulness and specifically in sleep disorders including those associated with aging.

Naidoo, Nirinjini

2010-01-01

87

Triptolide activates unfolded protein response leading to chronic ER stress in pancreatic cancer cells.  

PubMed

Pancreatic cancer is a devastating disease with a survival rate of <5%. Moreover, pancreatic cancer aggressiveness is closely related to high levels of prosurvival mediators, which can ultimately lead to rapid disease progression. One of the mechanisms that enables tumor cells to evade cellular stress and promote unhindered proliferation is the endoplasmic reticulum (ER) stress response. Disturbances in the normal functions of the ER lead to an evolutionarily conserved cell stress response, the unfolded protein response (UPR). The UPR initially compensates for damage, but it eventually triggers cell death if ER dysfunction is severe or prolonged. Triptolide, a diterpene triepoxide, has been shown to be an effective compound against pancreatic cancer. Our results show that triptolide induces the UPR by activating the PKR-like ER kinase-eukaryotic initiation factor 2? axis and the inositol-requiring enzyme 1?-X-box-binding protein 1 axis of the UPR and leads to chronic ER stress in pancreatic cancer. Our results further show that glucose-regulated protein 78 (GRP78), one of the major regulators of ER stress, is downregulated by triptolide, leading to cell death by apoptosis in MIA PaCa-2 cells and autophagy in S2-VP10 cells. PMID:24699326

Mujumdar, Nameeta; Banerjee, Sulagna; Chen, Zhiyu; Sangwan, Veena; Chugh, Rohit; Dudeja, Vikas; Yamamoto, Masato; Vickers, Selwyn M; Saluja, Ashok K

2014-06-01

88

Cigarette smoke induces an unfolded protein response in the human lung: a proteomic approach.  

PubMed

Cigarette smoking, which exposes the lung to high concentrations of reactive oxidant species (ROS) is the major risk factor for chronic obstructive pulmonary disease (COPD). Recent studies indicate that ROS interfere with protein folding in the endoplasmic reticulum and elicit a compensatory response termed the "unfolded protein response" (UPR). The importance of the UPR lies in its ability to alter expression of a variety of genes involved in antioxidant defense, inflammation, energy metabolism, protein synthesis, apoptosis, and cell cycle regulation. The present study used comparative proteomic technology to test the hypothesis that chronic cigarette smoking induces a UPR in the human lung. Studies were performed on lung tissue samples obtained from three groups of human subjects: nonsmokers, chronic cigarette smokers, and ex-smokers. Proteomes of lung samples from chronic cigarette smokers demonstrated 26 differentially expressed proteins (20 were up-regulated, 5 were down-regulated, and 1 was detected only in the smoking group) compared with nonsmokers. Several UPR proteins were up-regulated in smokers compared with nonsmokers and ex-smokers, including the chaperones, glucose-regulated protein 78 (GRP78) and calreticulin; a foldase, protein disulfide isomerase (PDI); and enzymes involved in antioxidant defense. In cultured human airway epithelial cells, GRP78 and the UPR-regulated basic leucine zipper, transcription factors, ATF4 and Nrf2, which enhance expression of important anti-oxidant genes, increased rapidly (< 24 h) with cigarette smoke extract. These data indicate that cigarette smoke induces a UPR response in the human lung that is rapid in onset, concentration dependent, and at least partially reversible with smoking cessation. We speculate that activation of a UPR by cigarette smoke may protect the lung from oxidant injury and the development of COPD. PMID:18079489

Kelsen, Steven G; Duan, Xunbao; Ji, Rong; Perez, Oscar; Liu, Chunli; Merali, Salim

2008-05-01

89

Tau accumulation activates the unfolded protein response by impairing endoplasmic reticulum-associated degradation  

PubMed Central

In Alzheimer’s disease (AD), the mechanisms of neuronal loss remain largely unknown. While tau pathology is closely correlated with neuronal loss, how its accumulation may lead to activation of neurotoxic pathways is unclear. Here we show that tau increased the levels of ubiquitinated proteins in the brain and that this triggered activation of the Unfolded Protein Response (UPR). This suggested that tau was interfering with protein quality control in the endoplasmic reticulum (ER). Consistent with this, ubiquitin was found to associate with the ER in human AD brains and rTg4510 tau transgenic mouse brains, but this was not always co-localized with tau. The increased levels of ubiquitinated protein were accompanied by increased levels of phosphorylated PERK, a marker that indicates UPR activation. Importantly, depleting soluble tau levels in cells and brain could reverse UPR activation. Tau accumulation facilitated its deleterious interaction with ER membrane and associated proteins that are essential for ER-associated degradation (ERAD), including VCP and Hrd1. Based on this, the effects of tau accumulation on ERAD efficiency were evaluated using the CD3? reporter, an ERAD substrate. Indeed, CD3? accumulated in both in vitro and in vivo models of tau over-expression and AD brains. These data suggest that soluble tau impairs ERAD, and the result is activation of the UPR. The reversibility of this process, however, suggests that tau-based therapeutics could significantly delay this type of cell death and consequently disease progression.

Abisambra, Jose F.; Jinwal, Umesh K.; Blair, Laura J.; O'Leary, John C.; Li, Qingyou; Brady, Sarah; Wang, Li; Guidi, Chantal E.; Zhang, Bo; Nordhues, Bryce A.; Cockman, Matthew; Suntharalingham, Amirthaa; Li, Pengfei; Jin, Ying; Atkins, Christopher A.; Dickey, Chad A.

2013-01-01

90

The unfolded protein response governs integrity of the haematopoietic stem-cell pool during stress.  

PubMed

The blood system is sustained by a pool of haematopoietic stem cells (HSCs) that are long-lived due to their capacity for self-renewal. A consequence of longevity is exposure to stress stimuli including reactive oxygen species (ROS), nutrient fluctuation and DNA damage. Damage that occurs within stressed HSCs must be tightly controlled to prevent either loss of function or the clonal persistence of oncogenic mutations that increase the risk of leukaemogenesis. Despite the importance of maintaining cell integrity throughout life, how the HSC pool achieves this and how individual HSCs respond to stress remain poorly understood. Many sources of stress cause misfolded protein accumulation in the endoplasmic reticulum (ER), and subsequent activation of the unfolded protein response (UPR) enables the cell to either resolve stress or initiate apoptosis. Here we show that human HSCs are predisposed to apoptosis through strong activation of the PERK branch of the UPR after ER stress, whereas closely related progenitors exhibit an adaptive response leading to their survival. Enhanced ER protein folding by overexpression of the co-chaperone ERDJ4 (also called DNAJB9) increases HSC repopulation capacity in xenograft assays, linking the UPR to HSC function. Because the UPR is a focal point where different sources of stress converge, our study provides a framework for understanding how stress signalling is coordinated within tissue hierarchies and integrated with stemness. Broadly, these findings reveal that the HSC pool maintains clonal integrity by clearance of individual HSCs after stress to prevent propagation of damaged stem cells. PMID:24776803

van Galen, Peter; Kreso, Antonija; Mbong, Nathan; Kent, David G; Fitzmaurice, Timothy; Chambers, Joseph E; Xie, Stephanie; Laurenti, Elisa; Hermans, Karin; Eppert, Kolja; Marciniak, Stefan J; Goodall, Jane C; Green, Anthony R; Wouters, Bradly G; Wienholds, Erno; Dick, John E

2014-06-12

91

Triggering unfolded protein response by 2-Deoxy-d-glucose inhibits porcine epidemic diarrhea virus propagation.  

PubMed

The unfolded protein response (UPR) is cyto-protective machinery elicited towards an influx of large amount of protein synthesis in the endoplasmic reticulum (ER). Extensive studies suggest that the UPR can also be activated during virus infection. In the present studies, we first evaluated if porcine epidemic diarrhea virus (PEDV) infection activated the UPR pathways. Electron microscopy analysis demonstrated the morphology changes of ER post-PEDV infection. Western blot and real-time PCR identified the differences of UPR genes in response to PEDV infection. The results suggested that PEDV infection induced UPR in Vero cells. Meanwhile, we silenced the expression of PKR-like ER kinase (PERK) by shRNA, we found that the knockdown of PERK increased virus loads in the cells, which was consistent with the result on 4-phenylbutyrate (4-PBA) treatment. We next determined whether 2-Deoxy-d-glucose (2-DG), an ER stress inducer, possessed antiviral activity against PEDV infection. Plaque formation assay, RT-PCR and Western blot analysis suggested that 2-DG might inhibit virus infection by affecting viral protein translation during the early stage of virus infection. Interestingly, we also found that 2-DG treatment could affect virus assembly, which is similar to previous studies on influenza virus. All these results support the therapeutic potential of using 2-DG or glucose/mannose analogs to induce the UPR to block virus replication. PMID:24681123

Wang, Yue; Li, Jia-Rong; Sun, Ming-Xia; Ni, Bo; Huan, Changchao; Huang, Li; Li, Chen; Fan, Hong-Jie; Ren, Xiao-Feng; Mao, Xiang

2014-06-01

92

Parkinsonian mimetics induce aspects of unfolded protein response in death of dopaminergic neurons.  

PubMed

Genes associated with Parkinson's disease (PD) have suggested a role for ubiquitin-proteasome dysfunction and aberrant protein degradation in this disorder. Inasmuch as oxidative stress has also been implicated in PD, the present study examined transcriptional changes mediated by the Parkinsonism-inducing neurotoxins 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenylpyridinium (MPP+) in a dopaminergic cell line. Microarray analysis of RNA isolated from toxin treated samples revealed that the stress-induced transcription factor CHOP/Gadd153 was dramatically up-regulated by both 6-OHDA and MPP+. Treatment with 6-OHDA also induced a large number of genes involved in endoplasmic reticulum stress and unfolded protein response (UPR) such as ER chaperones and elements of the ubiquitin-proteasome system. Reverse transcription-PCR, Western blotting, and immunocytochemical approaches were used to quantify and temporally order the UPR pathways involved in neurotoxin-induced cell death. 6-OHDA, but not MPP+, significantly increased hallmarks of UPR such as BiP, c-Jun, and processed Xbp1 mRNA. Both toxins increased the phosphorylation of UPR proteins, PERK and eIF2 alpha, but only 6-OHDA increased phosphorylation of c-Jun. Thus, 6-OHDA is capable of triggering multiple pathways associated with UPR, whereas MPP+ exhibits a more restricted response. The involvement of UPR in these widely used neurotoxin models supports the role of ubiquitin-proteasome pathway dysfunction in PD. PMID:12598533

Holtz, William Andrew; O'Malley, Karen Laurel

2003-05-23

93

Unfolded protein response (UPR) signaling regulates arsenic trioxide-mediated macrophage innate immune function disruption.  

PubMed

Arsenic exposure is known to disrupt innate immune functions in humans and in experimental animals. In this study, we provide a mechanism by which arsenic trioxide (ATO) disrupts macrophage functions. ATO treatment of murine macrophage cells diminished internalization of FITC-labeled latex beads, impaired clearance of phagocytosed fluorescent bacteria and reduced secretion of pro-inflammatory cytokines. These impairments in macrophage functions are associated with ATO-induced unfolded protein response (UPR) signaling pathway characterized by the enhancement in proteins such as GRP78, p-PERK, p-eIF2?, ATF4 and CHOP. The expression of these proteins is altered both at transcriptional and translational levels. Pretreatment with chemical chaperon, 4-phenylbutyric acid (PBA) attenuated the ATO-induced activation in UPR signaling and afforded protection against ATO-induced disruption of macrophage functions. This treatment also reduced ATO-mediated reactive oxygen species (ROS) generation. Interestingly, treatment with antioxidant N-acetylcysteine (NAC) prior to ATO exposure, not only reduced ROS production and UPR signaling but also improved macrophage functions. These data demonstrate that UPR signaling and ROS generation are interdependent and are involved in the arsenic-induced pathobiology of macrophage. These data also provide a novel strategy to block the ATO-dependent impairment in innate immune responses. PMID:23954561

Srivastava, Ritesh K; Li, Changzhao; Chaudhary, Sandeep C; Ballestas, Mary E; Elmets, Craig A; Robbins, David J; Matalon, Sadis; Deshane, Jessy S; Afaq, Farrukh; Bickers, David R; Athar, Mohammad

2013-11-01

94

Control of dopaminergic neuron survival by the unfolded protein response transcription factor XBP1.  

PubMed

Parkinson disease (PD) is characterized by the selective loss of dopaminergic neurons of the substantia nigra pars compacta (SNpc). Although growing evidence indicates that endoplasmic reticulum (ER) stress is a hallmark of PD, its exact contribution to the disease process is not well understood. Here we report that developmental ablation of X-Box binding protein 1 (XBP1) in the nervous system, a key regulator of the unfolded protein response (UPR), protects dopaminergic neurons against a PD-inducing neurotoxin. This survival effect was associated with a preconditioning condition that resulted from induction of an adaptive ER stress response in dopaminergic neurons of the SNpc, but not in other brain regions. In contrast, silencing XBP1 in adult animals triggered chronic ER stress and dopaminergic neuron degeneration. Supporting this finding, gene therapy to deliver an active form of XBP1 provided neuroprotection and reduced striatal denervation in animals injected with 6-hydroxydopamine. Our results reveal a physiological role of the UPR in the maintenance of protein homeostasis in dopaminergic neurons that may help explain the differential neuronal vulnerability observed in PD. PMID:24753614

Valdés, Pamela; Mercado, Gabriela; Vidal, Rene L; Molina, Claudia; Parsons, Geoffrey; Court, Felipe A; Martinez, Alexis; Galleguillos, Danny; Armentano, Donna; Schneider, Bernard L; Hetz, Claudio

2014-05-01

95

Endoplasmic Reticulum Stress and Unfolded Protein Response in Atm Deficient Thymocytes and Thymic Lymphoma Cells Are Attributable to Oxidative Stress  

Microsoft Academic Search

Both oxidative stress and endoplasmic reticulum (ER) stress have been implicated in carcinogenesis. It is well documented that cells deficient in the ataxia-telangiectasia mutated (ATM) gene undergo oxidative stress, which is critically involved in thymic lymphomagenesis in Atm?\\/? mice. Here we demonstrate that undifferentiated Atm?\\/? thymocytes show signs of ER stress and of the unfolded protein response (UPR). Using two-dimensional

Mingshan Yan; Jianjun Shen; Maria D. Person; Xianghong Kuang; William S. Lynn; Daphne Atlas; Paul K. Y. Wong

2008-01-01

96

Sorafenib enhances proteasome inhibitor-mediated cytotoxicity via inhibition of unfolded protein response and keratin phosphorylation.  

PubMed

Hepatocellular carcinoma (HCC) is highly resistant to conventional systemic therapies and prognosis for advanced HCC patients remains poor. Recent studies of the molecular mechanisms responsible for tumor initiation and progression have identified several potential molecular targets in HCC. Sorafenib is a multi-kinase inhibitor shown to have survival benefits in advanced HCC. It acts by inhibiting the serine/threonine kinases and the receptor type tyrosine kinases. In preclinical experiments sorafenib had anti-proliferative activity in hepatoma cells and it reduced tumor angiogenesis and increased apoptosis. Here, we demonstrate for the first time that the cytotoxic mechanisms of sorafenib include its inhibitory effects on protein ubiquitination, unfolded protein response (UPR) and keratin phosphorylation in response to endoplasmic reticulum (ER) stress. Moreover, we show that combined treatment with sorafenib and proteasome inhibitors (PIs) synergistically induced a marked increase in cell death in hepatoma- and hepatocyte-derived cells. These observations may open the way to potentially interesting treatment combinations that may augment the effect of sorafenib, possibly including drugs that promote ER stress. Because sorafenib blocked the cellular defense mechanisms against hepatotoxic injury not only in hepatoma cells but also in hepatocyte-derived cells, we must be careful to avoid severe liver injury. PMID:23727131

Honma, Yuichi; Harada, Masaru

2013-08-15

97

Metabolic stress promotes renal tubular inflammation by triggering the unfolded protein response.  

PubMed

The renal epithelium contributes to the development of inflammation during ischemic injury. Ischemia induces endoplasmic reticulum (ER) stress and activates the unfolded protein response (UPR). Ischemic tissues generate distress signals and inflammation that activates fibrogenesis and may promote adaptive immunity. Interestingly, the UPR may activate inflammation pathways. Our aim was to test whether the UPR is activated during metabolic stress and mediates a tubular inflammatory response. Glucose deprivation, not hypoxia and amino acids deprivation, activated the UPR in human renal cortical tubular cells in culture. This stress activated NF-?B and promoted the transcription of proinflammatory cytokines and chemokines, including IL-6, IL-8, TNF-?, RANTES and MCP-1. The protein kinase RNA (PKR)-like ER kinase signaling pathway was not required for the induction of inflammation but amplified cytokine. Inositol-requiring enzyme 1 activated NF-?B signaling and was required for the transcription of proinflammatory cytokines and chemokines following metabolic stress. Moreover, acute ischemia activated ER stress and inflammation in rat kidneys. Finally, the ER stress marker GRP78 and NF-?B p65/RelA were coexpressed in human kidney transplants biopsies performed before implantation, suggesting that ER stress activates tubular inflammation in human renal allografts. In conclusion, this study establishes a link between ischemic stress, the activation of the UPR and the generation of a tubular inflammatory response. PMID:21490675

Fougeray, S; Bouvier, N; Beaune, P; Legendre, C; Anglicheau, D; Thervet, E; Pallet, N

2011-01-01

98

Restoration of the unfolded protein response in pancreatic ? cells protects mice against type 1 diabetes.  

PubMed

Perturbations in endoplasmic reticulum (ER) homeostasis can evoke stress responses leading to aberrant glucose and lipid metabolism. ER dysfunction is linked to inflammatory disorders, but its role in the pathogenesis of autoimmune type 1 diabetes (T1D) remains unknown. We identified defects in the expression of unfolded protein response (UPR) mediators ATF6 (activating transcription factor 6) and XBP1 (X-box binding protein 1) in ? cells from two different T1D mouse models and then demonstrated similar defects in pancreatic ? cells from T1D patients. Administration of a chemical ER stress mitigator, tauroursodeoxycholic acid (TUDCA), at the prediabetic stage resulted in a marked reduction of diabetes incidence in the T1D mouse models. This reduction was accompanied by (i) a significant decrease in aggressive lymphocytic infiltration in the pancreas, (ii) improved survival and morphology of ? cells, (iii) reduced ? cell apoptosis, (iv) preserved insulin secretion, and (v) restored expression of UPR mediators. TUDCA's actions were dependent on ATF6 and were lost in mice with ? cell-specific deletion of ATF6. These data indicate that proper maintenance of the UPR is essential for the preservation of ? cells and that defects in this process can be chemically restored for preventive or therapeutic interventions in T1D. PMID:24225943

Engin, Feyza; Yermalovich, Alena; Nguyen, Truc; Ngyuen, Truc; Hummasti, Sarah; Fu, Wenxian; Eizirik, Decio L; Mathis, Diane; Hotamisligil, Gökhan S

2013-11-13

99

The role of endoplasmic reticulum stress and the unfolded protein response in fibrosis  

PubMed Central

Purpose of review To review the present knowledge of the role of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in the pathogenesis of fibrotic diseases. Recent findings ER stress and UPR occur in a number of diseases associated with organ fibrosis; however, the contribution of these pathways to the fibrotic process has not been systematically investigated. Current studies suggest that prolonged ER stress may lead to fibrosis through activation of CCAAT/enhancer-binding homologous protein-mediated apoptosis, followed by an inflammatory response and release of profibrotic cytokines. A direct profibrotic role of UPR mediators in activation of TGF-? signaling has been shown in lung fibroblasts. In addition, activation of ER stress and UPR pathways in immune cells contributes to increased production of proinflammatory cytokines. Summary Although limited in scope, current studies strongly suggest that ER stress and UPR may play an important role during development of fibrosis. Further studies are warranted to gain additional insights into the relationship between these processes.

Lenna, Stefania; Trojanowska, Maria

2013-01-01

100

Slower rescue of ER homeostasis by the unfolded protein response pathway associated with common variable immunodeficiency.  

PubMed

Common variable immunodeficiency (CVID) is a primary immunodeficiency characterized by hypogammaglobulinemia and recurrent infections. Herein we addressed the role of unfolded protein response (UPR) in the pathogenesis of the disease. Augmented unspliced X-box binding protein 1 (XBP-1) mRNA concurrent with co-localization of IgM and BiP/GRP78 were found in one CVID patient. At confocal microscopy analysis this patient's cells were enlarged and failed to present the typical surface distribution of IgM, which accumulated within an abnormally expanded endoplasmic reticulum. Sequencing did not reveal any mutation on XBP-1, neither on IRE-1alpha that could potentially prevent the splicing to occur. Analysis of spliced XBP-1, IRE-1alpha and BiP messages after LPS or Brefeldin A treatment showed that, unlike healthy controls that respond to these endoplasmic reticulum (ER) stressors by presenting waves of transcription of these three genes, this patient's cells presented lower rates of transcription, not reaching the same level of response of healthy subjects even after 48 h of ER stress. Treatment with DMSO rescued IgM and IgG secretion as well as the expression of spliced XBP-1. Our findings associate diminished splicing of XBP-1 mRNA with accumulation of IgM within the ER and lower rates of chaperone transcription, therefore providing a mechanism to explain the observed hypogammaglobulinemia. PMID:18325593

Kuribayashi, Juliana S; Bombardieri, Cíntia R; Baracho, Gisele V; Aliberti, Júlio; Machado, Fabiana S; Kalil, Jorge; Guilherme, Luiza; Kokron, Cristina M; Rizzo, Luiz V; de Camargo, Maristela M

2008-05-01

101

Pharmacological modulation of the retinal unfolded protein response in Bardet-Biedl syndrome reduces apoptosis and preserves light detection ability.  

PubMed

Ciliopathies, a class of rare genetic disorders, present often with retinal degeneration caused by protein transport defects between the inner segment and the outer segment of the photoreceptors. Bardet-Biedl syndrome is one such ciliopathy, genetically heterogeneous with 17 BBS genes identified to date, presenting early onset retinitis pigmentosa. By investigating BBS12-deprived retinal explants and the Bbs12(-/-) murine model, we show that the impaired intraciliary transport results in protein retention in the endoplasmic reticulum. The protein overload activates a proapoptotic unfolded protein response leading to a specific Caspase12-mediated death of the photoreceptors. Having identified a therapeutic window in the early postnatal retinal development and through optimized pharmacological modulation of the unfolded protein response, combining three specific compounds, namely valproic acid, guanabenz, and a specific Caspase12 inhibitor, achieved efficient photoreceptor protection, thereby maintaining light detection ability in vivo. PMID:22869374

Mockel, Anais; Obringer, Cathy; Hakvoort, Theodorus B M; Seeliger, Mathias; Lamers, Wouter H; Stoetzel, Corinne; Dollfus, Hélène; Marion, Vincent

2012-10-26

102

A Role for the Unfolded Protein Response (UPR) in Virulence and Antifungal Susceptibility in Aspergillus fumigatus  

PubMed Central

Filamentous fungi rely heavily on the secretory pathway, both for the delivery of cell wall components to the hyphal tip and the production and secretion of extracellular hydrolytic enzymes needed to support growth on polymeric substrates. Increased demand on the secretory system exerts stress on the endoplasmic reticulum (ER), which is countered by the activation of a coordinated stress response pathway termed the unfolded protein response (UPR). To determine the contribution of the UPR to the growth and virulence of the filamentous fungal pathogen Aspergillus fumigatus, we disrupted the hacA gene, encoding the major transcriptional regulator of the UPR. The ?hacA mutant was unable to activate the UPR in response to ER stress and was hypersensitive to agents that disrupt ER homeostasis or the cell wall. Failure to induce the UPR did not affect radial growth on rich medium at 37°C, but cell wall integrity was disrupted at 45°C, resulting in a dramatic loss in viability. The ?hacA mutant displayed a reduced capacity for protease secretion and was growth-impaired when challenged to assimilate nutrients from complex substrates. In addition, the ?hacA mutant exhibited increased susceptibility to current antifungal agents that disrupt the membrane or cell wall and had attenuated virulence in multiple mouse models of invasive aspergillosis. These results demonstrate the importance of ER homeostasis to the growth and virulence of A. fumigatus and suggest that targeting the UPR, either alone or in combination with other antifungal drugs, would be an effective antifungal strategy.

Richie, Daryl L.; Hartl, Lukas; Aimanianda, Vishukumar; Winters, Michael S.; Fuller, Kevin K.; Miley, Michael D.; White, Stephanie; McCarthy, Jason W.; Latge, Jean-Paul; Feldmesser, Marta; Rhodes, Judith C.; Askew, David S.

2009-01-01

103

The response to unfolded protein is involved in osmotolerance of Pichia pastoris  

PubMed Central

Background The effect of osmolarity on cellular physiology has been subject of investigation in many different species. High osmolarity is of importance for biotechnological production processes, where high cell densities and product titers are aspired. Several studies indicated that increased osmolarity of the growth medium can have a beneficial effect on recombinant protein production in different host organisms. Thus, the effect of osmolarity on the cellular physiology of Pichia pastoris, a prominent host for recombinant protein production, was studied in carbon limited chemostat cultures at different osmolarities. Transcriptome and proteome analyses were applied to assess differences upon growth at different osmolarities in both, a wild type strain and an antibody fragment expressing strain. While our main intention was to analyze the effect of different osmolarities on P. pastoris in general, this was complemented by studying it in context with recombinant protein production. Results In contrast to the model yeast Saccharomyces cerevisiae, the main osmolyte in P. pastoris was arabitol rather than glycerol, demonstrating differences in osmotic stress response as well as energy metabolism. 2D Fluorescence Difference Gel electrophoresis and microarray analysis were applied and demonstrated that processes such as protein folding, ribosome biogenesis and cell wall organization were affected by increased osmolarity. These data indicated that upon increased osmolarity less adaptations on both the transcript and protein level occurred in a P. pastoris strain, secreting the Fab fragment, compared with the wild type strain. No transcriptional activation of the high osmolarity glycerol (HOG) pathway was observed at steady state conditions. Furthermore, no change of the specific productivity of recombinant Fab was observed at increased osmolarity. Conclusion These data point out that the physiological response to increased osmolarity is different to S. cerevisiae. Increased osmolarity resulted in an unfolded protein response (UPR) like response in P. pastoris and lead to pre-conditioning of the recombinant Fab producing strain of P. pastoris to growth at high osmolarity. The current data demonstrate a strong similarity of environmental stress response mechanisms and recombinant protein related stresses. Therefore, these results might be used in future strain and bioprocess engineering of this biotechnologically relevant yeast.

2010-01-01

104

The Unfolded Protein Response in the Protozoan Parasite Toxoplasma gondii Features Translational and Transcriptional Control  

PubMed Central

The unfolded protein response (UPR) is an important regulatory network that responds to perturbations in protein homeostasis in the endoplasmic reticulum (ER). In mammalian cells, the UPR features translational and transcriptional mechanisms of gene expression aimed at restoring proteostatic control. A central feature of the UPR is phosphorylation of the ? subunit of eukaryotic initiation factor-2 (eIF2) by PERK (EIF2AK3/PEK), which reduces the influx of nascent proteins into the ER by lowering global protein synthesis, coincident with preferential translation of key transcription activators of genes that function to expand the processing capacity of this secretory organelle. Upon ER stress, the apicomplexan parasite Toxoplasma gondii is known to induce phosphorylation of Toxoplasma eIF2? and lower translation initiation. To characterize the nature of the ensuing UPR in this parasite, we carried out microarray analyses to measure the changes in the transcriptome and in translational control during ER stress. We determined that a collection of transcripts linked with the secretory process are induced in response to ER stress, supporting the idea that a transcriptional induction phase of the UPR occurs in Toxoplasma. Furthermore, we determined that about 500 gene transcripts showed enhanced association with translating ribosomes during ER stress. Many of these target genes are suggested to be involved in gene expression, including JmjC5, which continues to be actively translated during ER stress. This study indicates that Toxoplasma triggers a UPR during ER stress that features both translational and transcriptional regulatory mechanisms, which is likely to be important for parasite invasion and development.

Joyce, Bradley R.; Tampaki, Zoi; Kim, Kami

2013-01-01

105

Varicella-zoster virus glycoprotein expression differentially induces the unfolded protein response in infected cells  

PubMed Central

Varicella-zoster virus (VZV) is a human herpesvirus that spreads to children as varicella or chicken pox. The virus then establishes latency in the nervous system and re-emerges, typically decades later, as zoster or shingles. We have reported previously that VZV induces autophagy in infected cells as well as exhibiting evidence of the Unfolded Protein Response (UPR): XBP1 splicing, a greatly expanded Endoplasmic Reticulum (ER) and CHOP expression. Herein we report the results of a UPR specific PCR array that measures the levels of mRNA of 84 different components of the UPR in VZV infected cells as compared to tunicamycin treated cells as a positive control and uninfected, untreated cells as a negative control. Tunicamycin is a mixture of chemicals that inhibits N-linked glycosylation in the ER with resultant protein misfolding and the UPR. We found that VZV differentially induces the UPR when compared to tunicamycin treatment. For example, tunicamycin treatment moderately increased (8-fold) roughly half of the array elements while downregulating only three (one ERAD and two FOLD components). VZV infection on the other hand upregulated 33 components including a little described stress sensor CREB-H (64-fold) as well as ER membrane components INSIG and gp78, which modulate cholesterol synthesis while downregulating over 20 components mostly associated with ERAD and FOLD. We hypothesize that this expression pattern is associated with an expanding ER with downregulation of active degradation by ERAD and apoptosis as the cell attempts to handle abundant viral glycoprotein synthesis.

Carpenter, John E.; Grose, Charles

2014-01-01

106

Molecularly defined unfolded protein response subclasses have distinct correlations with fatty liver disease in zebrafish  

PubMed Central

The unfolded protein response (UPR) is a complex network of sensors and target genes that ensure efficient folding of secretory proteins in the endoplasmic reticulum (ER). UPR activation is mediated by three main sensors, which regulate the expression of hundreds of targets. UPR activation can result in outcomes ranging from enhanced cellular function to cell dysfunction and cell death. How this pathway causes such different outcomes is unknown. Fatty liver disease (steatosis) is associated with markers of UPR activation and robust UPR induction can cause steatosis; however, in other cases, UPR activation can protect against this disease. By assessing the magnitude of activation of UPR sensors and target genes in the liver of zebrafish larvae exposed to three commonly used ER stressors (tunicamycin, thapsigargin and Brefeldin A), we have identified distinct combinations of UPR sensors and targets (i.e. subclasses) activated by each stressor. We found that only the UPR subclass characterized by maximal induction of UPR target genes, which we term a stressed-UPR, induced steatosis. Principal component analysis demonstrated a significant positive association between UPR target gene induction and steatosis. The same principal component analysis showed significant correlation with steatosis in samples from patients with fatty liver disease. We demonstrate that an adaptive UPR induced by a short exposure to thapsigargin prior to challenging with tunicamycin reduced both the induction of a stressed UPR and steatosis incidence. We conclude that a stressed UPR causes steatosis and an adaptive UPR prevents it, demonstrating that this pathway plays dichotomous roles in fatty liver disease.

Vacaru, Ana M.; Di Narzo, Antonio Fabio; Howarth, Deanna L.; Tsedensodnom, Orkhontuya; Imrie, Dru; Cinaroglu, Ayca; Amin, Salma; Hao, Ke; Sadler, Kirsten C.

2014-01-01

107

Yokukansan Inhibits Neuronal Death during ER Stress by Regulating the Unfolded Protein Response  

PubMed Central

Background Recently, several studies have reported Yokukansan (Tsumura TJ-54), a traditional Japanese medicine, as a potential new drug for the treatment of Alzheimer's disease (AD). Endoplasmic reticulum (ER) stress is known to play an important role in the pathogenesis of AD, particularly in neuronal death. Therefore, we examined the effect of Yokukansan on ER stress-induced neurotoxicity and on familial AD-linked presenilin-1 mutation-associated cell death. Methods We employed the WST-1 assay and monitored morphological changes to evaluate cell viability following Yokukansan treatment or treatment with its components. Western blotting and PCR were used to observe the expression levels of GRP78/BiP, caspase-4 and C/EBP homologous protein. Results Yokukansan inhibited neuronal death during ER stress, with Cnidii Rhizoma (Senkyu), a component of Yokukansan, being particularly effective. We also showed that Yokukansan and Senkyu affect the unfolded protein response following ER stress and that these drugs inhibit the activation of caspase-4, resulting in the inhibition of ER stress-induced neuronal death. Furthermore, we found that the protective effect of Yokukansan and Senkyu against ER stress could be attributed to the ferulic acid content of these two drugs. Conclusions Our results indicate that Yokukansan, Senkyu and ferulic acid are protective against ER stress-induced neuronal cell death and may provide a possible new treatment for AD.

Miyata, Shingo; Kinoshita, Mitsuhiro; Kakehi, Kazuaki; Nishida, Shinji; Katayama, Taiichi; Tohyama, Masaya

2010-01-01

108

Oxytocin modulates markers of the unfolded protein response in Caco2BB gut cells.  

PubMed

We have shown that oxytocin receptor (OTR) expression in neonatal rat enterocytes is robust from birth to weaning, but OTR function during this period is unknown. We previously reported that oxytocin (OT) stimulation of Caco2BB cells (enterocytes in vitro) inhibits the mammalian target of rapamycin complex 1 (mTORC1) signaling. The unfolded protein response (UPR) is known to protectively reduce translation during endoplasmic reticulum (ER) stress. Because the mTORC1 pathway is linked to cellular stress, we investigated markers of UPR in OT-stimulated Caco2BB cells. We report that OT modulates several factors involved in sensing and translation of ER stress. High OT (62.5 nM) reduced translation initiation factor 4E-BP1 phosphorylation (Ser65), which is known to inhibit cap-dependent translation via its rate-limiting eukaryotic translation initiation factor 4E (eIF4E). Importantly, high OT increased phosphorylation of eukaryotic translation initiation factor 2a (eIF2a) phospho-Ser51, which inhibits eIF2a. High OT also increased protein kinase RNA-like endoplasmic reticulum kinase phosphorylation, a sensor of ER stress and a kinase of eIF2a. Both high and low OT activated inositol requiring enzyme1 (IRE1), which generates the transcription factor X-box binding protein 1 (XBP1) and induces the UPR. We also show that OT modulates XBP1 splicing and induces tribbles 3 (TRIB3; a negative regulator of Akt and protein involved in autophagy) and immunoglobulin binding protein (BiP; ER-chaperone). Taken together, these results indicate that OT modulates sensors of ER stress and autophagy. These findings support our hypothesis that transiently elevated OTR expression in neonatal gut may serve a protective function during a critical postnatal developmental period. PMID:24198165

Klein, Benjamin Y; Tamir, Hadassah; Hirschberg, David L; Glickstein, Sara B; Ludwig, Robert J; Welch, Martha G

2014-07-01

109

Hyperoxia resensitizes chemoresistant glioblastoma cells to temozolomide through unfolded protein response.  

PubMed

Background: Intratumoural hypoxia is associated with chemoresistance in glioblastoma multiforme (GBM), a highly malignant brain tumour. Adaptive response to endoplasmic reticulum stress induced by temozolomide is a major obstacle in recurrent GBM. We investigated whether hyperoxia resensitizes temozolomide-resistant GBM cells to temozolomide by abrogating the hypoxia-induced, unfolded protein response (UPR)-related protective mechanisms. Materials and Methods: We examined changes to key UPR modulators in temozolomide-sensitive and -resistant human GBM cells (D54 and U87) treated with/without temozolomide at different oxygen concentrations using western blotting, and cytotoxic benefits of overexpressing key chaperone, P4HB, in GBM cells (U87 and U251) under normoxia and hyperoxia. Results: Hyperoxia, alone or synergistically with temozolomide, activated the UPR in sensitive and resistant D54 and U87 cell lines. Hyperoxia also reduced survival benefit of U87 and U251 cells with P4HB overexpression through the UPR. Conclusion: Hyperoxia enhanced GBM cell sensitivity to temozolomide, likely through UPR, highlighting an important treatment modality targeting chemosensitive and -resistant GBM. PMID:24922660

Lee, Derek; Sun, Stella; Ho, Amy S W; Kiang, Karrie M Y; Zhang, Xiao Qin; Xu, Fei Fan; Leung, Gilberto K K

2014-06-01

110

The unfolded protein response in melanocytes: activation in response to chemical stressors of the endoplasmic reticulum and tyrosinase misfolding  

PubMed Central

Summary Accumulation of proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), comprising three signaling pathways initiated by Ire1, Perk and Atf6 respectively. UPR activation was compared in chemically stressed murine wildtype melanocytes and mutant melanocytes that retain tyrosinase in the ER. Thapsigargin, an ER stressor, activated all pathways in wildtype melanocytes, triggering Caspase 12-mediated apoptosis at toxic doses. Albino melanocytes expressing mutant tyrosinase showed evidence of ER stress with increased Ire1 expression; but the downstream effector, Xbp1, was not activated even following thapsigargin treatment. Attenuation of Ire1 signaling was recapitulated in wildtype melanocytes treated with thapsigargin for eight days, with diminished Xbp1 activation observed after four days. Atf6 was also activated in albino melanocytes, with no response to thapsigargin, while the Perk pathway was not activated and thapsigargin treatment elicited robust expression of the downstream effector CHOP. Thus, melanocytes adapt to ER stress by attenuating two UPR pathways.

Manga, Prashiela; Bis, Sabina; Knoll, Kristen; Perez, Beremis; Orlow, Seth J.

2010-01-01

111

Brucella Induces an Unfolded Protein Response via TcpB That Supports Intracellular Replication in Macrophages  

PubMed Central

Brucella melitensis is a facultative intracellular bacterium that causes brucellosis, the most prevalent zoonosis worldwide. The Brucella intracellular replicative niche in macrophages and dendritic cells thwarts immune surveillance and complicates both therapy and vaccine development. Currently, host-pathogen interactions supporting Brucella replication are poorly understood. Brucella fuses with the endoplasmic reticulum (ER) to replicate, resulting in dramatic restructuring of the ER. This ER disruption raises the possibility that Brucella provokes an ER stress response called the Unfolded Protein Response (UPR). In this study, B. melitensis infection up regulated expression of the UPR target genes BiP, CHOP, and ERdj4, and induced XBP1 mRNA splicing in murine macrophages. These data implicate activation of all 3 major signaling pathways of the UPR. Consistent with previous reports, XBP1 mRNA splicing was largely MyD88-dependent. However, up regulation of CHOP, and ERdj4 was completely MyD88 independent. Heat killed Brucella stimulated significantly less BiP, CHOP, and ERdj4 expression, but induced XBP1 splicing. Although a Brucella VirB mutant showed relatively intact UPR induction, a TcpB mutant had significantly compromised BiP, CHOP and ERdj4 expression. Purified TcpB, a protein recently identified to modulate microtubules in a manner similar to paclitaxel, also induced UPR target gene expression and resulted in dramatic restructuring of the ER. In contrast, infection with the TcpB mutant resulted in much less ER structural disruption. Finally, tauroursodeoxycholic acid, a pharmacologic chaperone that ameliorates the UPR, significantly impaired Brucella replication in macrophages. Together, these results suggest Brucella induces a UPR, via TcpB and potentially other factors, that enables its intracellular replication. Thus, the UPR may provide a novel therapeutic target for the treatment of brucellosis. These results also have implications for other intracellular bacteria that rely on host physiologic stress responses for replication.

Smith, Judith A.; Khan, Mike; Magnani, Diogo D.; Harms, Jerome S.; Durward, Marina; Radhakrishnan, Girish K.; Liu, Yi-Ping; Splitter, Gary A.

2013-01-01

112

Brucella induces an unfolded protein response via TcpB that supports intracellular replication in macrophages.  

PubMed

Brucella melitensis is a facultative intracellular bacterium that causes brucellosis, the most prevalent zoonosis worldwide. The Brucella intracellular replicative niche in macrophages and dendritic cells thwarts immune surveillance and complicates both therapy and vaccine development. Currently, host-pathogen interactions supporting Brucella replication are poorly understood. Brucella fuses with the endoplasmic reticulum (ER) to replicate, resulting in dramatic restructuring of the ER. This ER disruption raises the possibility that Brucella provokes an ER stress response called the Unfolded Protein Response (UPR). In this study, B. melitensis infection up regulated expression of the UPR target genes BiP, CHOP, and ERdj4, and induced XBP1 mRNA splicing in murine macrophages. These data implicate activation of all 3 major signaling pathways of the UPR. Consistent with previous reports, XBP1 mRNA splicing was largely MyD88-dependent. However, up regulation of CHOP, and ERdj4 was completely MyD88 independent. Heat killed Brucella stimulated significantly less BiP, CHOP, and ERdj4 expression, but induced XBP1 splicing. Although a Brucella VirB mutant showed relatively intact UPR induction, a TcpB mutant had significantly compromised BiP, CHOP and ERdj4 expression. Purified TcpB, a protein recently identified to modulate microtubules in a manner similar to paclitaxel, also induced UPR target gene expression and resulted in dramatic restructuring of the ER. In contrast, infection with the TcpB mutant resulted in much less ER structural disruption. Finally, tauroursodeoxycholic acid, a pharmacologic chaperone that ameliorates the UPR, significantly impaired Brucella replication in macrophages. Together, these results suggest Brucella induces a UPR, via TcpB and potentially other factors, that enables its intracellular replication. Thus, the UPR may provide a novel therapeutic target for the treatment of brucellosis. These results also have implications for other intracellular bacteria that rely on host physiologic stress responses for replication. PMID:24339776

Smith, Judith A; Khan, Mike; Magnani, Diogo D; Harms, Jerome S; Durward, Marina; Radhakrishnan, Girish K; Liu, Yi-Ping; Splitter, Gary A

2013-12-01

113

Role of Unfolded Protein Response Dysregulation in Oxidative Injury of Retinal Pigment Epithelial Cells  

PubMed Central

Abstract Aims: Age-related macular degeneration (AMD), a major cause of legal blindness in the elderly, is associated with genetic and environmental risk factors, such as cigarette smoking. Recent evidence shows that cigarette smoke (CS) that contains high levels of potent oxidants preferably targets retinal pigment epithelium (RPE) leading to oxidative damage and apoptosis; however, the mechanisms are poorly understood. The present study aimed to investigate the role of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in CS-related RPE apoptosis. Results: ER stress and proapoptotic gene C/EBP homologous protein (CHOP) were induced in the RPE/choroid complex from mice exposed to CS for 2 weeks and in human RPE cells treated with hydroquinone, a potent oxidant found at high concentrations in CS. Suppressing ER stress or inhibiting CHOP activation by pharmacological chaperones or genetic approaches attenuated hydroquinone-induced RPE cell apoptosis. In contrast to enhanced CHOP activation, protein level of active X-box binding protein 1 (XBP1), a major regulator of the adaptive UPR, was reduced in hydroquinone-treated cells. Conditional knockout of XBP1 gene in the RPE resulted in caspase-12 activation, increased CHOP expression, and decreased antiapoptotic gene Bcl-2. Furthermore, XBP1-deficient RPE cells are more sensitive to oxidative damage induced by hydroquinone or NaIO3, a CS-unrelated chemical oxidant. Conversely, overexpressing XBP1 protected RPE cells and attenuated oxidative stress-induced RPE apoptosis. Innovation and Conclusion: These findings provide strong evidence suggesting an important role of ER stress and the UPR in CS-related oxidative injury of RPE cells. Thus, the modulation of the UPR signaling may provide a promising target for the treatment of AMD. Antioxid. Redox Signal. 20, 2091–2106.

Chen, Chen; Cano, Marisol; Wang, Joshua J.; Li, Jingming; Huang, Chuangxin; Yu, Qiang; Herbert, Terence P.; Handa, James T.

2014-01-01

114

ADP ribosylation adapts an ER chaperone response to short-term fluctuations in unfolded protein load  

PubMed Central

Gene expression programs that regulate the abundance of the chaperone BiP adapt the endoplasmic reticulum (ER) to unfolded protein load. However, such programs are slow compared with physiological fluctuations in secreted protein synthesis. While searching for mechanisms that fill this temporal gap in coping with ER stress, we found elevated levels of adenosine diphosphate (ADP)–ribosylated BiP in the inactive pancreas of fasted mice and a rapid decline in this modification in the active fed state. ADP ribosylation mapped to Arg470 and Arg492 in the substrate-binding domain of hamster BiP. Mutations that mimic the negative charge of ADP-ribose destabilized substrate binding and interfered with interdomain allosteric coupling, marking ADP ribosylation as a rapid posttranslational mechanism for reversible inactivation of BiP. A kinetic model showed that buffering fluctuations in unfolded protein load with a recruitable pool of inactive chaperone is an efficient strategy to minimize both aggregation and costly degradation of unfolded proteins.

Petrova, Kseniya; Tomba, Giulia; Vendruscolo, Michele

2012-01-01

115

Small Molecule-Induced Mitochondrial Disruption Directs Prostate Cancer Inhibition via Unfolded Protein Response Signaling  

PubMed Central

We previously identified SMIP004 (N-(4-butyl-2-methyl-phenyl) acetamide) as a novel inducer of cancer-cell selective apoptosis of human prostate cancer cells. SMIP004 decreased the levels of positive cell cycle regulators, upregulated cyclin-dependent kinase inhibitors, and resulted in G1 arrest, inhibition of colony formation in soft agar, and cell death. However, the mechanism of SMIP004-induced cancer cell selective apoptosis remained unknown. Here, we used chemical genomic and proteomic profiling to unravel a SMIP004-induced pro-apoptotic pathway, which initiates with disruption of mitochondrial respiration leading to oxidative stress. This, in turn, activates two pathways, one eliciting cell cycle arrest by rapidly targeting cyclin D1 for proteasomal degradation and driving the transcriptional downregulation of the androgen receptor, and a second pathway that activates pro-apoptotic signaling through MAPK activation downstream of the unfolded protein response (UPR). SMIP004 potently inhibits the growth of prostate and breast cancer xenografts in mice. Our data suggest that SMIP004, by inducing mitochondrial ROS formation, targets specific sensitivities of prostate cancer cells to redox and bioenergetic imbalances that can be exploited in cancer therapy.

Rico-Bautista, Elizabeth; Zhu, Wenhong; Kitada, Shinichi; Ganapathy, Suthakar; Lau, Eric; Krajewski, Stan; Ramirez, Joel; Bush, Jason A.; Yuan, Zhimin; Wolf, Dieter A.

2013-01-01

116

Aberrant, differential and bidirectional regulation of the unfolded protein response towards cell survival by 3?-deoxyadenosine  

PubMed Central

The unfolded protein response (UPR) is involved in a diverse range of pathologies triggered by endoplasmic reticulum (ER) stress. Endeavor to seek selective regulators of the UPR is a promising challenge towards therapeutic intervention in ER stress-related disorders. In the present report, we describe aberrant, differential and bidirectional regulation of the UPR by 3?-deoxyadenosine (cordycepin) towards cell survival. 3?-Deoxyadenosine blocked ER stress-induced apoptosis via inhibiting the IRE1–JNK pro-apoptotic pathway. 3?-Deoxyadenosine also inhibited apoptosis through reinforcement of the pro-survival eIF2? signaling without affecting PERK activity. It was associated with depression of GADD34 that dephosphorylates eIF2?, and dephosphorylation of eIF2? by salubrinal mimicked the anti-apoptotic effect of 3?-deoxyadenosine. Unexpectedly, although 3?-deoxyadenosine caused activation of eIF2?, it inhibited downstream pro-apoptotic events including induction of ATF4 and expression of CHOP. Cooperation of adenosine transporter and A3 adenosine receptor, but not A1/A2 receptors, mediated the pluripotent effects of 3?-deoxyadenosine. In mice, ER stress caused activation of JNK, expression of CHOP and induction of apoptosis in renal tubules. The apoptosis was significantly attenuated by administration with 3?-deoxyadenosine, and it was correlated with blunted induction of JNK and CHOP in the kidney. These results disclosed atypical pro-survival regulation of the UPR by 3?-deoxyadenosine, which may be advantageous for the treatment of intractable, ER stress-related disorders.

Kitamura, M; Kato, H; Saito, Y; Nakajima, S; Takahashi, S; Johno, H; Gu, L; Katoh, R

2011-01-01

117

Intrauterine Growth Restriction Increases TNF? and Activates the Unfolded Protein Response in Male Rat Pups  

PubMed Central

Intrauterine growth restriction (IUGR) programs adult disease, including obesity and insulin resistance. Our group previously demonstrated that IUGR dysregulates adipose deposition in male, but not female, weanling rats. Dysregulated adipose deposition is often accompanied by the release of proinflammatory signaling molecules, such as tumor necrosis factor alpha (TNF?). TNF? contributes to adipocyte inflammation and impaired insulin signaling. TNF? has also been implicated in the activation of the unfolded protein response (UPR), which impairs insulin signaling. We hypothesized that, in male rat pups, IUGR would increase TNF?, TNFR1, and components of the UPR (Hspa5, ATF6, p-eIF2?, and Ddit3) prior to the onset of obesity. We further hypothesized that impaired glucose tolerance would occur after the onset of adipose dysfunction in male IUGR rats. To test this hypothesis, we used a well-characterized rat model of uteroplacental insufficiency-induced IUGR. Our primary findings are that, in male rats, IUGR (1) increased circulating and adipose TNF?, (2) increased mRNA levels of UPR components as well as p-eIF2a, and (3) impaired glucose tolerance after observed TNF? increased and after UPR activation. We speculate that programmed dysregulation of TNF? and UPR contributed to the development of glucose intolerance in male IUGR rats.

Riddle, Emily S.; Campbell, Michael S.; Lang, Brook Y.; Bierer, Ryann; Wang, Yan; Bagley, Heidi N.; Joss-Moore, Lisa A.

2014-01-01

118

Implication of unfolded protein response in resveratrol-induced inhibition of K562 cell proliferation  

SciTech Connect

Resveratrol (RES), a natural plant polyphenol, is an effective inducer of cell cycle arrest and apoptosis in a variety of carcinoma cell types. In addition, RES has been reported to inhibit tumorigenesis in several animal models suggesting that it functions as a chemopreventive and anti-tumor agent in vivo. The chemopreventive and chemotherapeutic properties associated with resveratrol offer promise for the design of new chemotherapeutic agents. However, the mechanisms by which RES mediates its effects are not yet fully understood. In this study, we showed that RES caused cell cycle arrest and proliferation inhibition via induction of unfolded protein response (UPR) in human leukemia K562 cell line. Treatment of K562 cells with RES induced a number of signature UPR markers, including transcriptional induction of GRP78 and CHOP, phosphorylation of eukaryotic initiation factor 2{alpha} (eIF2{alpha}), ER stress-specific XBP-1 splicing, suggesting the induction of UPR by RES. RES inhibited proliferation of K562 in a concentration-dependent manner. Flow cytometric analyses revealed that K562 cells were arrested in G1 phase upon RES treatment. Salubrinal, an eIF2{alpha} inhibitor, or overexpression of dominant negative mutants of PERK or eIF2{alpha}, effectively restored RES-induced cell cycle arrest, underscoring the important role of PERK/eIF2{alpha} branch of UPR in RES-induced inhibition of cell proliferation.

Liu, Bao-Qin; Gao, Yan-Yan; Niu, Xiao-Fang [Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001 (China)] [Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001 (China); Xie, Ji-Sheng [Youjiang Medical College for Nationalities, Guangxi 533000 (China)] [Youjiang Medical College for Nationalities, Guangxi 533000 (China); Meng, Xin; Guan, Yifu [Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001 (China)] [Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001 (China); Wang, Hua-Qin, E-mail: wanghq_doctor@hotmail.com [Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001 (China)] [Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001 (China)

2010-01-01

119

Modulation of the Unfolded Protein Response During Hepatocyte and Cardiomyocyte Apoptosis In Trauma/Hemorrhagic Shock  

PubMed Central

Trauma with hemorrhagic shock (T/HS), has been shown to result in liver injury marked by hepatocyte apoptosis and heart failure marked by cardiomyocyte apoptosis, both of which we have shown to be prevented by IL-6 administration at resuscitation, and Stat3 largely mediated this. As specific mediators have not been delineated, we investigated the unfolded protein response (UPR), which, with marked activation, can lead to apoptosis. Prior studies of hepatic and cardiac injury examined limited repertoires of UPR elements, making it difficult to assess the role of the UPR in T/HS. This study describes the first global examination of the UPR transcriptome in the liver and heart following T/HS, demonstrating organ-specific UPR transcriptome changes. The non-canonical UPR chaperone, Hsp70, was most dysregulated following T/HS and may contribute to hepatocyte protection via an IL-6-mediated pathway, identifying a potential new therapeutic strategy to prevent hepatocyte death and organ dysfunction in T/HS.

Thacker, Stephen A.; Robinson, Prema; Abel, Adam; Tweardy, David J.

2013-01-01

120

Endoplasmic reticulum stress, the unfolded protein response, and gene network modeling in antiestrogen resistant breast cancer  

PubMed Central

Lack of understanding of endocrine resistance remains one of the major challenges for breast cancer researchers, clinicians, and patients. Current reductionist approaches to understanding the molecular signaling driving resistance have offered mostly incremental progress over the past 10 years. As the field of systems biology has begun to mature, the approaches and network modeling tools being developed and applied therein offer a different way to think about how molecular signaling and the regulation of critical cellular functions are integrated. To gain novel insights, we first describe some of the key challenges facing network modeling of endocrine resistance, many of which arise from the properties of the data spaces being studied. We then use activation of the unfolded protein response (UPR) following induction of endoplasmic reticulum stress in breast cancer cells by antiestrogens, to illustrate our approaches to computational modeling. Activation of UPR is a key determinant of cell fate decision making and regulation of autophagy and apoptosis. These initial studies provide insight into a small subnetwork topology obtained using differential dependency network analysis and focused on the UPR gene XBP1. The XBP1 subnetwork topology incorporates BCAR3, BCL2, BIK, NF?B, and other genes as nodes; the connecting edges represent the dependency structures amongst these nodes. As data from ongoing cellular and molecular studies become available, we will build detailed mathematical models of this XBP1-UPR network.

Clarke, Robert; Shajahan, Ayesha N.; Wang, Yue; Tyson, John J.; Riggins, Rebecca B.; Weiner, Louis M.; Bauman, William T.; Xuan, Jianhua; Zhang, Bai; Facey, Caroline; Aiyer, Harini; Cook, Katherine; Hickman, F. Edward; Tavassoly, Iman; Verdugo, Anael; Chen, Chun; Zwart, Alan; Warri, Anni; Hilakivi-Clarke, Leena A.

2013-01-01

121

Resveratrol-induced cytotoxicity in human Burkitt's lymphoma cells is coupled to the unfolded protein response  

PubMed Central

Background Resveratrol (RES), a natural phytoalexin found at high levels in grapes and red wine, has been shown to induce anti-proliferation and apoptosis of human cancer cell lines. However, the underlying molecular mechanisms are at present only partially understood. Method The effects of RES on activation of unfolded protein responses (UPR) were evaluated using Western blotting, semi-quantitative and real-time RT-PCR. Cell death was evaluated using Annexin V/PI staining and subsequent FACS. Results Similar as tunicamycin, treatment with RES lead to the activation of all 3 branches of the UPR, with early splicing of XBP-1 indicative of IRE1 activation, phosphorylation of eIF2? consistent with ER resident kinase (PERK) activation, activating transcription factor 6 (ATF6) splicing, and increase in expression levels of the downstream molecules GRP78/BiP, GRP94 and CHOP/GADD153 in human Burkitt's lymphoma Raji and Daudi cell lines. RES was shown to induce cell death, which could be attenuated by thwarting upregulation of CHOP. Conclusions Our data suggest that activation of the apoptotic arm of the UPR and its downstream effector CHOP/GADD153 is involved, at least in part, in RES-induced apoptosis in Burkitt's lymphoma cells.

2010-01-01

122

Genes of the Unfolded Protein Response Pathway Harbor Risk Alleles for Primary Open Angle Glaucoma  

PubMed Central

The statistical power of genome-wide association (GWA) studies to detect risk alleles for human diseases is limited by the unfavorable ratio of SNPs to study subjects. This multiple testing problem can be surmounted with very large population sizes when common alleles of large effects give rise to disease status. However, GWA approaches fall short when many rare alleles may give rise to a common disease, or when the number of subjects that can be recruited is limited. Here, we demonstrate that this multiple testing problem can be overcome by a comparative genomics approach in which an initial genome-wide screen in a genetically amenable model organism is used to identify human orthologues that may harbor risk alleles for adult-onset primary open angle glaucoma (POAG). Glaucoma is a major cause of blindness, which affects over 60 million people worldwide. Several genes have been associated with juvenile onset glaucoma, but genetic factors that predispose to adult onset primary open angle glaucoma (POAG) remain largely unknown. Previous genome-wide analysis in a Drosophila ocular hypertension model identified transcripts with altered regulation and showed induction of the unfolded protein response (UPR) upon overexpression of transgenic human glaucoma-associated myocilin (MYOC). We selected 16 orthologous genes with 62 polymorphic markers and identified in two independent human populations two genes of the UPR that harbor POAG risk alleles, BIRC6 and PDIA5. Thus, effectiveness of the UPR in response to accumulation of misfolded or aggregated proteins may contribute to the pathogenesis of POAG and provide targets for early therapeutic intervention.

Carbone, Mary Anna; Chen, Yuhong; Hughes, Guy A.; Weinreb, Robert N.; Zabriskie, Norman A.; Zhang, Kang; Anholt, Robert R. H.

2011-01-01

123

Activation of the unfolded protein response is associated with pulmonary hypertension  

PubMed Central

Pulmonary hypertension remains an important cause of morbidity and mortality. Although there is currently no cure, descriptions of defective intracellular trafficking and protein misfolding in vascular cell models of pulmonary hypertension have been recently reported. We tested the hypothesis that activation of the unfolded protein response (UPR) would be associated with the development of severe PH. We investigated activation of the UPR in archival tissues from patients with severe PH, and in the monocrotaline-induced rat model of severe PH. We tested the ability of a pharmacologic agent capable of modulating the UPR to prevent and reverse pulmonary hypertension. We found evidence of an active UPR in archival tissue from humans with PH, but not in control lungs. Similarly, monocrotaline-treated rats demonstrated a significant difference in expression of each of the major arms of the UPR compared to controls. Interestingly, the UPR preceded the appearance of macrophages and the development of lung vascular remodeling in the rats. Treatment of monocrotaline rats with salubrinal, a modulator of the PERK arm of the UPR, attenuated PH and was associated with a decrease in lung macrophages. In culture, pulmonary artery smooth muscle cells with UPR induction produced IL-6 and CCL-2/MCP-1, and stimulated macrophage migration. These effects were abolished by pretreatment of cells with salubrinal. These data support the hypothesis that the UPR may play a role in the pathogenesis of inflammatory vascular remodeling and PH. As such, understanding the functional contributions of the UPR in the setting of PH may have important therapeutic implications.

Yeager, Michael E.; Reddy, Monica B.; Nguyen, Cecilia M.; Colvin, Kelley L.; Ivy, D. Dunbar; Stenmark, Kurt R.

2012-01-01

124

Mechanistic rationale for targeting the unfolded protein response in pre-B acute lymphoblastic leukemia.  

PubMed

The unfolded protein response (UPR) pathway, a stress-induced signaling cascade emanating from the endoplasmic reticulum (ER), regulates the expression and activity of molecules including BiP (HSPA5), IRE1 (ERN1), Blimp-1 (PRDM1), and X-box binding protein 1 (XBP1). These molecules are required for terminal differentiation of B cells into plasma cells and expressed at high levels in plasma cell-derived multiple myeloma. Although these molecules have no known role at early stages of B-cell development, here we show that their expression transiently peaks at the pre-B-cell receptor checkpoint. Inducible, Cre-mediated deletion of Hspa5, Prdm1, and Xbp1 consistently induces cellular stress and cell death in normal pre-B cells and in pre-B-cell acute lymphoblastic leukemia (ALL) driven by BCR-ABL1- and NRAS(G12D) oncogenes. Mechanistically, expression and activity of the UPR downstream effector XBP1 is regulated positively by STAT5 and negatively by the B-cell-specific transcriptional repressors BACH2 and BCL6. In two clinical trials for children and adults with ALL, high XBP1 mRNA levels at the time of diagnosis predicted poor outcome. A small molecule inhibitor of ERN1-mediated XBP1 activation induced selective cell death of patient-derived pre-B ALL cells in vitro and significantly prolonged survival of transplant recipient mice in vivo. Collectively, these studies reveal that pre-B ALL cells are uniquely vulnerable to ER stress and identify the UPR pathway and its downstream effector XBP1 as novel therapeutic targets to overcome drug resistance in pre-B ALL. PMID:24821775

Kharabi Masouleh, Behzad; Geng, Huimin; Hurtz, Christian; Chan, Lai N; Logan, Aaron C; Chang, Mi Sook; Huang, Chuanxin; Swaminathan, Srividya; Sun, Haibo; Paietta, Elisabeth; Melnick, Ari M; Koeffler, Phillip; Müschen, Markus

2014-05-27

125

Molecularly defined unfolded protein response subclasses have distinct correlations with fatty liver disease in zebrafish.  

PubMed

The unfolded protein response (UPR) is a complex network of sensors and target genes that ensure efficient folding of secretory proteins in the endoplasmic reticulum (ER). UPR activation is mediated by three main sensors, which regulate the expression of hundreds of targets. UPR activation can result in outcomes ranging from enhanced cellular function to cell dysfunction and cell death. How this pathway causes such different outcomes is unknown. Fatty liver disease (steatosis) is associated with markers of UPR activation and robust UPR induction can cause steatosis; however, in other cases, UPR activation can protect against this disease. By assessing the magnitude of activation of UPR sensors and target genes in the liver of zebrafish larvae exposed to three commonly used ER stressors (tunicamycin, thapsigargin and Brefeldin A), we have identified distinct combinations of UPR sensors and targets (i.e. subclasses) activated by each stressor. We found that only the UPR subclass characterized by maximal induction of UPR target genes, which we term a stressed-UPR, induced steatosis. Principal component analysis demonstrated a significant positive association between UPR target gene induction and steatosis. The same principal component analysis showed significant correlation with steatosis in samples from patients with fatty liver disease. We demonstrate that an adaptive UPR induced by a short exposure to thapsigargin prior to challenging with tunicamycin reduced both the induction of a stressed UPR and steatosis incidence. We conclude that a stressed UPR causes steatosis and an adaptive UPR prevents it, demonstrating that this pathway plays dichotomous roles in fatty liver disease. PMID:24973751

Vacaru, Ana M; Di Narzo, Antonio Fabio; Howarth, Deanna L; Tsedensodnom, Orkhontuya; Imrie, Dru; Cinaroglu, Ayca; Amin, Salma; Hao, Ke; Sadler, Kirsten C

2014-07-01

126

Conservation between animals and plants of the cis-acting element involved in the unfolded protein response  

Microsoft Academic Search

Using Arabidopsis thaliana, we identified the cis-element involved in the plant unfolded protein response (UPR). In transgenic plants, tunicamycin stimulated expression of a reporter gene under the control of the BiP promoter and promoter analysis identified a 24bp sequence crucial to this induction. When fused with a minimal promoter, a hexamer of this sequence was sufficient for induction of a

Dong-Ha Oh; Chang-Seob Kwon; Hiroshi Sano; Won-Il Chung; Nozomu Koizumi

2003-01-01

127

The Unfolded Protein Response Transducer Ire1p Contains a Nuclear Localization Sequence Recognized by Multiple Importins  

Microsoft Academic Search

The Ire1p transmembrane receptor kinase\\/endonuclease transduces the unfolded protein response (UPR) from the endoplasmic reticulum (ER) to the nucleus in Saccharomyces cerevisiae. In this study, we analyzed the capacity of a highly basic sequence in the linker region of Ire1p to function as a nuclear localization sequence (NLS) both in vivo and in vitro. This 18-residue sequence is capable of

Laurence Goffin; Sadanand Vodala; Christine Fraser; Joanne Ryan; Mark Timms; Sarina Meusburger; Bruno Catimel; Edouard C. Nice; Pamela A. Silver; Chong-Yun Xiao; David A. Jans; Mary-Jane H. Gething

2006-01-01

128

Overexpression of Myocilin in the Drosophila Eye Activates the Unfolded Protein Response: Implications for Glaucoma  

PubMed Central

Background Glaucoma is the world's second leading cause of bilateral blindness with progressive loss of vision due to retinal ganglion cell death. Myocilin has been associated with congenital glaucoma and 2–4% of primary open angle glaucoma (POAG) cases, but the pathogenic mechanisms remain largely unknown. Among several hypotheses, activation of the unfolded protein response (UPR) has emerged as a possible disease mechanism. Methodology / Principal Findings We used a transgenic Drosophila model to analyze whole-genome transcriptional profiles in flies that express human wild-type or mutant MYOC in their eyes. The transgenic flies display ocular fluid discharge, reflecting ocular hypertension, and a progressive decline in their behavioral responses to light. Transcriptional analysis shows that genes associated with the UPR, ubiquitination, and proteolysis, as well as metabolism of reactive oxygen species and photoreceptor activity undergo altered transcriptional regulation. Following up on the results from these transcriptional analyses, we used immunoblots to demonstrate the formation of MYOC aggregates and showed that the formation of such aggregates leads to induction of the UPR, as evident from activation of the fluorescent UPR marker, xbp1-EGFP. Conclusions / Significance Our results show that aggregation of MYOC in the endoplasmic reticulum activates the UPR, an evolutionarily conserved stress pathway that culminates in apoptosis. We infer from the Drosophila model that MYOC-associated ocular hypertension in the human eye may result from aggregation of MYOC and induction of the UPR in trabecular meshwork cells. This process could occur at a late age with wild-type MYOC, but might be accelerated by MYOC mutants to account for juvenile onset glaucoma.

Carbone, Mary Anna; Ayroles, Julien F.; Yamamoto, Akihiko; Morozova, Tatiana V.; West, Steven A.; Magwire, Michael M.; Mackay, Trudy F. C.; Anholt, Robert R. H.

2009-01-01

129

Dinaciclib (SCH727965) inhibits the unfolded protein response through a CDK1- and 5-dependent mechanism.  

PubMed

Evidence implicating dysregulation of the IRE1/XBP-1s arm of the unfolded protein response (UPR) in cancer pathogenesis (e.g., multiple myeloma) has prompted the development of IRE1 RNase inhibitors. Here, effects of cyclin-dependent kinase (CDK) inhibitor SCH727965 (dinaciclib) on the IRE1 arm of the UPR were examined in human leukemia and myeloma cells. Exposure of cells to extremely low (e.g., nmol/L) concentrations of SCH727965, a potent inhibitor of CDKs 1/2/5/9, diminished XBP-1s and Grp78 induction by the endoplasmic reticulum (ER) stress-inducers thapsigargin and tunicamycin, while sharply inducing cell death. SCH727965, in contrast to IRE1 RNase inhibitors, inhibited the UPR in association with attenuation of XBP-1s nuclear localization and accumulation rather than transcription, translation, or XBP-1 splicing. Notably, in human leukemia cells, CDK1 and 5 short hairpin RNA (shRNA) knockdown diminished Grp78 and XBP-1s upregulation while increasing thapsigargin lethality, arguing for a functional role for CDK1/5 in activation of the cytoprotective IRE1/XBP-1s arm of the UPR. In contrast, CDK9 or 2 inhibitors or shRNA knockdown failed to downregulate XBP-1s or Grp78. Furthermore, IRE1, XBP-1, or Grp78 knockdown significantly increased thapsigargin lethality, as observed with CDK1/5 inhibition/knockdown. Finally, SCH727965 diminished myeloma cell growth in vivo in association with XBP-1s downregulation. Together, these findings demonstrate that SCH727965 acts at extremely low concentrations to attenuate XBP-1s nuclear accumulation and Grp78 upregulation in response to ER stress inducers. They also highlight a link between specific components of the cell-cycle regulatory apparatus (e.g., CDK1/5) and the cytoprotective IRE1/XBP-1s/Grp78 arm of the UPR that may be exploited therapeutically in UPR-driven malignancies. PMID:24362465

Nguyen, Tri K; Grant, Steven

2014-03-01

130

Aging and sleep deprivation induce the unfolded protein response in the pancreas: implications for metabolism.  

PubMed

Sleep disruption has detrimental effects on glucose metabolism through pathways that remain poorly defined. Although numerous studies have examined the consequences of sleep deprivation (SD) in the brain, few have directly tested its effects on peripheral organs. We examined several tissues in mice for induction of the unfolded protein response (UPR) following acute SD. In young animals, we found a robust induction of BiP in the pancreas, indicating an active UPR. At baseline, pancreata from aged animals exhibited a marked increase in a pro-apoptotic transcription factor, CHOP, that was amplified by SD, whereas BiP induction was not observed, suggesting a maladaptive response to cellular stress with age. Acute SD increased plasma glucose levels in both young and old animals. However, this change was not overtly related to stress in the pancreatic beta cells, as plasma insulin levels were not lower following acute SD. Accordingly, animals subjected to acute SD remained tolerant to a glucose challenge. In a chronic SD experiment, young mice were found to be sensitized to insulin and have improved glycemic control, whereas aged animals became hyperglycemic and failed to maintain appropriate plasma insulin concentrations. Our results show that both age and SD cooperate to induce the UPR in pancreatic tissue. While changes in insulin secretion are unlikely to play a major role in the acute effects of SD, CHOP induction in pancreatic tissues suggests that chronic SD may contribute to the loss or dysfunction of endocrine cells and that these effects may be exacerbated by normal aging. PMID:24102714

Naidoo, Nirinjini; Davis, James G; Zhu, Jingxu; Yabumoto, Maya; Singletary, Kristan; Brown, Marishka; Galante, Raymond; Agarwal, Beamon; Baur, Joseph A

2014-02-01

131

Effects of inactivation and constitutive expression of the unfolded- protein response pathway on protein production in the yeast Saccharomyces cerevisiae.  

PubMed

One strategy to obtain better yields of secreted proteins has been overexpression of single endoplasmic reticulum-resident foldases or chaperones. We report here that manipulation of the unfolded-protein response (UPR) pathway regulator, HAC1, affects production of both native and foreign proteins in the yeast Saccharomyces cerevisiae. The effects of HAC1 deletion and overexpression on the production of a native protein, invertase, and two foreign proteins, Bacillus amyloliquefaciens alpha-amylase and Trichoderma reesei endoglucanase EGI, were studied. Disruption of HAC1 caused decreases in the secretion of both alpha-amylase (70 to 75% reduction) and EGI (40 to 50% reduction) compared to the secretion by the parental strain. Constitutive overexpression of HAC1 caused a 70% increase in alpha-amylase secretion but had no effect on EGI secretion. The invertase levels were twofold higher in the strain overexpressing HAC1. Also, the effect of the active form of T. reesei hac1 was tested in S. cerevisiae. hac1 expression caused a 2.4-fold increase in the secretion of alpha-amylase in S. cerevisiae and also slight increases in invertase and total protein production. Overexpression of both S. cerevisiae HAC1 and T. reesei hac1 caused an increase in the expression of the known UPR target gene KAR2 at early time points during cultivation. PMID:12676684

Valkonen, Mari; Penttilä, Merja; Saloheimo, Markku

2003-04-01

132

Effects of Inactivation and Constitutive Expression of the Unfolded- Protein Response Pathway on Protein Production in the Yeast Saccharomyces cerevisiae  

PubMed Central

One strategy to obtain better yields of secreted proteins has been overexpression of single endoplasmic reticulum-resident foldases or chaperones. We report here that manipulation of the unfolded-protein response (UPR) pathway regulator, HAC1, affects production of both native and foreign proteins in the yeast Saccharomyces cerevisiae. The effects of HAC1 deletion and overexpression on the production of a native protein, invertase, and two foreign proteins, Bacillus amyloliquefaciens ?-amylase and Trichoderma reesei endoglucanase EGI, were studied. Disruption of HAC1 caused decreases in the secretion of both ?-amylase (70 to 75% reduction) and EGI (40 to 50% reduction) compared to the secretion by the parental strain. Constitutive overexpression of HAC1 caused a 70% increase in ?-amylase secretion but had no effect on EGI secretion. The invertase levels were twofold higher in the strain overexpressing HAC1. Also, the effect of the active form of T. reesei hac1 was tested in S. cerevisiae. hac1 expression caused a 2.4-fold increase in the secretion of ?-amylase in S. cerevisiae and also slight increases in invertase and total protein production. Overexpression of both S. cerevisiae HAC1 and T. reesei hac1 caused an increase in the expression of the known UPR target gene KAR2 at early time points during cultivation.

Valkonen, Mari; Penttila, Merja; Saloheimo, Markku

2003-01-01

133

Activation of the Cellular Unfolded Protein Response by Recombinant Adeno-Associated Virus Vectors  

PubMed Central

The unfolded protein response (UPR) is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER). In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the three major UPR axes, namely, endoribonuclease inositol-requiring enzyme-1 (IRE1?), activating transcription factor 6 (ATF6) and PKR-like ER kinase (PERK) in AAV infected cells. Total RNA from mock or AAV infected HeLa cells were used to determine the levels of 8 different ER-stress responsive transcripts from these pathways. We observed a significant up-regulation of IRE1? (up to 11 fold) and PERK (up to 8 fold) genes 12–48 hours after infection with self-complementary (sc)AAV2 but less prominent with single-stranded (ss)AAV2 vectors. Further studies demonstrated that scAAV1 and scAAV6 also induce cellular UPR in vitro, with AAV1 vectors activating the PERK pathway (3 fold) while AAV6 vectors induced a significant increase on all the three major UPR pathways [6–16 fold]. These data suggest that the type and strength of UPR activation is dependent on the viral capsid. We then examined if transient inhibition of UPR pathways by RNA interference has an effect on AAV transduction. siRNA mediated silencing of PERK and IRE1? had a modest effect on AAV2 and AAV6 mediated gene expression (?1.5–2 fold) in vitro. Furthermore, hepatic gene transfer of scAAV2 vectors in vivo, strongly elevated IRE1? and PERK pathways (2 and 3.5 fold, respectively). However, when animals were pre-treated with a pharmacological UPR inhibitor (metformin) during scAAV2 gene transfer, the UPR signalling and its subsequent inflammatory response was attenuated concomitant to a modest 2.8 fold increase in transgene expression. Collectively, these data suggest that AAV vectors activate the cellular UPR pathways and their selective inhibition may be beneficial during AAV mediated gene transfer.

Balakrishnan, Balaji; Sen, Dwaipayan; Hareendran, Sangeetha; Roshini, Vaani; David, Sachin; Srivastava, Alok; Jayandharan, Giridhara R.

2013-01-01

134

Effect of Exercise Intensity on Unfolded Protein Response in Skeletal Muscle of Rat  

PubMed Central

Endoplasmic reticulum (ER) stress, unfolded protein response (UPR), and mitochondrial biogenesis were assessed following varying intensities of exercise training. The animals were randomly assigned to receive either low- (LIT, n=7) or high intensity training (HIT, n=7), or were assigned to a control group (n=7). Over 5 weeks, the animals in the LIT were exercised on a treadmill with a 10° incline for 60 min at a speed of 20 m/min group, and in the HIT group at a speed of 34 m/min for 5 days a week. No statistically significant differences were found in the body weight, plasma triglyceride, and total cholesterol levels across the three groups, but fasting glucose and insulin levels were significantly lower in the exercise-trained groups. Additionally, no statistically significant differences were observed in the levels of PERK phosphorylation in skeletal muscles between the three groups. However, compared to the control and LIT groups, the level of BiP was lower in the HIT group. Compared to the control group, the levels of ATF4 in skeletal muscles and CHOP were significantly lower in the HIT group. The HIT group also showed increased PGC-1? mRNA expression in comparison with the control group. Furthermore, both of the trained groups showed higher levels of mitochondrial UCP3 than the control group. In summary, we found that a 5-week high-intensity exercise training routine resulted in increased mitochondrial biogenesis and decreased ER stress and apoptotic signaling in the skeletal muscle tissue of rats.

Kim, Kihoon; Kim, Yun-Hye; Lee, Sung-Hye; Jeon, Man-Joong; Park, So-Young

2014-01-01

135

The Yeast Rab GTPase Ypt1 Modulates Unfolded Protein Response Dynamics by Regulating the Stability of HAC1 RNA  

PubMed Central

The unfolded protein response (UPR) is a conserved mechanism that mitigates accumulation of unfolded proteins in the ER. The yeast UPR is subject to intricate post-transcriptional regulation, involving recruitment of the RNA encoding the Hac1 transcription factor to the ER and its unconventional splicing. To investigate the mechanisms underlying regulation of the UPR, we screened the yeast proteome for proteins that specifically interact with HAC1 RNA. Protein microarray experiments revealed that HAC1 interacts specifically with small ras GTPases of the Ypt family. We characterized the interaction of HAC1 RNA with one of these proteins, the yeast Rab1 homolog Ypt1. We found that Ypt1 protein specifically associated in vivo with unspliced HAC1 RNA. This association was disrupted by conditions that impaired protein folding in the ER and induced the UPR. Also, the Ypt1-HAC1 interaction depended on IRE1 and ADA5, the two genes critical for UPR activation. Decreasing expression of the Ypt1 protein resulted in a reduced rate of HAC1 RNA decay, leading to significantly increased levels of both unspliced and spliced HAC1 RNA, and delayed attenuation of the UPR, when ER stress was relieved. Our findings establish that Ypt1 contributes to regulation of UPR signaling dynamics by promoting the decay of HAC1 RNA, suggesting a potential regulatory mechanism for linking vesicle trafficking to the UPR and ER homeostasis.

Tsvetanova, Nikoleta G.; Riordan, Daniel P.; Brown, Patrick O.

2012-01-01

136

The novel MMS-inducible gene Mif1/KIAA0025 is a target of the unfolded protein response pathway.  

PubMed

In a search for genes induced by DNA-damaging agents, we identified two genes that are activated by methyl methanesulfonate (MMS). Expression of both genes is regulated after endoplasmic reticulum (ER) stress via the unfolded protein response (UPR) pathway. The first gene of those identified is the molecular chaperone BiP/GRP78. The second gene, Mif1, is identical to the anonymous cDNA KIAA0025. Treatment with the glycosylation inhibitor tunicamycin both enhances the synthesis of Mif1 mRNA and protein. The Mif1 5' flanking region contains a functional ER stress-responsive element which is sufficient for induction by tunicamycin. MMS, on the other hand, activates Mif1 via an UPR-independent pathway. The gene encodes a 52 kDa protein with homology to the human DNA repair protein HHR23A and contains an ubiquitin-like domain. Overexpressed Mif1 protein is localized in the ER. PMID:10708769

van Laar, T; Schouten, T; Hoogervorst, E; van Eck, M; van der Eb, A J; Terleth, C

2000-03-01

137

Endoplasmic Reticulum Protein BI-1 Modulates Unfolded Protein Response Signaling and Protects Against Stroke and Traumatic Brain Injury  

PubMed Central

Bax-Inhibitor-1 (BI-1) is an evolutionarily conserved cytoprotective protein that resides in membranes of the endoplasmic reticulum (ER). BI-1’s cytoprotective activity is manifested in the context of ER stress, with previous studies showing that BI-1 modulates several ER-associated functions, including Unfolded Protein Response (UPR) signaling. Here we investigated the role of BI-1 in neuroprotection by generating transgenic mice in which BI-1 was constitutively expressed from a neuronal-specific promoter. Cultured primary cortical neurons from BI-1 transgenic mouse embryos exhibited greater resistance to cell death induced by agents known to cause ER stress compared to their non-transgenic counterparts. While brain morphology and vasculature of BI-1 mice appeared to be unchanged from normal non-transgenic mice, BI-1 transgenic mice showed reduced brain/lesion volumes and better performance in motoric tests, compared with non-transgenic littermates, in two models of acute brain injury – stroke caused by middle cerebral artery occlusion (MCAO) and traumatic brain injury (TBI) caused by controlled cortical impact. Furthermore, brain tissue from BI-1 transgenic mice showed reduced levels of apoptotic cells and reduced induction of markers of ER stress after brain injury, including CHOP protein expression. In summary, our findings demonstrate that enforced neuronal expression of BI-1 reduces ER stress and provides protection from acute brain injury, suggesting that strategies for enhancing BI-1 expression or activity should be considered for development of new therapies for counteracting the consequences of stroke and acute brain trauma.

Krajewska, Maryla; Xu, Lucy; Xu, Wenjie; Krajewski, Stan; Kress, Christina L.; Cui, Janice; Yang, Li; Irie, Fumitoshi; Yamaguchi, Yu; Lipton, Stuart A.; Reed, John C.

2010-01-01

138

Loss of Subcellular Lipid Transport Due to ARV1 Deficiency Disrupts Organelle Homeostasis and Activates the Unfolded Protein Response*  

PubMed Central

The ARV1-encoded protein mediates sterol transport from the endoplasmic reticulum (ER) to the plasma membrane. Yeast ARV1 mutants accumulate multiple lipids in the ER and are sensitive to pharmacological modulators of both sterol and sphingolipid metabolism. Using fluorescent and electron microscopy, we demonstrate sterol accumulation, subcellular membrane expansion, elevated lipid droplet formation, and vacuolar fragmentation in ARV1 mutants. Motif-based regression analysis of ARV1 deletion transcription profiles indicates activation of Hac1p, an integral component of the unfolded protein response (UPR). Accordingly, we show constitutive splicing of HAC1 transcripts, induction of a UPR reporter, and elevated expression of UPR targets in ARV1 mutants. IRE1, encoding the unfolded protein sensor in the ER lumen, exhibits a lethal genetic interaction with ARV1, indicating a viability requirement for the UPR in cells lacking ARV1. Surprisingly, ARV1 mutants expressing a variant of Ire1p defective in sensing unfolded proteins are viable. Moreover, these strains also exhibit constitutive HAC1 splicing that interacts with DTT-mediated perturbation of protein folding. These data suggest that a component of UPR induction in arv1? strains is distinct from protein misfolding. Decreased ARV1 expression in murine macrophages also results in UPR induction, particularly up-regulation of activating transcription factor-4, CHOP (C/EBP homologous protein), and apoptosis. Cholesterol loading or inhibition of cholesterol esterification further elevated CHOP expression in ARV1 knockdown cells. Thus, loss or down-regulation of ARV1 disturbs membrane and lipid homeostasis, resulting in a disruption of ER integrity, one consequence of which is induction of the UPR.

Shechtman, Caryn F.; Henneberry, Annette L.; Seimon, Tracie A.; Tinkelenberg, Arthur H.; Wilcox, Lisa J.; Lee, Eunjee; Fazlollahi, Mina; Munkacsi, Andrew B.; Bussemaker, Harmen J.; Tabas, Ira; Sturley, Stephen L.

2011-01-01

139

Wolfram Syndrome protein, Miner1, regulates sulphydryl redox status, the unfolded protein response, and Ca2+ homeostasis  

PubMed Central

Miner1 is a redox-active 2Fe2S cluster protein. Mutations in Miner1 result in Wolfram Syndrome, a metabolic disease associated with diabetes, blindness, deafness, and a shortened lifespan. Embryonic fibroblasts from Miner1?/? mice displayed ER stress and showed hallmarks of the unfolded protein response. In addition, loss of Miner1 caused a depletion of ER Ca2+ stores, a dramatic increase in mitochondrial Ca2+ load, increased reactive oxygen and nitrogen species, an increase in the GSSG/GSH and NAD+/NADH ratios, and an increase in the ADP/ATP ratio consistent with enhanced ATP utilization. Furthermore, mitochondria in fibroblasts lacking Miner1 displayed ultrastructural alterations, such as increased cristae density and punctate morphology, and an increase in O2 consumption. Treatment with the sulphydryl anti-oxidant N-acetylcysteine reversed the abnormalities in the Miner1 deficient cells, suggesting that sulphydryl reducing agents should be explored as a treatment for this rare genetic disease.

Wiley, Sandra E; Andreyev, Alexander Y; Divakaruni, Ajit S; Karisch, Robert; Perkins, Guy; Wall, Estelle A; van der Geer, Peter; Chen, Yi-Fan; Tsai, Ting-Fen; Simon, Melvin I; Neel, Benjamin G; Dixon, Jack E; Murphy, Anne N

2013-01-01

140

Antigen Retrieval Causes Protein Unfolding  

PubMed Central

Antigen retrieval (AR), in which formalin-fixed paraffin-embedded tissue sections are briefly heated in buffers at high temperature, often greatly improves immunohistochemical staining. An important unresolved question regarding AR is how formalin treatment affects the conformation of protein epitopes and how heating unmasks these epitopes for subsequent antibody binding. The objective of the current study was to use model proteins to determine the effect of formalin treatment on protein conformation and thermal stability in relation to the mechanism of AR. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to identify the presence of protein formaldehyde cross-links, and circular dichroism spectropolarimetry was used to determine the effect of formalin treatment and high-temperature incubation on the secondary and tertiary structure of the model proteins. Results revealed that for some proteins, formalin treatment left the native protein conformation unaltered, whereas for others, formalin denatured tertiary structure, yielding a molten globule protein. In either case, heating to temperatures used in AR methods led to irreversible protein unfolding, which supports a linear epitope model of recovered protein immunoreactivity. Consequently, the core mechanism of AR likely centers on the restoration of normal protein chemical composition coupled with improved accessibility to linear epitopes through protein unfolding.

Fowler, Carol B.; Evers, David L.; O'Leary, Timothy J.; Mason, Jeffrey T.

2011-01-01

141

Protein Oxidation Triggers the Unfolded Protein Response and Neuronal Injury in Chemically Induced Parkinson Disease  

Microsoft Academic Search

The recent identification of genetic mutations linked to Parkinson’s disease (PD), such as ?-synuclein, parkin, and LRRK2,\\u000a has highlighted the role of aberrant protein handling and degradation in this disorder. Moreover, a growing body of data suggests\\u000a that environmental toxins that mimic PD also exhibit faulty protein handling, providing a mechanistic link between toxicity\\u000a and the identified PD mutations. In

Alison I. Bernstein; Karen L. O’Malley

142

Yeast Bax Inhibitor, Bxi1p, Is an ER-Localized Protein That Links the Unfolded Protein Response and Programmed Cell Death in Saccharomyces cerevisiae  

PubMed Central

Bax inhibitor-1 (BI-1) is an anti-apoptotic gene whose expression is upregulated in a wide range of human cancers. Studies in both mammalian and plant cells suggest that the BI-1 protein resides in the endoplasmic reticulum and is involved in the unfolded protein response (UPR) that is triggered by ER stress. It is thought to act via a mechanism involving altered calcium dynamics. In this paper, we provide evidence that the Saccharomyces cerevisiae protein encoded by the open reading frame, YNL305C, is a bona fide homolog for BI-1. First, we confirm that yeast cells from two different strain backgrounds lacking YNL305C, which we have renamed BXI1, are more sensitive to heat-shock induced cell death than wildtype controls even though they have indistinguishable growth rates at 30°C. They are also more susceptible both to ethanol-induced and to glucose-induced programmed cell death. Significantly, we show that Bxi1p-GFP colocalizes with the ER localized protein Sec63p-RFP. We have also discovered that ?bxi1 cells are not only more sensitive to drugs that induce ER stress, but also have a decreased unfolded protein response as measured with a UPRE-lacZ reporter. Finally, we have discovered that deleting BXI1 diminishes the calcium signaling response in response to the accumulation of unfolded proteins in the ER as measured by a calcineurin-dependent CDRE-lacZ reporter. In toto, our data suggests that the Bxi1p, like its metazoan homologs, is an ER-localized protein that links the unfolded protein response and programmed cell death.

Cascio, Vincent; Austriaco, Nicanor

2011-01-01

143

Myc-Driven Overgrowth Requires Unfolded Protein Response-Mediated Induction of Autophagy and Antioxidant Responses in Drosophila melanogaster  

PubMed Central

Autophagy, a lysosomal self-degradation and recycling pathway, plays dual roles in tumorigenesis. Autophagy deficiency predisposes to cancer, at least in part, through accumulation of the selective autophagy cargo p62, leading to activation of antioxidant responses and tumor formation. While cell growth and autophagy are inversely regulated in most cells, elevated levels of autophagy are observed in many established tumors, presumably mediating survival of cancer cells. Still, the relationship of autophagy and oncogenic signaling is poorly characterized. Here we show that the evolutionarily conserved transcription factor Myc (dm), a proto-oncogene involved in cell growth and proliferation, is also a physiological regulator of autophagy in Drosophila melanogaster. Loss of Myc activity in null mutants or in somatic clones of cells inhibits autophagy. Forced expression of Myc results in cell-autonomous increases in cell growth, autophagy induction, and p62 (Ref2P)-mediated activation of Nrf2 (cnc), a transcription factor promoting antioxidant responses. Mechanistically, Myc overexpression increases unfolded protein response (UPR), which leads to PERK-dependent autophagy induction and may be responsible for p62 accumulation. Genetic or pharmacological inhibition of UPR, autophagy or p62/Nrf2 signaling prevents Myc-induced overgrowth, while these pathways are dispensable for proper growth of control cells. In addition, we show that the autophagy and antioxidant pathways are required in parallel for excess cell growth driven by Myc. Deregulated expression of Myc drives tumor progression in most human cancers, and UPR and autophagy have been implicated in the survival of Myc-dependent cancer cells. Our data obtained in a complete animal show that UPR, autophagy and p62/Nrf2 signaling are required for Myc-dependent cell growth. These novel results give additional support for finding future approaches to specifically inhibit the growth of cancer cells addicted to oncogenic Myc.

Nagy, Peter; Varga, Agnes; Pircs, Karolina; Hegedus, Krisztina; Juhasz, Gabor

2013-01-01

144

Activation of the unfolded protein response pathway causes ceramide accumulation in yeast and INS-1E insulinoma cells.  

PubMed

Sphingolipids are not only important components of membranes but also have functions in protein trafficking and intracellular signaling. The LCB1 gene encodes a subunit of the serine palmitoyltransferase, which is responsible for the first step of sphingolipid synthesis. Here, we show that activation of the unfolded protein response (UPR) can restore normal ceramide levels and viability in yeast cells with a conditional defect in LCB1. Dependence on UPR was demonstrated by showing the HAC1-dependence of the suppression. A similar induction of ceramides by UPR seems to take place in mammalian cells. In rat pancreatic INS-1E cells, UPR activation induces the transcription of the CerS6 gene, which encodes a ceramide synthase. This correlates with the specific accumulation of ceramide with a C16 fatty acyl chain upon UPR activation. Therefore, our study reveals a novel connection between UPR induction and ceramide synthesis that seems to be conserved between yeast and mammalian cells. PMID:22210926

Epstein, Sharon; Kirkpatrick, Clare L; Castillon, Guillaume A; Muñiz, Manuel; Riezman, Isabelle; David, Fabrice P A; Wollheim, Claes B; Riezman, Howard

2012-03-01

145

Residual structure in unfolded proteins  

PubMed Central

The denatured state ensemble (DSE) of unfolded proteins, once considered to be well-modeled by an energetically featureless random coil, is now well-known to contain flickering elements of residual structure. The position and nature of DSE residual structure may provide clues toward deciphering the protein folding code. This review focuses on recent advances in our understanding of the nature of DSE collapse under folding conditions, the quantification of the stability of residual structure in the DSE, the determination of the location and types of residues involved in thermodynamically significant residual structure and advances in detection of long-range interactions in the DSE.

Bowler, Bruce E.

2012-01-01

146

Disease-related amyloidogenic variants of human lysozyme trigger the unfolded protein response and disturb eye development in Drosophila melanogaster  

PubMed Central

We have created a Drosophila model of lysozyme amyloidosis to investigate the in vivo behavior of disease-associated variants. To achieve this objective, wild-type (WT) protein and the amyloidogenic variants F57I and D67H were expressed in Drosophila melanogaster using the UAS-gal4 system and both the ubiquitous and retinal expression drivers Act5C-gal4 and gmr-gal4. The nontransgenic w1118 Drosophila line was used as a control throughout. We utilized ELISA experiments to probe lysozyme protein levels, scanning electron microscopy for eye phenotype classification, and immunohistochemistry to detect the unfolded protein response (UPR) activation. We observed that expressing the destabilized F57I and D67H lysozymes triggers UPR activation, resulting in degradation of these variants, whereas the WT lysozyme is secreted into the fly hemolymph. Indeed, the level of WT was up to 17 times more abundant than the variant proteins. In addition, the F57I variant gave rise to a significant disruption of the eye development, and this correlated to pronounced UPR activation. These results support the concept that the onset of familial amyloid disease is linked to an inability of the UPR to degrade completely the amyloidogenic lysozymes prior to secretion, resulting in secretion of these destabilized variants, thereby leading to deposition and associated organ damage.—Kumita, J. R., Helmfors, L., Williams, J., Luheshi, L. M., Menzer, L., Dumoulin, M., Lomas, D. A., Crowther, D. C., Dobson, C. M., Brorsson, A.-C. Disease-related amyloidogenic variants of human lysozyme trigger the unfolded protein response and disturb eye development in Drosophila melanogaster.

Kumita, Janet R.; Helmfors, Linda; Williams, Jocy; Luheshi, Leila M.; Menzer, Linda; Dumoulin, Mireille; Lomas, David A.; Crowther, Damian C.; Dobson, Christopher M.; Brorsson, Ann-Christin

2012-01-01

147

The unfolded protein response mediates adaptation to exercise in skeletal muscle through a PGC-1? /ATF6? complex  

PubMed Central

SUMMARY Exercise has been shown to be effective for treating obesity and type 2 diabetes. However, the molecular mechanisms for adaptation to exercise training are not fully understood. Endoplasmic reticulum (ER) stress has been linked to metabolic dysfunction. Here we show that the unfolded protein response (UPR), an adaptive response pathway that maintains ER homeostasis upon luminal stress, is activated in skeletal muscle during exercise and adapts skeletal muscle to exercise training. The transcriptional coactivator PGC-1?, which regulates several exercise-associated aspects of skeletal muscle function, mediates the UPR in myotubes and skeletal muscle through coactivation of ATF6?. Efficient recovery from acute exercise is compromised in ATF6??/? mice. Blocking ER-stress related cell death via deletion of CHOP partially rescues the exercise intolerance phenotype in muscle-specific PGC-1? KO mice. These findings suggest that modulation of the UPR through PGC1? represents an alternative avenue to improve skeletal muscle function and achieve metabolic benefits.

Wu, Jun; Ruas, Jorge L.; Estall, Jennifer L.; Rasbach, Kyle A.; Choi, Jang Hyun; Ye, Li; Bostrom, Pontus; Tyra, Heather M.; Crawford, Robert W.; Campbell, Kevin P.; Rutkowski, D. Thomas; Kaufman, Randal J.; Spiegelman, Bruce M.

2011-01-01

148

Inhibition of the unfolded protein response by ricin a-chain enhances its cytotoxicity in mammalian cells.  

PubMed

Ricin is a highly toxic type II ribosome-inactivating protein that has potential as a biochemical weapon and as the toxic component of immunotoxins. The unfolded protein response (UPR) is a survival response that helps cells to recover from endoplasmic reticulum (ER) stress. Failure to recover from ER stress leads to apoptosis. In yeast, ricin-A-chain (RTA), the enzymatic component of ricin, inhibits UPR. Our goals were to determine if RTA inhibits UPR in two epithelial cell lines and if this affects RTA cytotoxicity. RTA alone did not induce UPR. However, RTA inhibited both phosphorylation of inositol-requiring enzyme 1 (IRE1) and splicing of X-box binding protein1 mRNA by the UPR-inducing agent tunicamycin (Tm). The ability of dithiothreitol (DTT) to activate eukaryotic translation initiation factor 2 alpha (eIF2?), a component of the PERK pathway, was also inhibited by RTA. Treatment with RTA in combination with Tm or DTT inhibited protein synthesis more than either agent did alone in one cell line, while caspase cleavage was enhanced by the treatment combination in both cell lines. These data indicate that RTA is more cytotoxic when UPR is inhibited. This ability to inhibit UPR may enhance the potential of RTA as a therapeutic immunotoxin in solid tumors. PMID:22069719

Wang, Chao-Ting; Jetzt, Amanda E; Cheng, Ju-Shun; Cohick, Wendie S

2011-05-01

149

Disruption of Crosstalk Between the Fatty Acid Synthesis and Proteasome Pathways Enhances Unfolded Protein Response Signaling and Cell Death  

PubMed Central

Fatty Acid Synthase (FASN) is the terminal enzyme responsible for fatty acid synthesis and is upregulated in tumors of various origins to facilitate their growth and progression. Because of several reports linking the fatty acid synthase and proteasome pathways, we asked whether FASN inhibitors could combine with bortezomib, the FDA-approved proteasome inhibitor, to amplify cell death. Indeed, bortezomib treatment augmented sub-optimal FASN inhibitor concentrations to reduce clonogenic survival, which was paralleled by an increase in apoptotic markers. Interestingly, FASN inhibitors induced accumulation of ubiquinated proteins and enhanced the effects of bortezomib treatment. In turn, bortezomib increased fatty acid synthesis, suggesting crosstalk between the pathways. We hypothesized that cell death resulting from crosstalk perturbation was mediated by increased unfolded protein response (UPR) signaling. Indeed, disruption of crosstalk activated and saturated the adaptation arm of UPR signaling, including eIF2? phosphorylation, activating transcription factor 4 (ATF4) expression, and X-box binding protein 1 (XBP-1) splicing. Furthermore, while single agents did not activate the alarm phase of the UPR, crosstalk interruption resulted in activated JNK and C-EBP homologous protein (CHOP)-dependent cell death. Combined, the data support the concept that the UPR balance between adaptive to stress signaling can be exploited to mediate increased cell death and suggests novel applications of FASN inhibitors for clinical use.

Little, Joy L.; Wheeler, Frances B.; Koumenis, Constantinos; Kridel, Steven J.

2008-01-01

150

Oxidative stress-triggered unfolded protein response is upstream of intrinsic cell death evoked by parkinsonian mimetics.  

PubMed

Oxidative stress is a key player in a variety of neurodegenerative disorders including Parkinson's disease. Widely used as a parkinsonian mimetic, 6-hydroxydopamine (6-OHDA) generates reactive oxygen species (ROS) as well as coordinated changes in gene transcription associated with the unfolded protein response (UPR) and apoptosis. Whether 6-OHDA-induced UPR activation is dependent on ROS has not yet been determined. The present study used molecular indicators of oxidative stress to place 6-OHDA-generated ROS upstream of the appearance of UPR markers such as activating transcription factor 3 (ATF3) and phosphorylated stress-activated protein kinase (SAPK/JNK) signaling molecules. Antioxidants completely blocked 6-OHDA-mediated UPR activation and rescued cells from toxicity. Moreover, cytochrome c release from mitochondria was observed after the appearance of early UPR markers, suggesting that cellular stress pathways are responsible for its release. Mechanistically, the 6-OHDA-induced UPR was independent of intracellular calcium changes. Rather, evidence of protein oxidation was observed before the expression of UPR markers, suggesting that the rapid accumulation of damaged proteins triggered cell stress/UPR. Taken together, 6-OHDA-mediated cell death in dopaminergic cells proceeds via ROS-dependent UPR up-regulation which leads to an interaction with the intrinsic mitochondrial pathway and downstream caspase activation. PMID:16987235

Holtz, William A; Turetzky, Jay M; Jong, Yuh-Jiin I; O'Malley, Karen L

2006-10-01

151

Computational model for protein unfolding simulation  

NASA Astrophysics Data System (ADS)

The protein folding problem is one of the fundamental and important questions in molecular biology. However, the all-atom molecular dynamics studies of protein folding and unfolding are still computationally expensive and severely limited by the time scale of simulation. In this paper, a simple and fast protein unfolding method is proposed based on the conformational stability analyses and structure modeling. In this method, two structure-based conditions are considered to identify the unstable regions of proteins during the unfolding processes. The protein unfolding trajectories are mimicked through iterative structure modeling according to conformational stability analyses. Two proteins, chymotrypsin inhibitor 2 (CI2) and ? -spectrin SH3 domain (SH3) were simulated by this method. Their unfolding pathways are consistent with the previous molecular dynamics simulations. Furthermore, the transition states of the two proteins were identified in unfolding processes and the theoretical ? values of these transition states showed significant correlations with the experimental data (the correlation coefficients are >0.8). The results indicate that this method is effective in studying protein unfolding. Moreover, we analyzed and discussed the influence of parameters on the unfolding simulation. This simple coarse-grained model may provide a general and fast approach for the mechanism studies of protein folding.

Tian, Xu-Hong; Zheng, Ye-Han; Jiao, Xiong; Liu, Cai-Xing; Chang, Shan

2011-06-01

152

A mathematical model of the unfolded protein stress response reveals the decision mechanism for recovery, adaptation and apoptosis  

PubMed Central

Background The unfolded protein response (UPR) is a major signalling cascade acting in the quality control of protein folding in the endoplasmic reticulum (ER). The cascade is known to play an accessory role in a range of genetic and environmental disorders including neurodegenerative and cardiovascular diseases, diabetes and kidney diseases. The three major receptors of the ER stress involved with the UPR, i.e. IRE1 ?, PERK and ATF6, signal through a complex web of pathways to convey an appropriate response. The emerging behaviour ranges from adaptive to maladaptive depending on the severity of unfolded protein accumulation in the ER; however, the decision mechanism for the switch and its timing have so far been poorly understood. Results Here, we propose a mechanism by which the UPR outcome switches between survival and death. We compose a mathematical model integrating the three signalling branches, and perform a comprehensive bifurcation analysis to investigate possible responses to stimuli. The analysis reveals three distinct states of behaviour, low, high and intermediate activity, associated with stress adaptation, tolerance, and the initiation of apoptosis. The decision to adapt or destruct can, therefore, be understood as a dynamic process where the balance between the stress and the folding capacity of the ER plays a pivotal role in managing the delivery of the most appropriate response. The model demonstrates for the first time that the UPR is capable of generating oscillations in translation attenuation and the apoptotic signals, and this is supplemented with a Bayesian sensitivity analysis identifying a set of parameters controlling this behaviour. Conclusions This work contributes largely to the understanding of one of the most ubiquitous signalling pathways involved in protein folding quality control in the metazoan ER. The insights gained have direct consequences on the management of many UPR-related diseases, revealing, in addition, an extended list of candidate disease modifiers. Demonstration of stress adaptation sheds light to how preconditioning might be beneficial in manifesting the UPR outcome to prevent untimely apoptosis, and paves the way to novel approaches for the treatment of many UPR-related conditions.

2013-01-01

153

Compound C Prevents the Unfolded Protein Response during Glucose Deprivation through a Mechanism Independent of AMPK and BMP Signaling  

PubMed Central

Inhibiting the unfolded protein response (UPR) can be a therapeutic approach, especially for targeting the tumor microenvironment. Here, we show that compound C (also known as dorsomorphin), a small-molecule inhibitor of AMP-activated protein kinase (AMPK) and bone morphogenetic protein (BMP) signaling, inhibit the UPR-induced transcription program depending on the glucose deprivation conditions. We found that compound C prevented UPR marker glucose-regulated protein 78 (GRP78) accumulation and exerted enhanced cytotoxicity during glucose deprivation. Gene expression profiling, together with biochemical analysis, revealed that compound C had a unique mode of action to suppress the transcriptional activation of UPR-targeted genes, as compared with the classic UPR inhibitors versipelostatin and biguanides. Surprisingly, the UPR-inhibiting activity of compound C was not associated with either AMPK or BMP signaling inhibition. We further found that combination treatments of compound C and the classic UPR inhibitors resulted in synergistic cell death with UPR suppression during glucose deprivation. Our findings demonstrate that compound C could be a unique tool for developing a UPR-targeted antitumor therapy.

Saito, Sakae; Furuno, Aki; Sakurai, Junko; Park, Hae-Ryong; Shin-ya, Kazuo; Tomida, Akihiro

2012-01-01

154

Activation of the unfolded protein response is associated with impaired granulopoiesis in transgenic mice expressing mutant Elane  

PubMed Central

Severe congenital neutropenia (SCN) is an inborn disorder of granulopoiesis that in many cases is caused by mutations of the ELANE gene, which encodes neutrophil elastase (NE). Recent data suggest a model in which ELANE mutations result in NE protein misfolding, induction of endoplasmic reticulum (ER) stress, activation of the unfolded protein response (UPR), and ultimately a block in granulocytic differentiation. To test this model, we generated transgenic mice carrying a targeted mutation of Elane (G193X) reproducing a mutation found in SCN. The G193X Elane allele produces a truncated NE protein that is rapidly degraded. Granulocytic precursors from G193X Elane mice, though without significant basal UPR activation, are sensitive to chemical induction of ER stress. Basal and stress granulopoiesis after myeloablative therapy are normal in these mice. Moreover, inaction of protein kinase RNA-like ER kinase (Perk), one of the major sensors of ER stress, either alone or in combination with G193X Elane, had no effect on basal granulopoiesis. However, inhibition of the ER-associated degradation (ERAD) pathway using a proteosome inhibitor resulted in marked neutropenia in G193X Elane. The selective sensitivity of G913X Elane granulocytic cells to ER stress provides new and strong support for the UPR model of disease patho-genesis in SCN.

Nanua, Suparna; Murakami, Mark; Xia, Jun; Grenda, David S.; Woloszynek, Jill; Strand, Marie

2011-01-01

155

The critical roles of endoplasmic reticulum chaperones and unfolded protein response in tumorigenesis and anti-cancer therapies  

PubMed Central

Cancer progression is characterized by rapidly proliferating cancer cells that are in need of increased protein synthesis. Therefore, enhanced endoplasmic reticulum (ER) activity is required to facilitate the folding, assembly and transportation of membrane and secretory proteins. These functions are carried out by ER chaperones. It is now becoming clear that the ER chaperones have critical functions outside of simply facilitating protein folding. For example, cancer progression requires GRP78 for cancer cell survival and proliferation, as well as angiogenesis in the microenvironment. GRP78 can translocate to the cell surface acting as a receptor regulating oncogenic signaling and cell viability. Calreticulin, another ER chaperone, can translocate to the cell surface of apoptotic cancer cells and induce immunogenic cancer cell death and antitumor responses in vivo. Tumor-secreted GRP94 has been shown to elicit antitumor immune responses when used as antitumor vaccines. Protein disulfide isomerase is another ER chaperone that demonstrates pro-oncogenic and pro-survival functions. Due to intrinsic alterations of cellular metabolism and extrinsic factors in the tumor microenvironment, cancer cells are under ER stress, and they respond to this stress by activating the unfolded protein response (UPR). Depending on the severity and duration of ER stress, the signaling branches of the UPR can activate adaptive and pro-survival signals, or induce apoptotic cell death. The PERK signaling branch of the UPR has a dual role in cancer proliferation and survival, and is also required for ER stress-induced autophagy. The activation of the IRE1? branch promotes tumorigenesis, cancer cell survival, and regulates tumor invasion. In summary, perturbance of ER homeostasis plays critical roles in tumorigenesis, and therapeutic modulation of ER chaperones and/or UPR components presents potential antitumor treatments.

Luo, Biquan; Lee, Amy S.

2013-01-01

156

The Specialized Unfolded Protein Response of B Lymphocytes: ATF6?-Independent Development of Antibody-Secreting B cells  

PubMed Central

B lymphocytes, like all mammalian cells, are equipped with the unfolded protein response (UPR), a complex signaling system allowing for both pro- and mal-adaptive responses to increased demands on the endoplasmic reticulum (ER). The UPR is comprised of three signaling pathways initiated by the ER transmembrane stress sensors, IRE1?/?, PERK and ATF6?/?. Activation of IRE1 yields XBP1(S), a transcription factor that directs expansion of the ER and enhances protein biosynthetic and secretory machinery. XBP1(S) is essential for the differentiation of B lymphocytes into antibody-secreting cells. In contrast, the PERK pathway, a regulator of translation and transcription, is dispensable for the generation of antibody-secreting cells. Functioning as a transcription factor, ATF6? can augment ER quality control processes and drive ER expansion, but the potential role of this UPR pathway in activated B cells has not been investigated. Here, we report studies of ATF6?-deficient B cells demonstrating that ATF6? is not required for the development of antibody-secreting cells. Thus, when B cells are stimulated to secrete antibody, a specialized UPR relies exclusively on the IRE1-XBP1 pathway to remodel the ER and expand cellular secretory capacity.

Aragon, Ileana V.; Barrington, Robert A.; Jackowski, Suzanne; Mori, Kazutoshi; Brewer, Joseph W.

2012-01-01

157

Sterol metabolism regulates neuroserpin polymer degradation in the absence of the unfolded protein response in the dementia FENIB  

PubMed Central

Mutants of neuroserpin are retained as polymers within the endoplasmic reticulum (ER) of neurones to cause the autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. The cellular consequences are unusual in that the ordered polymers activate the ER overload response (EOR) in the absence of the canonical unfolded protein response. We use both cell lines and Drosophila models to show that the G392E mutant of neuroserpin that forms polymers is degraded by UBE2j1 E2 ligase and Hrd1 E3 ligase while truncated neuroserpin, a protein that lacks 132 amino acids, is degraded by UBE2g2 (E2) and gp78 (E3) ligases. The degradation of G392E neuroserpin results from SREBP-dependent activation of the cholesterol biosynthetic pathway in cells that express polymers of neuroserpin (G392E). Inhibition of HMGCoA reductase, the limiting enzyme of the cholesterol biosynthetic pathway, reduced the ubiquitination of G392E neuroserpin in our cell lines and increased the retention of neuroserpin polymers in both HeLa cells and primary neurones. Our data reveal a reciprocal relationship between cholesterol biosynthesis and the clearance of mutant neuroserpin. This represents the first description of a link between sterol metabolism and modulation of the proteotoxicity mediated by the EOR.

Roussel, Benoit D.; Newton, Timothy M.; Malzer, Elke; Simecek, Nikol; Haq, Imran; Thomas, Sally E.; Burr, Marian L.; Lehner, Paul J.; Crowther, Damian C.; Marciniak, Stefan J.; Lomas, David A.

2013-01-01

158

Gamma-tocotrienol induced apoptosis is associated with unfolded protein response in human breast cancer cells.  

PubMed

Gamma-tocotrienol (?-T3) is a member of the vitamin E family. Tocotrienols (T3s) are powerful antioxidants and possess anticancer, neuroprotective and cholesterol-lowering properties. Tocotrienols inhibit the growth of various cancer cell lines without affecting normal cells. Less is known about the exact mechanisms of action of T3s on cell death and other growth inhibitory pathways. In the present study, we demonstrate that ?-T3 induces apoptosis in MDA-MB 231 and MCF-7 breast cancer cells as evident by PARP cleavage and caspase-7 activation. Gene expression analysis of MCF-7 cells treated with ?-T3 revealed alterations in the expression of multiple genes involved in cell growth and proliferation, cell death, cell cycle, cellular development, cellular movement and gene expression. Further analysis of differentially modulated genes using Ingenuity Pathway Analysis software suggested modulation of canonical signal transduction or metabolic pathways such as NRF-2-mediated oxidative stress response, TGF-? signaling and endoplasmic reticulum (ER) stress response. Analysis of ER-stress-related proteins in MCF-7 and MDA-MB 231 cells treated with ?-T3 demonstrated activation of PERK and pIRE1? pathway to induce ER stress. Activating transcription factor 3 (ATF3) was identified as the most up-regulated gene (16.8-fold) in response to ?-T3. Activating transcription factor 3 knockdown using siRNA suggested an essential role of ATF3 in ?-T3-induced apoptosis. In summary, we demonstrate that ?-T3 modulates ER stress signaling and have identified ATF3 as a molecular target for ?-T3 in breast cancer cells. PMID:21429729

Patacsil, Dorrelyn; Tran, Anh Thu; Cho, Youn Sook; Suy, Simeng; Saenz, Francisco; Malyukova, Irina; Ressom, Habtom; Collins, Sean P; Clarke, Robert; Kumar, Deepak

2012-01-01

159

The unfolded protein response in a pair of sensory neurons promotes entry of C. elegans into dauer diapause.  

PubMed

In response to unfavorable environmental conditions such as starvation, crowding, and elevated temperature, Caenorhabditis elegans larvae enter an alternative developmental stage known as dauer, which is characterized by adaptive changes in stress resistance and metabolism. The genetic dissection of the molecular mechanisms of the C. elegans dauer developmental decision has defined evolutionarily conserved signaling pathways of organismal neuroendocrine physiology. Here, we have identified a mechanism by which a dominant mutation in a neuronal insulin gene, daf-28(sa191), causes constitutive entry into dauer diapause. We demonstrate that expression of the mutant DAF-28 insulin peptide results in endoplasmic reticulum (ER) stress in the ASI pair of chemosensory neurons. The neuronal ER stress does not compromise cellular survival but activates PEK-1, the C. elegans ortholog of the mammalian eIF2? kinase PERK, which in turn phosphorylates Ser49 of eIF2?, specifically in the ASI neuron pair, to promote entry into dauer diapause. Our data establish a novel role for ER stress and the unfolded protein response (UPR) in promoting entry into dauer diapause and suggest that, in addition to cell-autonomous activities in the maintenance of ER homeostasis, the UPR may act in a non-cell-autonomous manner to promote organismal adaptation to stress during larval development. PMID:24316205

Kulalert, Warakorn; Kim, Dennis H

2013-12-16

160

Unfolded protein response signaling by transcription factor XBP-1 regulates ADAM10 and is affected in Alzheimer's disease.  

PubMed

In Alzheimer's disease (AD), disturbed homeostasis of the proteases competing for amyloid precursor protein processing has been reported: a disintegrin and metalloproteinase 10 (ADAM10), the physiological ?-secretase, is decreased in favor of the amyloid-?-generating enzyme BACE-1. To identify transcription factors that modulate the expression of either protease, we performed a screening approach: 48 transcription factors significantly interfered with ADAM10/BACE-1-promoter activity. One selective inducer of ADAM10 gene expression is the X-box binding protein-1 (XBP-1). This protein regulates the unfolded protein-response pathway. We demonstrate that particularly the spliced XBP-1 variant dose dependently regulates ADAM10 expression, which can be synergistically enhanced by 100 nM insulin. Analysis of 2 different transgenic mouse models (APP/PS1 and 5xFAD) revealed that at early time points in pathology XBP-1 metabolism is induced. This is accompanied by a 2-fold augmented ADAM10 amount as compared with nontransgenic littermates (P=0.011). Along with aging of the mice, the system is counterregulated, and XBP-1 together with ADAM10 expression level decreased to ?50% as compared with control animals. Analyses of expression levels in human AD brains showed that ADAM10 mRNA correlated with active XBP-1 (r=0.3120), but expression did not reach levels of healthy age-matched controls, suggesting deregulation of XBP-1 signaling. Our results demonstrate that XBP-1 is a driver of ADAM10 gene expression and that disturbance of this pathway might contribute to development or progression of AD. PMID:24165480

Reinhardt, Sven; Schuck, Florian; Grösgen, Sven; Riemenschneider, Matthias; Hartmann, Tobias; Postina, Rolf; Grimm, Marcus; Endres, Kristina

2014-02-01

161

Cavities determine the pressure unfolding of proteins  

PubMed Central

It has been known for nearly 100 years that pressure unfolds proteins, yet the physical basis of this effect is not understood. Unfolding by pressure implies that the molar volume of the unfolded state of a protein is smaller than that of the folded state. This decrease in volume has been proposed to arise from differences between the density of bulk water and water associated with the protein, from pressure-dependent changes in the structure of bulk water, from the loss of internal cavities in the folded states of proteins, or from some combination of these three factors. Here, using 10 cavity-containing variants of staphylococcal nuclease, we demonstrate that pressure unfolds proteins primarily as a result of cavities that are present in the folded state and absent in the unfolded one. High-pressure NMR spectroscopy and simulations constrained by the NMR data were used to describe structural and energetic details of the folding landscape of staphylococcal nuclease that are usually inaccessible with existing experimental approaches using harsher denaturants. Besides solving a 100-year-old conundrum concerning the detailed structural origins of pressure unfolding of proteins, these studies illustrate the promise of pressure perturbation as a unique tool for examining the roles of packing, conformational fluctuations, and water penetration as determinants of solution properties of proteins, and for detecting folding intermediates and other structural details of protein-folding landscapes that are invisible to standard experimental approaches.

Roche, Julien; Caro, Jose A.; Norberto, Douglas R.; Barthe, Philippe; Roumestand, Christian; Schlessman, Jamie L.; Garcia, Angel E.; Garcia-Moreno E., Bertrand; Royer, Catherine A.

2012-01-01

162

The Unfolded Protein Response Induces the Angiogenic Switch in Human Tumor Cells through the PERK/ATF4 Pathway  

PubMed Central

Neovascularization is a limiting factor in tumor growth and progression. It is well known that changes in the tumor microenvironment, such as hypoxia and glucose deprivation (GD), can induce VEGF production. However, the mechanism linking GD to tumor growth and angiogenesis is unclear. We hypothesize that GD induces the angiogenic switch in tumors through activation of the unfolded protein response (UPR). We report that UPR activation in human tumors results in elevated expression of proangiogenic mediators and a concomitant decrease in angiogenesis inhibitors. cDNA microarray results showed that GD-induced UPR activation promoted upregulation of a number of proangiogenic mediators (VEGF, FGF2, IL6, etc.) and downregulation of several angiogenic inhibitors (THBS1, CXCL14 and CXCL10). In vitro studies revealed that partially blocking UPR signaling by silencing PERK or ATF4 significantly reduced the production of angiogenesis mediators induced by GD. However, suppressing the alpha subunit of hypoxia-inducible factors had no effect on this process. Chromatin immunoprecipitation confirmed binding of ATF4 to a regulatory site in the VEGF gene. In vivo results confirmed that knockdown of PERK in tumor cells slows down tumor growth and decreases tumor blood vessel density. Collectively, these results demonstrate that the PERK/ATF4 arm of UPR mediates the angiogenic switch and is a potential target for antiangiogenic cancer therapy.

Wang, Yugang; Alam, Goleeta N.; Ning, Yu; Visioli, Fernanda; Dong, Zhihong; Nor, Jacques E.; Polverini, Peter J.

2013-01-01

163

Attenuation of unfolded protein response and apoptosis by mReg2 induced GRP78 in mouse insulinoma cells.  

PubMed

Murine regenerating (mReg) genes have been implicated in preserving islet cell biology. Expanding on our previous work showing that overexpression of mReg2 protects MIN6 insulinoma cells against streptozotocin-induced apoptosis, we now demonstrate that mReg2 induces glucose-regulated peptide 78 (GRP78) expression via the Akt-mTORC1 axis and protects MIN6 cells against ER stress induced by thapsigargin and glucolipotoxicity. Activation of mTORC1 activity results from both mReg2-induced increased mTOR phosphorylation as well as increased expression of Raptor and G?L. Inhibition of Akt and mTORC1 blunted the ability of mReg2 to induce GRP78 and attenuate unfolded protein response (UPR). Knockdown of GRP78 sensitized the cells overexpressing mReg2 to UPR without affecting its ability to activate Akt-mTORC1 signaling. Induced expression of mReg2 may protect insulin producing cells from ER stress in diabetes. PMID:24801175

Liu, Lu; Chowdhury, Subrata; Fang, Xin; Liu, Jun-Li; Srikant, Coimbatore B

2014-05-29

164

Ouabain targets the unfolded protein response for selective killing of HepG2 cells during glucose deprivation.  

PubMed

Ouabain is a cardiotonic steroid and specific inhibitor of the Na(+)/K(+)-ATPase. The relationship between ouabain treatment and the unfolded protein response (UPR) in cells is not precisely understood. Therefore, we studied the possible effects of ouabain on proliferation, apoptosis, and the UPR. HepG2 cells were cultured overnight and then treated with various concentrations of ouabain (0.75 to 750?nM) in the absence or presence of 10?mM 2-deoxyglucose (2-DG) for 48 hours. We also used real-time polymerase chain reaction to obtain quantitative measurements of expression levels of Grp78, Grp94, CHOP, MTJ-1, HKII, MDR-1, MRP-1, HO-1, and Par-4. Cell number, viability, and proliferation of HepG2 cells were monitored with a real-time cell analyzer system (xCELLigence). We show that ouabain modulates the UPR transcription program and induces cell death in glucose-deprived tumor cells. Ouabain at all concentrations showed no cytotoxicity whereas all concentrations were very effective under 2-DG stress conditions. Our findings show that disruption of the UPR during glucose deprivation could be an attractive approach for selective cancer cell killing and could provide a chemical basis for developing UPR-targeting drugs against solid tumors. Ouabain use as an adjunct to conventional cancer therapy also warrants vigorous investigation. PMID:22757644

Ozdemir, Tulay; Nar, Rukiye; Kilinc, Veli; Alacam, Hasan; Salis, Osman; Duzgun, Aynur; Gulten, Sedat; Bedir, Abdulkerim

2012-10-01

165

Ethanol metabolism and oxidative stress are required for unfolded protein response activation and steatosis in zebrafish with alcoholic liver disease  

PubMed Central

SUMMARY Secretory pathway dysfunction and lipid accumulation (steatosis) are the two most common responses of hepatocytes to ethanol exposure and are major factors in the pathophysiology of alcoholic liver disease (ALD). However, the mechanisms by which ethanol elicits these cellular responses are not fully understood. Recent data indicates that activation of the unfolded protein response (UPR) in response to secretory pathway dysfunction can cause steatosis. Here, we examined the relationship between alcohol metabolism, oxidative stress, secretory pathway stress and steatosis using zebrafish larvae. We found that ethanol was immediately internalized and metabolized by larvae, such that the internal ethanol concentration in 4-day-old larvae equilibrated to 160 mM after 1 hour of exposure to 350 mM ethanol, with an average ethanol metabolism rate of 56 ?mol/larva/hour over 32 hours. Blocking alcohol dehydrogenase 1 (Adh1) and cytochrome P450 2E1 (Cyp2e1), the major enzymes that metabolize ethanol, prevented alcohol-induced steatosis and reduced induction of the UPR in the liver. Thus, we conclude that ethanol metabolism causes ALD in zebrafish. Oxidative stress generated by Cyp2e1-mediated ethanol metabolism is proposed to be a major culprit in ALD pathology. We found that production of reactive oxygen species (ROS) increased in larvae exposed to ethanol, whereas inhibition of the zebrafish CYP2E1 homolog or administration of antioxidants reduced ROS levels. Importantly, these treatments also blocked ethanol-induced steatosis and reduced UPR activation, whereas hydrogen peroxide (H2O2) acted as a pro-oxidant that synergized with low doses of ethanol to induce the UPR. Collectively, these data demonstrate that ethanol metabolism and oxidative stress are conserved mechanisms required for the development of steatosis and hepatic dysfunction in ALD, and that these processes contribute to ethanol-induced UPR activation and secretory pathway stress in hepatocytes.

Tsedensodnom, Orkhontuya; Vacaru, Ana M.; Howarth, Deanna L.; Yin, Chunyue; Sadler, Kirsten C.

2013-01-01

166

The Unfolded Protein Response in Human Corneal Endothelial Cells Following Hypothermic Storage: Implications of a Novel Stress Pathway  

PubMed Central

Human corneal endothelial cells (HCEC) have become increasingly important for a range of eye disease treatment therapies. Accordingly, a more detailed understanding of the processing and preservation associated stresses experienced by corneal cells might contribute to improved therapeutic outcomes. To this end, the unfolded protein response (UPR) pathway was investigated as a potential mediator of corneal cell death in response to hypothermic storage. Once preservation-induced failure had begun in HCECs stored at 4°C, it was noted that necrosis accounted for the majority of cell death but with significant apoptotic involvement, peaking at several hours post-storage (4-8 hours). Western blot analysis demonstrated changes associated with apoptotic activation (caspase 9, caspase 3, and PARP cleavage). Further, the activation of the UPR pathway was observed through increased and sustained levels of ER folding and chaperone proteins (Bip, PDI, and ERO1-L?) in samples experiencing significant cell death. Modulation of the UPR pathway using the specific inhibitor, salubrinal, resulted in a 2-fold increase in cell survival in samples experiencing profound cold-induced failure. Furthermore, this increased cell survival was associated with increased membrane integrity, cell attachment, and decreased necrotic cell death populations. Conversely, addition of the UPR inducer, tunicamycin, during cold exposure resulted in a significant decrease in HCEC survival during the recovery period. These data implicate for the first time that this novel cell stress pathway may be activated in HCEC as a result of the complex stresses associated with hypothermic exposure. The data suggest that the targeted control of the UPR pathway during both processing and preservation protocols may improve cell survival and function of HCEC thus improving the clinical utility of these cells as well as whole human corneas.

Corwin, William L.; Baust, John M.; Baust, John G.; Van Buskirk, Robert G.

2012-01-01

167

Deficient Notch signaling associated with neurogenic pecanex is compensated for by the unfolded protein response in Drosophila.  

PubMed

The Notch (N) signaling machinery is evolutionarily conserved and regulates a broad spectrum of cell-specification events, through local cell-cell communication. pecanex (pcx) encodes a multi-pass transmembrane protein of unknown function, widely found from Drosophila to humans. The zygotic and maternal loss of pcx in Drosophila causes a neurogenic phenotype (hyperplasia of the embryonic nervous system), suggesting that pcx might be involved in N signaling. Here, we established that Pcx is a component of the N-signaling pathway. Pcx was required upstream of the membrane-tethered and the nuclear forms of activated N, probably in N signal-receiving cells, suggesting that pcx is required prior to or during the activation of N. pcx overexpression revealed that Pcx resides in the endoplasmic reticulum (ER). Disruption of pcx function resulted in enlargement of the ER that was not attributable to the reduced N signaling activity. In addition, hyper-induction of the unfolded protein response (UPR) by the expression of activated Xbp1 or dominant-negative Heat shock protein cognate 3 suppressed the neurogenic phenotype and ER enlargement caused by the absence of pcx. A similar suppression of these phenotypes was induced by overexpression of O-fucosyltransferase 1, an N-specific chaperone. Taking these results together, we speculate that the reduction in N signaling in embryos lacking pcx function might be attributable to defective ER functions, which are compensated for by upregulation of the UPR and possibly by enhancement of N folding. Our results indicate that the ER plays a previously unrecognized role in N signaling and that this ER function depends on pcx activity. PMID:22190636

Yamakawa, Tomoko; Yamada, Kenta; Sasamura, Takeshi; Nakazawa, Naotaka; Kanai, Maiko; Suzuki, Emiko; Fortini, Mark E; Matsuno, Kenji

2012-02-01

168

Multistep protein unfolding during nanopore translocation  

NASA Astrophysics Data System (ADS)

Cells are divided into compartments and separated from the environment by lipid bilayer membranes. Essential molecules are transported back and forth across the membranes. We have investigated how folded proteins use narrow transmembrane pores to move between compartments. During this process, the proteins must unfold. To examine co-translocational unfolding of individual molecules, we tagged protein substrates with oligonucleotides to enable potential-driven unidirectional movement through a model protein nanopore, a process that differs fundamentally from extension during force spectroscopy measurements. Our findings support a four-step translocation mechanism for model thioredoxin substrates. First, the DNA tag is captured by the pore. Second, the oligonucleotide is pulled through the pore, causing local unfolding of the C terminus of the thioredoxin adjacent to the pore entrance. Third, the remainder of the protein unfolds spontaneously. Finally, the unfolded polypeptide diffuses through the pore into the recipient compartment. The unfolding pathway elucidated here differs from those revealed by denaturation experiments in solution, for which two-state mechanisms have been proposed.

Rodriguez-Larrea, David; Bayley, Hagan

2013-04-01

169

Unfolded Protein Response Is Required in nu/nu Mice Microvasculature for Treating Breast Tumor with Tunicamycin*  

PubMed Central

Up-regulation of the dolichol pathway, a “hallmark” of asparagine-linked protein glycosylation, enhances angiogenesis in vitro. The dynamic relationship between these two processes is now evaluated with tunicamycin. Capillary endothelial cells treated with tunicamycin were growth inhibited and could not be reversed with exogenous VEGF165. Inhibition of angiogenesis is supported by down-regulation of (i) phosphorylated VEGFR1 and VEGFR2 receptors; (ii) VEGF165-specific phosphotyrosine kinase activity; and (iii) MatrigelTM invasion and chemotaxis. In vivo, tunicamycin prevented the vessel development in MatrigelTM implants in athymic Balb/c (nu/nu) mice. Immunohistochemical analysis of CD34 (p < 0.001) and CD144 (p < 0.001) exhibited reduced vascularization. A 3.8-fold increased expression of TSP-1, an endogenous angiogenesis inhibitor in MatrigelTM implants correlated with that in tunicamycin (32 h)-treated capillary endothelial cells. Intravenous injection of tunicamycin (0.5 mg/kg to 1.0 mg/kg) per week slowed down a double negative (MDA-MB-435) grade III breast adenocarcinoma growth by ?50–60% in 3 weeks. Histopathological analysis of the paraffin sections indicated significant reduction in vessel size, the microvascular density and tumor mitotic index. Ki-67 and VEGF expression in tumor tissue were also reduced. A significant reduction of N-glycan expression in tumor microvessel was also observed. High expression of GRP-78 in CD144-positive cells supported unfolded protein response-mediated ER stress in tumor microvasculature. ?65% reduction of a triple negative (MDA-MB-231) breast tumor xenograft in 1 week with tunicamycin (0.25 mg/kg) given orally and the absence of systemic and/or organ failure strongly supported tunicamycin's potential for a powerful glycotherapeutic treatment of breast cancer in the clinic.

Banerjee, Aditi; Lang, Jing-Yu; Hung, Mien-Chie; Sengupta, Krishanu; Banerjee, Sushanta K.; Baksi, Krishna; Banerjee, Dipak K.

2011-01-01

170

The p53/HSP70 inhibitor, 2-phenylethynesulfonamide, causes oxidative stress, unfolded protein response and apoptosis in rainbow trout cells.  

PubMed

The effect of 2-phenylethynesulfonamide (PES), which is a p53 and HSP70 inhibitor in mammalian cells, was studied on the rainbow trout (Oncorhynchus mykiss) gill epithelial cell line, RTgill-W1, in order to evaluate PES as a tool for understanding the cellular survival pathways operating in fish. As judged by three viability assays, fish cells were killed by 24h exposures to PES, but cell death was blocked by the anti-oxidant N-acetylcysteine (NAC). Cell death had several hallmarks of apoptosis: DNA laddering, nuclear fragmentation, Annexin V staining, mitochondrial membrane potential decline, and caspases activation. Reactive oxygen species (ROS) production peaked in several hours after the addition of PES and before cell death. HSP70 and BiP levels were higher in cultures treated with PES for 24h, but this was blocked by NAC. As well, PES treatment caused HSP70, BiP and p53 to accumulate in the detergent-insoluble fraction, and this too was prevented by NAC. Of several possible scenarios to explain the results, the following one is the simplest. PES enhances the generation of ROS, possibly by inhibiting the anti-oxidant actions of p53 and HSP70. ER stress arises from the ROS and from PES inhibiting the chaperone activities of HSP70. The ER stress in turn initiates the unfolded protein response (UPR), but this fails to restore ER homeostasis so proteins aggregate and cells die. Despite these multiple actions, PES should be useful for studying fish cellular survival pathways. PMID:24270669

Zeng, Fanxing; Tee, Catherine; Liu, Michelle; Sherry, James P; Dixon, Brian; Duncker, Bernard P; Bols, Niels C

2014-01-01

171

HLA-B27 misfolding in transgenic rats is associated with activation of the unfolded protein response.  

PubMed

The mechanism by which the MHC class I allele, HLA-B27, contributes to spondyloarthritis pathogenesis is unknown. In contrast to other alleles that have been examined, HLA-B27 has a tendency to form high m.w. disulfide-linked H chain complexes in the endoplasmic reticulum (ER), bind the ER chaperone BiP/Grp78, and undergo ER-associated degradation. These aberrant characteristics have provided biochemical evidence that HLA-B27 is prone to misfold. Recently, similar biochemical characteristics of HLA-B27 were reported in cells from HLA-B27/human beta2-microglobulin transgenic (HLA-B27 transgenic) rats, an animal model of spondyloarthritis, and correlated with disease susceptibility. In this study, we demonstrate that the unfolded protein response (UPR) is activated in macrophages derived from the bone marrow of HLA-B27 transgenic rats with inflammatory disease. Microarray analysis of these cells also reveals an IFN response signature. In contrast, macrophages derived from premorbid rats do not exhibit a strong UPR or evidence of IFN exposure. Activation of macrophages from premorbid HLA-B27 transgenic rats with IFN-gamma increases HLA-B27 expression and leads to UPR induction, while no UPR is seen in cells from nondisease-prone HLA-B7 transgenic or wild-type (nontransgenic) animals. This is the first demonstration, to our knowledge, that HLA-B27 misfolding is associated with ER stress that results in activation of the UPR. These observations link HLA-B27 expression with biological effects that are independent of immunological recognition, but nevertheless may play an important role in the pathogenesis of inflammatory diseases associated with this MHC class I allele. PMID:16081815

Turner, Matthew J; Sowders, Dawn P; DeLay, Monica L; Mohapatra, Rajashree; Bai, Shuzhen; Smith, Judith A; Brandewie, Jaclyn R; Taurog, Joel D; Colbert, Robert A

2005-08-15

172

A Role for the Unfolded Protein Response (UPR) in Virulence and Antifungal Susceptibility in Aspergillus fumigatus  

Microsoft Academic Search

Filamentous fungi rely heavily on the secretory pathway, both for the delivery of cell wall components to the hyphal tip and the production and secretion of extracellular hydrolytic enzymes needed to support growth on polymeric substrates. Increased demand on the secretory system exerts stress on the endoplasmic reticulum (ER), which is countered by the activation of a coordinated stress response

Daryl L. Richie; Lukas Hartl; Vishukumar Aimanianda; Michael S. Winters; Kevin K. Fuller; Michael D. Miley; Stephanie White; Jason W. McCarthy; Jean-Paul Latgé; Marta Feldmesser; Judith C. Rhodes; David S. Askew

2009-01-01

173

Activation of the unfolded protein response enhances motor recovery after spinal cord injury  

Microsoft Academic Search

Spinal cord injury (SCI) is a major cause of paralysis, and involves multiple cellular and tissular responses including demyelination, inflammation, cell death and axonal degeneration. Recent evidence suggests that perturbation on the homeostasis of the endoplasmic reticulum (ER) is observed in different SCI models; however, the functional contribution of this pathway to this pathology is not known. Here we demonstrate

V Valenzuela; E Collyer; D Armentano; G B Parsons; F A Court; C Hetz

2012-01-01

174

Roles of Endoplasmic Reticulum Stress and Unfolded Protein Response Associated Genes in Seed Stratification and Bud Endodormancy during Chilling Accumulation in Prunus persica  

PubMed Central

Dormancy mechanisms in seeds and buds arrest growth until environmental conditions are optimal for development. A genotype-specific period of chilling is usually required to release dormancy, but the underlying molecular mechanisms are still not fully understood. To discover transcriptional pathways associated with dormancy release common to seed stratification and bud endodormancy, we explored the chilling-dependent expression of 11 genes involved in endoplasmic reticulum stress and the unfolded protein response signal pathways. We propose that endoplasmic reticulum stress and the unfolded protein response impact on seed as well as bud germination and development by chilling-dependent mechanisms. The emerging discovery of similarities between seed stratification and bud endodormancy status indicate that these two processes are probably regulated by common endoplasmic reticulum stress and unfolded protein response signalling pathways. Clarification of regulatory pathways common to both seed and bud dormancy may enhance understanding of the mechanisms underlying dormancy and breeding programs may benefit from earlier prediction of chilling requirements for uniform blooming of novel genotypes of deciduous fruit tree species.

Fu, Xi Ling; Xiao, Wei; Wang, Dong Ling; Chen, Min; Tan, Qiu Ping; Li, Ling; De Chen, Xiu; Gao, Dong Sheng

2014-01-01

175

Altered unfolded protein response is implicated in the age-related exacerbation of proteinuria-induced proximal tubular cell damage.  

PubMed

Aging is a dominant risk factor for end-stage renal disease. We analyzed the mechanism involved in age-related exacerbation of proteinuria-induced proximal tubular cell (PTC) damage by focusing on endoplasmic reticulum-related unfolded protein response (UPR). After equal-degree induction of proteinuria in 24-month-old (aged) and 3-month-old (young) mice by intraperitoneal free fatty acid-bound albumin overload, tubulointerstitial lesions were more severe in aged than in young mice. In aged PTCs, proteinuria-induced cell-adaptive UPR resulting from induction of the molecular chaperone BiP was significantly suppressed, whereas proapoptotic UPR with CHOP overexpression was enhanced. Treatment with the exogenous molecular chaperone tauroursodeoxycholic acid (TUDCA) ameliorated proteinuria-induced tubulointerstitial lesions and PTC apoptosis in aged mice. Among the three UPR branches, alterations in the inositol-requiring 1? (IRE1?) pathway, but not the activating transcription factor 6 or PERK pathway, were associated with impaired BiP induction in aged kidneys. Moreover, siRNA-mediated suppression of BiP and IRE1? exacerbated free fatty acid-bound albumin-induced apoptosis in cultured PTCs, whereas siRNA-mediated CHOP suppression ameliorated apoptosis. Finally, proteinuria-induced BiP induction in PTCs was diminished in kidney specimens from elderly patients. These results indicate that maladaptive UPRs are involved in proteinuria-induced tubulointerstitial lesions exacerbation in aged kidneys, and that supplementation of chaperones may be used to treat elderly patients with persistent proteinuria. These results should improve understanding of cell vulnerability in aged kidneys. PMID:23871833

Takeda, Naoko; Kume, Shinji; Tanaka, Yuki; Morita, Yoshikata; Chin-Kanasaki, Masami; Araki, Hisazumi; Isshiki, Keiji; Araki, Shin-ichi; Haneda, Masakazu; Koya, Daisuke; Kashiwagi, Atsunori; Maegawa, Hiroshi; Uzu, Takashi

2013-09-01

176

PARP16 is a tail-anchored endoplasmic reticulum protein required for the PERK and IRE1?-mediated unfolded protein response  

PubMed Central

Summary Poly(ADP-ribose) polymerases (PARPs) (also known as ADP-ribosyl transferase D proteins) modify acceptor proteins with ADP-ribose modifications of varying length (reviewed in refs 1–3). PARPs regulate key stress response pathways, including DNA damage repair and the cytoplasmic stress response2,3,4,5,6. Here, we show that PARPs also regulate the unfolded protein response (UPR) of the endoplasmic reticulum (ER). Human PARP16/ARTD15 is a tail-anchored ER transmembrane protein required for activation of the functionally related ER stress sensors PERK and IRE1? during the UPR. The third identified ER stress sensor, ATF6, is not regulated by PARP16. Similar to other PARPs that function during stress, PARP16 enzymatic activity is up-regulated during ER stress when it (ADP-ribosyl)ates itself, PERK and IRE1?. (ADP-ribosyl)ation by PARP16 is sufficient for activating PERK and IRE1? in the absence of ER stress, and is required for PERK and IRE1 ? activation during the UPR. Modification of PERK and IRE1? by PARP16 increases their kinase activities and the endonuclease activity of IRE1?. Interestingly, the C-terminal luminal tail of PARP16 is required for PARP16 function during ER stress, suggesting that it transduces stress signals to the cytoplasmic PARP catalytic domain.

Jwa, Miri; Chang, Paul

2012-01-01

177

Mechanical Unfolding of an Ankyrin Repeat Protein  

PubMed Central

Ankryin repeat proteins comprise tandem arrays of a 33-residue, predominantly ?-helical motif that stacks roughly linearly to produce elongated and superhelical structures. They function as scaffolds mediating a diverse range of protein-protein interactions, and some have been proposed to play a role in mechanical signal transduction processes in the cell. Here we use atomic force microscopy and molecular-dynamics simulations to investigate the natural 7-ankyrin repeat protein gankyrin. We find that gankyrin unfolds under force via multiple distinct pathways. The reactions do not proceed in a cooperative manner, nor do they always involve fully stepwise unfolding of one repeat at a time. The peeling away of half an ankyrin repeat, or one or more ankyrin repeats, occurs at low forces; however, intermediate species are formed that are resistant to high forces, and the simulations indicate that in some instances they are stabilized by nonnative interactions. The unfolding of individual ankyrin repeats generates a refolding force, a feature that may be more easily detected in these proteins than in globular proteins because the refolding of a repeat involves a short contraction distance and incurs a low entropic cost. We discuss the origins of the differences between the force- and chemical-induced unfolding pathways of ankyrin repeat proteins, as well as the differences between the mechanics of natural occurring ankyrin repeat proteins and those of designed consensus ankyin repeat and globular proteins.

Serquera, David; Lee, Whasil; Settanni, Giovanni; Marszalek, Piotr E.; Paci, Emanuele; Itzhaki, Laura S.

2010-01-01

178

The unfolded protein response is required to maintain the integrity of the endoplasmic reticulum, prevent oxidative stress and preserve differentiation in ?-cells  

PubMed Central

Diabetes is an epidemic of worldwide proportions caused by ?-cell failure. Nutrient fluctuations and insulin resistance drive ?-cells to synthesize insulin beyond their capacity for protein folding and secretion and thereby activate the unfolded protein response (UPR), an adaptive signalling pathway to promote cell survival upon accumulation of unfolded protein in the endoplasmic reticulum (ER). Protein kinase-like endoplasmic reticulum kinase (PERK) signals one component of the UPR through phosphorylation of eukaryotic initiation factor 2 on the ?-subunit (eIF2?) to attenuate protein synthesis, thereby reducing the biosynthetic burden. ?-Cells uniquely require PERK-mediated phosphorylation of eIF2? to preserve cell function. Unabated protein synthesis in ?-cells is sufficient to initiate a cascade of events, including oxidative stress, that are characteristic of ?-cell failure observed in type 2 diabetes. In contrast to acute adaptive UPR activation, chronic activation increases expression of the proapoptotic transcription factor CAAT/enhancer-binding protein homologous protein (CHOP). Chop deletion in insulin-resistant mice profoundly increases ?-cell mass and prevents ?-cell failure to forestall the progression of diabetes. The findings suggest an unprecedented link by which protein synthesis and/or misfolding in the ER causes oxidative stress and should encourage the development of novel strategies to treat diabetes.

Kaufman, R J; Back, S H; Song, B; Han, J; Hassler, J

2010-01-01

179

CHAC1/MGC4504 Is a Novel Proapoptotic Component of the Unfolded Protein Response, Downstream of the ATF4-ATF3-CHOP Cascade1  

PubMed Central

To understand pathways mediating the inflammatory responses of human aortic endothelial cells to oxidized phospholipids, we previously used a combination of genetics and genomics to model a coexpression network encompassing >1000 genes. CHAC1 (cation transport regulator-like protein 1), a novel gene regulated by ox-PAPC (oxidized 1-palmitoyl-2-arachidonyl-sn-3-glycero-phosphorylcholine), was identified in a co-regulated group of genes enriched for components of the ATF4 (activating transcription factor 4) arm of the unfolded protein response pathway. Herein, we characterize the role of CHAC1 and validate the network model. We first define the activation of CHAC1 mRNA by chemical unfolded protein response-inducers, but not other cell stressors. We then define activation of CHAC1 by the ATF4-ATF3-CHOP (C/EBP homologous protein), and not parallel XBP1 (X box-binding protein 1) or ATF6 pathways, using siRNA and/or overexpression plasmids. To examine the subset of genes downstream of CHAC1, we used expression microarray analysis to identify a list of 227 differentially regulated genes. We validated the activation of TNFRSF6B (tumor necrosis factor receptor superfamily, member 6b), a FASL decoy receptor, in cells treated with CHAC1 small interfering RNA. Finally, we showed that CHAC1 overexpression enhanced apoptosis, while CHAC1 small interfering RNA suppressed apoptosis, as determined by TUNEL, PARP (poly(ADP-ribose) polymerase) cleavage, and AIF (apoptosis-inducing factor) nuclear translocation.

Mungrue, Imran N.; Pagnon, Joanne; Kohannim, Omid; Gargalovic, Peter S.; Lusis, Aldons J.

2010-01-01

180

BH3-only proteins are part of a regulatory network that control the sustained signalling of the unfolded protein response sensor IRE1?  

PubMed Central

Adaptation to endoplasmic reticulum (ER) stress depends on the activation of the unfolded protein response (UPR) stress sensor inositol-requiring enzyme 1? (IRE1?), which functions as an endoribonuclease that splices the mRNA of the transcription factor XBP-1 (X-box-binding protein-1). Through a global proteomic approach we identified the BCL-2 family member PUMA as a novel IRE1? interactor. Immun oprecipitation experiments confirmed this interaction and further detected the association of IRE1? with BIM, another BH3-only protein. BIM and PUMA double-knockout cells failed to maintain sustained XBP-1 mRNA splicing after prolonged ER stress, resulting in early inactivation. Mutation in the BH3 domain of BIM abrogated the physical interaction with IRE1?, inhibiting its effects on XBP-1 mRNA splicing. Unexpectedly, this regulation required BCL-2 and was antagonized by BAD or the BH3 domain mimetic ABT-737. The modulation of IRE1? RNAse activity by BH3-only proteins was recapitulated in a cell-free system suggesting a direct regulation. Moreover, BH3-only proteins controlled XBP-1 mRNA splicing in vivo and affected the ER stress-regulated secretion of antibodies by primary B cells. We conclude that a subset of BCL-2 family members participates in a new UPR-regulatory network, thus assuming apoptosis-unrelated functions.

Rodriguez, Diego A; Zamorano, Sebastian; Lisbona, Fernanda; Rojas-Rivera, Diego; Urra, Hery; Cubillos-Ruiz, Juan R; Armisen, Ricardo; Henriquez, Daniel R; H Cheng, Emily; Letek, Michal; Vaisar, Tomas; Irrazabal, Thergiory; Gonzalez-Billault, Christian; Letai, Anthony; Pimentel-Muinos, Felipe X; Kroemer, Guido; Hetz, Claudio

2012-01-01

181

Nodulin 22, a Novel Small Heat-Shock Protein of the Endoplasmic Reticulum, Is Linked to the Unfolded Protein Response in Common Bean  

PubMed Central

The importance of plant small heat shock proteins (sHsp) in multiple cellular processes has been evidenced by their unusual abundance and diversity; however, little is known about their biological role. Here, we characterized the in vitro chaperone activity and subcellular localization of nodulin 22 of Phaseolus vulgaris (PvNod22; common bean) and explored its cellular function through a virus-induced gene silencing–based reverse genetics approach. We established that PvNod22 facilitated the refolding of a model substrate in vitro, suggesting that it acts as a molecular chaperone in the cell. Through microscopy analyses of PvNod22, we determined its localization in the endoplasmic reticulum (ER). Furthermore, we found that silencing of PvNod22 resulted in necrotic lesions in the aerial organs of P. vulgaris plants cultivated under optimal conditions and that downregulation of PvNod22 activated the ER-unfolded protein response (UPR) and cell death. We also established that PvNod22 expression in wild-type bean plants was modulated by abiotic stress but not by chemicals that trigger the UPR, indicating PvNod22 is not under UPR control. Our results suggest that the ability of PvNod22 to suppress protein aggregation contributes to the maintenance of ER homeostasis, thus preventing the induction of cell death via UPR in response to oxidative stress during plant-microbe interactions.

Rodriguez-Lopez, Jonathan; Martinez-Centeno, Cynthia; Padmanaban, Annamalai; Guillen, Gabriel; Olivares, Juan Elias; Stefano, Giovanni; Lledias, Fernando; Ramos, Fernando; Ghabrial, Said A.; Brandizzi, Federica; Rocha-Sosa, Mario; Diaz-Camino, Claudia; Sanchez, Federico

2014-01-01

182

Nodulin 22, a novel small heat-shock protein of the endoplasmic reticulum, is linked to the unfolded protein response in common bean.  

PubMed

The importance of plant small heat shock proteins (sHsp) in multiple cellular processes has been evidenced by their unusual abundance and diversity; however, little is known about their biological role. Here, we characterized the in vitro chaperone activity and subcellular localization of nodulin 22 of Phaseolus vulgaris (PvNod22; common bean) and explored its cellular function through a virus-induced gene silencing-based reverse genetics approach. We established that PvNod22 facilitated the refolding of a model substrate in vitro, suggesting that it acts as a molecular chaperone in the cell. Through microscopy analyses of PvNod22, we determined its localization in the endoplasmic reticulum (ER). Furthermore, we found that silencing of PvNod22 resulted in necrotic lesions in the aerial organs of P. vulgaris plants cultivated under optimal conditions and that downregulation of PvNod22 activated the ER-unfolded protein response (UPR) and cell death. We also established that PvNod22 expression in wild-type bean plants was modulated by abiotic stress but not by chemicals that trigger the UPR, indicating PvNod22 is not under UPR control. Our results suggest that the ability of PvNod22 to suppress protein aggregation contributes to the maintenance of ER homeostasis, thus preventing the induction of cell death via UPR in response to oxidative stress during plant-microbe interactions. PMID:24073881

Rodriguez-López, Jonathan; Martínez-Centeno, Cynthia; Padmanaban, Annamalai; Guillén, Gabriel; Olivares, Juan Elías; Stefano, Giovanni; Lledías, Fernando; Ramos, Fernando; Ghabrial, Said A; Brandizzi, Federica; Rocha-Sosa, Mario; Díaz-Camino, Claudia; Sanchez, Federico

2014-01-01

183

Proteomic and biochemical analyses reveal the activation of unfolded protein response, ERK-1/2 and ribosomal protein S6 signaling in experimental autoimmune myocarditis rat model  

PubMed Central

Background To investigate the molecular and cellular pathogenesis underlying myocarditis, we used an experimental autoimmune myocarditis (EAM)-induced heart failure rat model that represents T cell mediated postinflammatory heart disorders. Results By performing unbiased 2-dimensional electrophoresis of protein extracts from control rat heart tissues and EAM rat heart tissues, followed by nano-HPLC-ESI-QIT-MS, 67 proteins were identified from 71 spots that exhibited significantly altered expression levels. The majority of up-regulated proteins were confidently associated with unfolded protein responses (UPR), while the majority of down-regulated proteins were involved with the generation of precursor metabolites and energy metabolism in mitochondria. Although there was no difference in AKT signaling between EAM rat heart tissues and control rat heart tissues, the amounts and activities of extracellular signal-regulated kinase (ERK)-1/2 and ribosomal protein S6 (rpS6) were significantly increased. By comparing our data with the previously reported myocardial proteome of the Coxsackie viruses of group B (CVB)-mediated myocarditis model, we found that UPR-related proteins were commonly up-regulated in two murine myocarditis models. Even though only two out of 29 down-regulated proteins in EAM rat heart tissues were also dysregulated in CVB-infected rat heart tissues, other proteins known to be involved with the generation of precursor metabolites and energy metabolism in mitochondria were also dysregulated in CVB-mediated myocarditis rat heart tissues, suggesting that impairment of mitochondrial functions may be a common underlying mechanism of the two murine myocarditis models. Conclusions UPR, ERK-1/2 and S6RP signaling were activated in both EAM- and CVB-induced myocarditis murine models. Thus, the conserved components of signaling pathways in two murine models of acute myocarditis could be targets for developing new therapeutic drugs or methods aimed at treating enigmatic myocarditis.

2011-01-01

184

siRNA Silencing of Proteasome Maturation Protein (POMP) Activates the Unfolded Protein Response and Constitutes a Model for KLICK Genodermatosis  

PubMed Central

Keratosis linearis with ichthyosis congenita and keratoderma (KLICK) is an autosomal recessive skin disorder associated with a single-nucleotide deletion in the 5?untranslated region of the proteasome maturation protein (POMP) gene. The deletion causes a relative switch in transcription start sites for POMP, predicted to decrease levels of POMP protein in terminally differentiated keratinocytes. To investigate the pathophysiology behind KLICK we created an in vitro model of the disease using siRNA silencing of POMP in epidermal air-liquid cultures. Immunohistochemical analysis of the tissue constructs revealed aberrant staining of POMP, proteasome subunits and the skin differentiation marker filaggrin when compared to control tissue constructs. The staining patterns of POMP siRNA tissue constructs showed strong resemblance to those observed in skin biopsies from KLICK patients. Western blot analysis of lysates from the organotypic tissue constructs revealed an aberrant processing of profilaggrin to filaggrin in samples transfected with siRNA against POMP. Knock-down of POMP expression in regular cell cultures resulted in decreased amounts of proteasome subunits. Prolonged silencing of POMP in cultured cells induced C/EBP homologous protein (CHOP) expression consistent with an activation of the unfolded protein response and increased endoplasmic reticulum (ER) stress. The combined results indicate that KLICK is caused by reduced levels of POMP, leading to proteasome insufficiency in differentiating keratinocytes. Proteasome insufficiency disturbs terminal epidermal differentiation, presumably by increased ER stress, and leads to perturbed processing of profilaggrin. Our findings underline a critical role for the proteasome in human epidermal differentiation.

Dahlqvist, Johanna; Torma, Hans; Badhai, Jitendra; Dahl, Niklas

2012-01-01

185

On the nature of the unfolded state: competing mechanisms in the unfolding of anhydrous protein ions  

Microsoft Academic Search

Protein ions undergo folding–unfolding transitions unaided by water. Experiment and simulations suggest a critical protonation state below which the native fold might be metastable. Here, we use molecular dynamics simulations of charged lysozyme to explore the underlying mechanism for the unfolding transition. Two processes are found to work simultaneously in vacuo: the unfolding bias associated with the repulsion between charged

Gustavo A. Arteca; O. Tapia

2004-01-01

186

The unfolded protein response element IRE1? senses bacterial proteins invading the ER to activate RIG-I and innate immune signaling.  

PubMed

The plasma membrane and all membrane-bound organelles except for the Golgi and endoplasmic reticulum (ER) are equipped with pattern-recognition molecules to sense microbes or their products and induce innate immunity for host defense. Here, we report that inositol-requiring-1? (IRE1?), an ER protein that signals in the unfolded protein response (UPR), is activated to induce inflammation by binding a portion of cholera toxin as it co-opts the ER to cause disease. Other known UPR transducers, including the IRE1?-dependent transcription factor XBP1, are dispensable for this signaling. The inflammatory response depends instead on the RNase activity of IRE1? to degrade endogenous mRNA, a process termed regulated IRE1?-dependent decay (RIDD) of mRNA. The mRNA fragments produced engage retinoic-acid inducible gene 1 (RIG-I), a cytosolic sensor of RNA viruses, to activate NF-?B and interferon pathways. We propose IRE1? provides for a generalized mechanism of innate immune surveillance originating within the ER lumen. PMID:23684307

Cho, Jin A; Lee, Ann-Hwee; Platzer, Barbara; Cross, Benedict C S; Gardner, Brooke M; De Luca, Heidi; Luong, Phi; Harding, Heather P; Glimcher, Laurie H; Walter, Peter; Fiebiger, Edda; Ron, David; Kagan, Jonathan C; Lencer, Wayne I

2013-05-15

187

The Unfolded Protein Response Element IRE1? Senses Bacterial Proteins Invading the ER to Activate RIG-I and Innate Immune Signaling  

PubMed Central

SUMMARY The plasma membrane and all membrane-bound organelles except for the Golgi and endoplasmic reticulum (ER) are equipped with pattern-recognition molecules to sense microbes or their products and induce innate immunity for host defense. Here, we report that inositol-requiring-1? (IRE1?), an ER protein that signals in the unfolded protein response (UPR), is activated to induce inflammation by binding a portion of cholera toxin as it co-opts the ER to cause disease. Other known UPR transducers, including the IRE1?-dependent transcription factor XBP1, are dispensable for this signaling. The inflammatory response depends instead on the RNase activity of IRE1? to degrade endogenous mRNA, a process termed regulated IRE1?-dependent decay (RIDD) of mRNA. The mRNA fragments produced engage retinoic-acid inducible gene 1 (RIG-I), a cyto-solic sensor of RNA viruses, to activate NF-?B and interferon pathways. We propose IRE1? provides for a generalized mechanism of innate immune surveillance originating within the ER lumen.

Cho, Jin A.; Lee, Ann-Hwee; Platzer, Barbara; Cross, Benedict C.S.; Gardner, Brooke M.; De Luca, Heidi; Luong, Phi; Harding, Heather P.; Glimcher, Laurie H.; Walter, Peter; Fiebiger, Edda; Ron, David; Kagan, Jonathan C.; Lencer, Wayne I.

2013-01-01

188

The unfolded protein response transducer ATF6 represents a novel transmembrane-type endoplasmic reticulum-associated degradation substrate requiring both mannose trimming and SEL1L protein.  

PubMed

Proteins misfolded in the endoplasmic reticulum (ER) are cleared by the ubiquitin-dependent proteasome system in the cytosol, a series of events collectively termed ER-associated degradation (ERAD). It was previously shown that SEL1L, a partner protein of the E3 ubiquitin ligase HRD1, is required for degradation of misfolded luminal proteins (ERAD-Ls substrates) but not misfolded transmembrane proteins (ERAD-Lm substrates) in both mammalian and chicken DT40 cells. Here, we analyzed ATF6, a type II transmembrane glycoprotein that serves as a sensor/transducer of the unfolded protein response, as a potential ERAD-Lm substrate in DT40 cells. Unexpectedly, degradation of endogenous ATF6 and exogenously expressed chicken and human ATF6 by the proteasome required SEL1L. Deletion analysis revealed that the luminal region of ATF6 is a determinant for SEL1L-dependent degradation. Chimeric analysis showed that the luminal region of ATF6 confers SEL1L dependence on type I transmembrane protein as well. In contrast, degradation of other known type I ERAD-Lm substrates (BACE457, T-cell receptor-?, CD3-?, and CD147) did not require SEL1L. Thus, ATF6 represents a novel type of ERAD-Lm substrate requiring SEL1L for degradation despite its transmembrane nature. In addition, endogenous ATF6 was markedly stabilized in wild-type cells treated with kifunensine, an inhibitor of ?1,2-mannosidase in the ER, indicating that degradation of ATF6 requires proper mannose trimming. Our further analyses revealed that the five ERAD-Lm substrates examined are classified into three subgroups based on their dependence on mannose trimming and SEL1L. Thus, ERAD-Lm substrates are degraded through much more diversified mechanisms in higher eukaryotes than previously thought. PMID:24043630

Horimoto, Satoshi; Ninagawa, Satoshi; Okada, Tetsuya; Koba, Hibiki; Sugimoto, Takehiro; Kamiya, Yukiko; Kato, Koichi; Takeda, Shunichi; Mori, Kazutoshi

2013-11-01

189

Mechanistic explanation of different unfolding behaviors observed for transmembrane and soluble ?-barrel proteins.  

PubMed

In response to mechanical stress, membrane proteins progress through sequences of major unfolding barriers, whereas soluble proteins usually must overcome only one major unfolding barrier. To gain insight into these markedly different unfolding behaviors, we applied force-probe molecular dynamics simulations and unfolded two ?-barrel proteins, the transmembrane outer membrane protein G (OmpG) and the water-soluble green fluorescent protein (GFP). The simulations mimic with high precision the unfolding experiments and show that OmpG in the absence of a membrane and GFP circumvent high unfolding barriers by rotations and explore alternative unfolding pathways. Embedding OmpG in the lipid membrane restricts this search for pathways and forces the protein to cross high unfolding barriers. Likewise, restricting the rotation forces GFP to traverse high unfolding barriers in a similar manner to membrane-embedded OmpG. These results indicate that mechanically stressed proteins search alternative unfolding pathways by rotations and explain why membrane proteins generally show higher mechanical stability compared to water-soluble proteins. PMID:23830738

Hensen, Ulf; Müller, Daniel J

2013-08-01

190

Functional analysis of the impact of ORMDL3 expression on inflammation and activation of the unfolded protein response in human airway epithelial cells  

PubMed Central

Background The gene ORMDL3 was shown to be associated with early-onset asthma susceptibility in multiple independent genome-wide and candidate-gene association studies. Asthmatic patients have elevated expression levels of this gene. ORMDL3 encodes a transmembrane protein localized in the endoplasmic reticulum (ER) that may be involved in ER stress and inflammation. It is essential to validate the genetic associations linking ORMDL3 with asthma through functional studies that confirm the biological relevance of this gene in disease. We investigated the effects of manipulating ORMDL3 expression levels in vitro in airway cells on innate immune inflammatory responses, ER stress and activation of the unfolded protein response (UPR). Methods ORMDL3 expression levels were manipulated in airway cells using an overexpression plasmid and siRNA technologies. Successful modulation of ORMDL3 was confirmed at both the gene and protein level. The functional impact of modulation of ORMDL3 expression levels on inflammatory responses and activation of the UPR were quantified using complementary cellular and molecular immunology techniques. Results Cells with altered ORMDL3 levels responded equally well to innate immune stimuli and produced similar levels of pro-inflammatory cytokines compared to wild-type cells. Treatment with ER stress inducers, thapsigargin and tunicamycin, resulted in activation of the unfolded protein response (UPR). However, we observed no difference in UPR activation in cells with ORMDL3 knockdown compared to cells with normal ORMDL3 levels. Conclusions Our results suggest that ORMDL3 variation in the airway epithelium is unlikely to play a significant role in modulating innate immune responses and the UPR in the lung.

2013-01-01

191

An unfolded protein response is the initial cellular response to the expression of mutant matrilin-3 in a mouse model of multiple epiphyseal dysplasia  

PubMed Central

Multiple epiphyseal dysplasia (MED) can result from mutations in matrilin-3, a structural protein of the cartilage extracellular matrix. We have previously shown that in a mouse model of MED the tibia growth plates were normal at birth but developed a progressive dysplasia characterised by the intracellular retention of mutant matrilin-3 and abnormal chondrocyte morphology. By 3 weeks of age, mutant mice displayed a significant decrease in chondrocyte proliferation and dysregulated apoptosis. The aim of this current study was to identify the initial post-natal stages of the disease. We confirmed that the disease phenotype is seen in rib and xiphoid cartilage and, like tibia growth plate cartilage is characterised by the intracellular retention of mutant matrilin-3. Gene expression profiling showed a significant activation of classical unfolded protein response (UPR) genes in mutant chondrocytes at 5 days of age, which was still maintained by 21 days of age. Interestingly, we also noted the upregulation of arginine-rich, mutated in early stage of tumours (ARMET) and cysteine-rich with EGF-like domain protein 2 (CRELD2) are two genes that have only recently been implicated in the UPR. This endoplasmic reticulum (ER) stress and UPR did not lead to increased chondrocyte apoptosis in mutant cartilage by 5 days of age. In an attempt to alleviate ER stress, mutant mice were fed with a chemical chaperone, 4-sodium phenylbutyrate (SPB). SPB at the dosage used had no effect on chaperone expression at 5 days of age but modestly decreased levels of chaperone proteins at 3 weeks. However, this did not lead to increased secretion of mutant matrilin-3 and in the long term did not improve the disease phenotype. We performed similar studies with a mouse model of Schmid metaphyseal chondrodysplasia, but again this treatment did not improve the phenotype. Electronic supplementary material The online version of this article (doi:10.1007/s12192-010-0193-y) contains supplementary material, which is available to authorized users.

Nundlall, Seema; Rajpar, M. Helen; Bell, Peter A.; Clowes, Christopher; Zeeff, Leo A. H.; Gardner, Benjamin; Thornton, David J.; Boot-Handford, Raymond P.

2010-01-01

192

An unfolded protein response is the initial cellular response to the expression of mutant matrilin-3 in a mouse model of multiple epiphyseal dysplasia.  

PubMed

Multiple epiphyseal dysplasia (MED) can result from mutations in matrilin-3, a structural protein of the cartilage extracellular matrix. We have previously shown that in a mouse model of MED the tibia growth plates were normal at birth but developed a progressive dysplasia characterised by the intracellular retention of mutant matrilin-3 and abnormal chondrocyte morphology. By 3 weeks of age, mutant mice displayed a significant decrease in chondrocyte proliferation and dysregulated apoptosis. The aim of this current study was to identify the initial post-natal stages of the disease. We confirmed that the disease phenotype is seen in rib and xiphoid cartilage and, like tibia growth plate cartilage is characterised by the intracellular retention of mutant matrilin-3. Gene expression profiling showed a significant activation of classical unfolded protein response (UPR) genes in mutant chondrocytes at 5 days of age, which was still maintained by 21 days of age. Interestingly, we also noted the upregulation of arginine-rich, mutated in early stage of tumours (ARMET) and cysteine-rich with EGF-like domain protein 2 (CRELD2) are two genes that have only recently been implicated in the UPR. This endoplasmic reticulum (ER) stress and UPR did not lead to increased chondrocyte apoptosis in mutant cartilage by 5 days of age. In an attempt to alleviate ER stress, mutant mice were fed with a chemical chaperone, 4-sodium phenylbutyrate (SPB). SPB at the dosage used had no effect on chaperone expression at 5 days of age but modestly decreased levels of chaperone proteins at 3 weeks. However, this did not lead to increased secretion of mutant matrilin-3 and in the long term did not improve the disease phenotype. We performed similar studies with a mouse model of Schmid metaphyseal chondrodysplasia, but again this treatment did not improve the phenotype. PMID:20428984

Nundlall, Seema; Rajpar, M Helen; Bell, Peter A; Clowes, Christopher; Zeeff, Leo A H; Gardner, Benjamin; Thornton, David J; Boot-Handford, Raymond P; Briggs, Michael D

2010-11-01

193

siRNA silencing of proteasome maturation protein (POMP) activates the unfolded protein response and constitutes a model for KLICK genodermatosis.  

PubMed

Keratosis linearis with ichthyosis congenita and keratoderma (KLICK) is an autosomal recessive skin disorder associated with a single-nucleotide deletion in the 5'untranslated region of the proteasome maturation protein (POMP) gene. The deletion causes a relative switch in transcription start sites for POMP, predicted to decrease levels of POMP protein in terminally differentiated keratinocytes. To investigate the pathophysiology behind KLICK we created an in vitro model of the disease using siRNA silencing of POMP in epidermal air-liquid cultures. Immunohistochemical analysis of the tissue constructs revealed aberrant staining of POMP, proteasome subunits and the skin differentiation marker filaggrin when compared to control tissue constructs. The staining patterns of POMP siRNA tissue constructs showed strong resemblance to those observed in skin biopsies from KLICK patients. Western blot analysis of lysates from the organotypic tissue constructs revealed an aberrant processing of profilaggrin to filaggrin in samples transfected with siRNA against POMP. Knock-down of POMP expression in regular cell cultures resulted in decreased amounts of proteasome subunits. Prolonged silencing of POMP in cultured cells induced C/EBP homologous protein (CHOP) expression consistent with an activation of the unfolded protein response and increased endoplasmic reticulum (ER) stress. The combined results indicate that KLICK is caused by reduced levels of POMP, leading to proteasome insufficiency in differentiating keratinocytes. Proteasome insufficiency disturbs terminal epidermal differentiation, presumably by increased ER stress, and leads to perturbed processing of profilaggrin. Our findings underline a critical role for the proteasome in human epidermal differentiation. PMID:22235297

Dahlqvist, Johanna; Törmä, Hans; Badhai, Jitendra; Dahl, Niklas

2012-01-01

194

Unfolding Kinetics of Egg Protein  

NASA Astrophysics Data System (ADS)

This study explores denaturing kinetics of egg white using high resolution calorimetric technique. Fresh egg was scanned fro heating and cooling to see the thermodynamics 10^o C to 100^o C at different heating ramp rates varying from 1 to 20^o C/min. An endothermic peak was found on heating scan showing denaturing of protein which was found absent at the cooling indicating the absence of any residue after heating. The denature peak shifted towards higher temperature as ramp rate increases following Arrhenius behavior and shows an activated denaturing kinetics of the egg protein. This peak was also compared with the water to avoid water effects. Behavior of denaturing peak can be explained in terms of Arrhenius theory.

Sharma, Dipti

2011-03-01

195

Airway mesenchymal cell death by mevalonate cascade inhibition: Integration of autophagy, unfolded protein response and apoptosis focusing on Bcl2 family proteins.  

PubMed

HMG-CoA reductase, the proximal rate-limiting enzyme in the mevalonate pathway, is inhibited by statins. Beyond their cholesterol lowering impact, statins have pleiotropic effects and their use is linked to improved lung health. We have shown that mevalonate cascade inhibition induces apoptosis and autophagy in cultured human airway mesenchymal cells. Here, we show that simvastatin also induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in these cells. We tested whether coordination of ER stress, autophagy and apoptosis determines survival or demise of human lung mesenchymal cells exposed to statin. We observed that simvastatin exposure activates UPR (activated transcription factor 4, activated transcription factor 6 and IRE1?) and caspase-4 in primary human airway fibroblasts and smooth muscle cells. Exogenous mevalonate inhibited apoptosis, autophagy and UPR, but exogenous cholesterol was without impact, indicating that sterol intermediates are involved with mechanisms mediating statin effects. Caspase-4 inhibition decreased simvastatin-induced apoptosis, whereas inhibition of autophagy by ATG7 or ATG3 knockdown significantly increased cell death. In BAX(-/-)/BAK(-/-) murine embryonic fibroblasts, simvastatin-triggered apoptotic and UPR events were abrogated, but autophagy flux was increased leading to cell death via necrosis. Our data indicate that mevalonate cascade inhibition, likely associated with depletion of sterol intermediates, can lead to cell death via coordinated apoptosis, autophagy, and ER stress. The interplay between these pathways appears to be principally regulated by autophagy and Bcl-2-family pro-apoptotic proteins. These findings uncover multiple mechanisms of action of statins that could contribute to refining the use of such agent in treatment of lung disease. PMID:24637330

Ghavami, Saeid; Sharma, Pawan; Yeganeh, Behzad; Ojo, Oluwaseun O; Jha, Aruni; Mutawe, Mark M; Kashani, Hessam H; Los, Marek J; Klonisch, Thomas; Unruh, Helmut; Halayko, Andrew J

2014-07-01

196

Increase in gene-transcript levels as indicators of up-regulation of the unfolded protein response in spontaneous canine tumors.  

PubMed

The unfolded protein response (UPR), a conserved cellular response to stressors such as hypoxia and nutrient deprivation, is associated with angiogenesis and metastasis in tumor cells. This article discusses a pilot study conducted to determine whether components of the UPR could be identified in spontaneous canine tumors and whether they were up-regulated within tumor tissue compared with adjacent normal tissue. Tissue samples of various spontaneous canine neoplasms were taken from 13 dogs shortly after surgical excision or euthanasia; control samples were taken from adjacent normal tissue. RNA purification and real-time quantitative reverse-transcription polymerase chain reaction were done to measure the expression of 4 genes associated with the UPR (HERP, CHOP, GRP78, and XBP1s). The results indicated that UPR gene expression can be identified in spontaneous canine tumors and that the UPR is up-regulated, as indicated by significantly increased expression of CHOP and GRP78 within the tumor. PMID:24982546

Elliot, Kirsten; MacDonald-Dickinson, Valerie; Linn, Kathleen; Simko, Elemir; Misra, Vikram

2014-07-01

197

A subcytotoxic dose of subtilase cytotoxin prevents lipopolysaccharide-induced inflammatory responses, depending on its capacity to induce the unfolded protein response.  

PubMed

Subtilase cytotoxin (SubAB) is the prototype of a newly identified family of AB(5) cytotoxins produced by Shiga toxigenic Escherichia coli. SubAB specifically cleaves the essential endoplasmic reticulum (ER) chaperone BiP (GRP78), resulting in the activation of ER stress-induced unfolded protein response (UPR). We have recently shown that the UPR following ER stress can suppress cellular responses to inflammatory stimuli during the later phase, in association with inhibition of NF-kappaB activation. These findings prompted us to hypothesize that SubAB, as a selective UPR inducer, might have beneficial effects on inflammation-associated pathology via a UPR-dependent inhibition of NF-kappaB activation. The pretreatment of a mouse macrophage cell line, RAW264.7, with a subcytotoxic dose of SubAB-triggered UPR and inhibited LPS-induced MCP-1 and TNF-alpha production associated with inhibition of NF-kappaB activation. SubA(A272)B, a SubAB active site mutant that cannot induce UPR, did not show such effects. In addition, pretreatment with a sublethal dose of SubAB, but not SubA(A272)B, protected the mice from LPS-induced endotoxic lethality associated with reduced serum MCP-1 and TNF-alpha levels and also prevented the development of experimental arthritis induced by LPS in mice. Collectively, although SubAB has been identified originally as a toxin associated with the pathogenesis of hemolytic uremic syndrome, the unique ability of SubAB to selectively induce the UPR may have the potential to prevent LPS-associated inflammatory pathology under subcytotoxic conditions. PMID:19553530

Harama, Daisuke; Koyama, Kensuke; Mukai, Mai; Shimokawa, Naomi; Miyata, Masanori; Nakamura, Yuki; Ohnuma, Yuko; Ogawa, Hideoki; Matsuoka, Shuji; Paton, Adrienne W; Paton, James C; Kitamura, Masanori; Nakao, Atsuhito

2009-07-15

198

Solvent Effects on Protein Folding/Unfolding  

NASA Astrophysics Data System (ADS)

Pressure effects on the hydrophobic potential of mean force led Hummer et al. to postulate a model for pressure denaturation of proteins in which denaturation occurs by means of water penetration into the protein interior, rather than by exposing the protein hydrophobic core to the solvent --- commonly used to describe temperature denaturation. We study the effects of pressure in protein folding/unfolding kinetics in an off-lattice minimalist model of a protein in which pressure effects have been incorporated by means of the pair-wise potential of mean force of hydrophobic groups in water. We show that pressure slows down the kinetics of folding by decreasing the reconfigurational diffusion coefficient and moves the location of the folding transition state.

García, A. E.; Hillson, N.; Onuchic, J. N.

199

Translational and posttranslational regulation of XIAP by eIF2? and ATF4 promotes ER stress-induced cell death during the unfolded protein response  

PubMed Central

Endoplasmic reticulum (ER) protein misfolding activates the unfolded protein response (UPR) to help cells cope with ER stress. If ER homeostasis is not restored, UPR promotes cell death. The mechanisms of UPR-mediated cell death are poorly understood. The PKR-like endoplasmic reticulum kinase (PERK) arm of the UPR is implicated in ER stress–induced cell death, in part through up-regulation of proapoptotic CCAAT/enhancer binding protein homologous protein (CHOP). Chop?/? cells are partially resistant to ER stress–induced cell death, and CHOP overexpression alone does not induce cell death. These findings suggest that additional mechanisms regulate cell death downstream of PERK. Here we find dramatic suppression of antiapoptosis XIAP proteins in response to chronic ER stress. We find that PERK down-regulates XIAP synthesis through eIF2? and promotes XIAP degradation through ATF4. Of interest, PERK's down-regulation of XIAP occurs independently of CHOP activity. Loss of XIAP leads to increased cell death, whereas XIAP overexpression significantly enhances resistance to ER stress–induced cell death, even in the absence of CHOP. Our findings define a novel signaling circuit between PERK and XIAP that operates in parallel with PERK to CHOP induction to influence cell survival during ER stress. We propose a “two-hit” model of ER stress–induced cell death involving concomitant CHOP up-regulation and XIAP down-regulation both induced by PERK.

Hiramatsu, Nobuhiko; Messah, Carissa; Han, Jaeseok; LaVail, Matthew M.; Kaufman, Randal J.; Lin, Jonathan H.

2014-01-01

200

Translational and posttranslational regulation of XIAP by eIF2? and ATF4 promotes ER stress-induced cell death during the unfolded protein response.  

PubMed

Endoplasmic reticulum (ER) protein misfolding activates the unfolded protein response (UPR) to help cells cope with ER stress. If ER homeostasis is not restored, UPR promotes cell death. The mechanisms of UPR-mediated cell death are poorly understood. The PKR-like endoplasmic reticulum kinase (PERK) arm of the UPR is implicated in ER stress-induced cell death, in part through up-regulation of proapoptotic CCAAT/enhancer binding protein homologous protein (CHOP). Chop((-)/(-)) cells are partially resistant to ER stress-induced cell death, and CHOP overexpression alone does not induce cell death. These findings suggest that additional mechanisms regulate cell death downstream of PERK. Here we find dramatic suppression of antiapoptosis XIAP proteins in response to chronic ER stress. We find that PERK down-regulates XIAP synthesis through eIF2? and promotes XIAP degradation through ATF4. Of interest, PERK's down-regulation of XIAP occurs independently of CHOP activity. Loss of XIAP leads to increased cell death, whereas XIAP overexpression significantly enhances resistance to ER stress-induced cell death, even in the absence of CHOP. Our findings define a novel signaling circuit between PERK and XIAP that operates in parallel with PERK to CHOP induction to influence cell survival during ER stress. We propose a "two-hit" model of ER stress-induced cell death involving concomitant CHOP up-regulation and XIAP down-regulation both induced by PERK. PMID:24623724

Hiramatsu, Nobuhiko; Messah, Carissa; Han, Jaeseok; Lavail, Matthew M; Kaufman, Randal J; Lin, Jonathan H

2014-05-01

201

Biotin Supplementation Decreases the Expression of the SERCA3 Gene (ATP2A3) in Jurkat Cells, thus Triggering Unfolded Protein Response*  

PubMed Central

Protein folding in the endoplasmic reticulum (ER) depends on Ca2+; uptake of Ca2+ into the ER is mediated by SERCA3. The 5?-flanking region of the SERCA3 gene (ATP2A3) contains numerous binding sites for the transcription factors Sp1 and Sp3. Biotin affects the nuclear abundance of Sp1 and Sp3, which may act as transcriptional activators or repressors. Here we determined whether biotin affects the expression of the SERCA3 gene and, thus, protein folding in human lymphoid cells. Jurkat cells were cultured in media containing 0.025 nmol/L biotin (denoted “deficient”) or 10 nmol/L biotin (“supplemented”). The transcriptional activity of the full-length human SERCA3 promoter was 50% lower in biotin-supplemented cells compared to biotin-deficient cells. Biotin-dependent repressors bind to elements located 731 to 1312 basepairs upstream from the transcription start site in the SERCA3 gene. The following lines of evidence suggest that low expression of SERCA3 in biotin-supplemented cells impaired folding of secretory proteins in the ER, triggering Unfolded Protein Response: (i) sequestration of Ca2+ in the ER decreased by 14–24% in response to biotin supplementation; (ii) secretion of interleukin-2 into the extracellular space decreased by 75% in response to biotin supplementation; (iii) the nuclear abundance of stress-induced transcription factors increased in response to biotin supplementation; and (iv) the abundance of stress-related proteins such UBE1, GADD153, XBP1, and phosphorylated eIF2? increased in response to biotin supplementation. Collectively, this study suggests that supplements containing pharmacological doses of biotin may cause cell stress by impairing protein folding in the ER.

Griffin, Jacob B.; Rodriguez-Melendez, Rocio; Dode, Leonard; Wuytack, Frank; Zempleni, Janos

2006-01-01

202

Cross-talk unfolded: MARCKS proteins.  

PubMed Central

The proteins of the MARCKS (myristoylated alanine-rich C kinase substrate) family were first identified as prominent substrates of protein kinase C (PKC). Since then, these proteins have been implicated in the regulation of brain development and postnatal survival, cellular migration and adhesion, as well as endo-, exo- and phago-cytosis, and neurosecretion. The effector domain of MARCKS proteins is phosphorylated by PKC, binds to calmodulin and contributes to membrane binding. This multitude of mutually exclusive interactions allows cross-talk between the signal transduction pathways involving PKC and calmodulin. This review focuses on recent, mostly biophysical and biochemical results renewing interest in this protein family. MARCKS membrane binding is now understood at the molecular level. From a structural point of view, there is a consensus emerging that MARCKS proteins are "natively unfolded". Interestingly, domains similar to the effector domain have been discovered in other proteins. Furthermore, since the effector domain enhances the polymerization of actin in vitro, MARCKS proteins have been proposed to mediate regulation of the actin cytoskeleton. However, the recent observations that MARCKS might serve to sequester phosphatidylinositol 4,5-bisphosphate in the plasma membrane of unstimulated cells suggest an alternative model for the control of the actin cytoskeleton. While myristoylation is classically considered to be a co-translational, irreversible event, new reports on MARCKS proteins suggest a more dynamic picture of this protein modification. Finally, studies with mice lacking MARCKS proteins have investigated the functions of these proteins during embryonic development in the intact organism.

Arbuzova, Anna; Schmitz, Arndt A P; Vergeres, Guy

2002-01-01

203

Role of partial protein unfolding in alcohol-induced protein aggregation  

PubMed Central

Proteins aggregate in response to various stresses including changes in solvent conditions. Addition of alcohols has been recently shown to induce aggregation of disease-related as well as non-disease-related proteins. Here we probed the biophysical mechanisms underlying alcohol-induced protein aggregation, in particular the role of partial protein unfolding in aggregation. We have studied aggregation mechanisms due to benzyl alcohol which is used in numerous biochemical and biotechnological applications. We chose cytochrome c as a model protein, for the reason that various optical and structural probes are available to monitor its global and partial unfolding reactions. Benzyl alcohol induced the aggregation of cytochrome c in isothermal conditions and decreased the temperature at which the protein aggregates. However, benzyl alcohol did not perturb the overall native conformation of cytochrome c. Instead, it caused partial unfolding of a local protein region around the methionine residue at position 80. Site-specific optical probes, two-dimensional NMR titrations, and hydrogen exchange all support this conclusion. The protein aggregation temperature varied linearly with the melting temperature of the Met80 region. Stabilizing the Met80 region by heme iron reduction drastically decreased protein aggregation, which confirmed that the local unfolding of this region causes protein aggregation. These results indicate that a possible mechanism by which alcohols induce protein aggregation is through partial rather than complete unfolding of native proteins.

Singh, Surinder M.; Cabello-Villegas, Javier; Hutchings, Regina L.; Mallela, Krishna M.G.

2010-01-01

204

Tick-borne encephalitis virus triggers inositol-requiring enzyme 1 (IRE1) and transcription factor 6 (ATF6) pathways of unfolded protein response.  

PubMed

Tick-borne encephalitis (TBE) is a serious human neurological disease caused by TBE virus (TBEV). However, the mechanisms of TBEV-caused pathogenesis remain unclear. The endoplasmic reticulum (ER) stress response, also defined as the unfolded protein response (UPR), is an important conserved molecular signaling pathway that modulates many biological functions including innate immunity and viral pathogenesis. Here, we investigated the effects of the two UPR signaling pathways upon TBEV infection in Vero E6 cells. We showed that the amount of heat shock protein 72 (Hsp72) increased in the course of TBEV infection. We then confirmed that TBEV infection activates the IRE1 pathway, leading to RNA and protein expression of the spliced X box binding protein 1 (sXBP1). Furthermore, we observed the translocation of ATF6 during TBEV infection and expression of cleaved transcription factor 6 (ATF6) which suggest activation of ATF6 pathway. Finally, we examined whether inhibition of the IRE1 pathway has an effect on TBEV infection. Cell treatment with 3,5-Dibromosalicylaldehyde (IRE1 inhibitor) and tauroursodeoxycholic acid (TUDCA) showed that TBEV replication was significantly limited. These findings provide the first evidence that TBEV infection activates the two UPR signaling pathways. Moreover, inhibition of TBEV replication by UPR inhibitors may provide a novel therapeutic strategy against TBE. PMID:24177270

Yu, Chao; Achazi, Katharina; Niedrig, Matthias

2013-12-26

205

Electrospray ionization-induced protein unfolding.  

PubMed

Electrospray ionization mass spectrometry (ESI-MS) measurements were performed under a variety of solution conditions on a highly acidic sub-fragment (B3C) of the C-terminal carbohydrate-binding repeat region of Clostridium difficile toxin B, and two mutants (B4A and B4B) containing fewer acidic residues. ESI-MS measurements performed in negative ion mode on aqueous ammonium acetate solutions of B3C at low ionic strength (I?protein unfolding. In contrast, no evidence of unfolding was detected from ESI-MS measurements made in positive ion mode at low I or in either mode at higher I. The results of proton nuclear magnetic resonance and circular dichroism spectroscopy measurements and gel filtration chromatography performed on solutions of B3C under low and high I conditions suggest that the protein exists predominantly in a folded state in neutral aqueous solutions with I?>?10 mM. The results of ESI-MS measurements performed on B3C in a series of solutions with high I at pH 5 to 9 rule out the possibility that the structural changes are related to ESI-induced changes in pH. It is proposed that unfolding of B3C, observed in negative mode for solutions with low I, occurs during the ESI process and arises due to Coulombic repulsion between the negatively charged residues and liquid/droplet surface charge. ESI-MS measurements performed in negative ion mode on B4A and B4B also reveal a shift to higher charge states at low I but the magnitude of the changes are smaller than observed for B3C. PMID:22993046

Lin, Hong; Kitova, Elena N; Johnson, Margaret A; Eugenio, Luiz; Ng, Kenneth K S; Klassen, John S

2012-12-01

206

Activation of the unfolded protein response by 2-deoxy-D-glucose inhibits Kaposi's sarcoma-associated herpesvirus replication and gene expression.  

PubMed

Lytic replication of the Kaposi's sarcoma-associated herpesvirus (KSHV) is essential for the maintenance of both the infected state and characteristic angiogenic phenotype of Kaposi's sarcoma and thus represents a desirable therapeutic target. During the peak of herpesvirus lytic replication, viral glycoproteins are mass produced in the endoplasmic reticulum (ER). Normally, this leads to ER stress which, through an unfolded protein response (UPR), triggers phosphorylation of the ? subunit of eukaryotic initiation factor 2 (eIF2?), resulting in inhibition of protein synthesis to maintain ER and cellular homeostasis. However, in order to replicate, herpesviruses have acquired the ability to prevent eIF2? phosphorylation. Here we show that clinically achievable nontoxic doses of the glucose analog 2-deoxy-d-glucose (2-DG) stimulate ER stress, thereby shutting down eIF2? and inhibiting KSHV and murine herpesvirus 68 replication and KSHV reactivation from latency. Viral cascade genes that are involved in reactivation, including the master transactivator (RTA) gene, glycoprotein B, K8.1, and angiogenesis-regulating genes are markedly decreased with 2-DG treatment. Overall, our data suggest that activation of UPR by 2-DG elicits an early antiviral response via eIF2? inactivation, which impairs protein synthesis required to drive viral replication and oncogenesis. Thus, induction of ER stress by 2-DG provides a new antiherpesviral strategy that may be applicable to other viruses. PMID:22926574

Leung, Howard J; Duran, Elda M; Kurtoglu, Metin; Andreansky, Samita; Lampidis, Theodore J; Mesri, Enrique A

2012-11-01

207

Overexpression of MHC class I in muscle of lymphocyte-deficient mice causes a severe myopathy with induction of the unfolded protein response.  

PubMed

Muscle fibers do not normally express major histocompatibility complex class I (MHC-I) molecules, and their reexpression is a hallmark of inflammatory myopathies. It has been shown in mice that overexpression of MHC-I induces a poorly inflammatory myositis accompanied by the unfolded protein response (UPR), but it is unclear whether it is attributable to T-cell-mediated MHC-I-dependent immune responses or to MHC-I forced expression per se. Indeed, besides presenting antigenic peptides to CD8(+) T cells, MHC-I may also possibly exert nonimmunologic, yet poorly understood pathogenic effects. Thus, we investigated the pathogenicity of MHC-I expression in muscle independently of its immune functions. HT transgenic mice that conditionally overexpress H-2K(b) in muscle were bred to an immunodeficient Rag2(-/-) background. The muscle proteome was analyzed by label-free high-resolution protein quantitation and Western blot. Despite the absence of adaptive immunity, HT Rag2(-/-) mice developed a very severe myopathy associated with the cytoplasmic accumulation of H-2K(b) molecules. The UPR was manifest by up-regulation of characteristic proteins. In humans, we found that HLA class I molecules not only were expressed at the sarcolemma but also could accumulate intracellularly in some patients with inclusion body myositis. Accordingly, the UPR was triggered as a function of the degree of HLA accumulation in myofibers. Hence, reexpression of MHC-I in normally negative myofibers exerts pathogenic effects independently of its immune function. PMID:23850081

Fréret, Manuel; Drouot, Laurent; Obry, Antoine; Ahmed-Lacheheb, Sandra; Dauly, Claire; Adriouch, Sahil; Cosette, Pascal; Authier, François-Jérôme; Boyer, Olivier

2013-09-01

208

Cell surface expression of bacterial esterase A by Saccharomyces cerevisiae and its enhancement by constitutive activation of the cellular unfolded protein response.  

PubMed

Yeast cell surface display is a powerful tool for expression and immobilization of biocatalytically active proteins on a unicellular eukaryote. Here bacterial carboxylesterase EstA from Burkholderia gladioli was covalently anchored into the cell wall of Saccharomyces cerevisiae by in-frame fusion to the endogenous yeast proteins Kre1p, Cwp2p, and Flo1p. When p-nitrophenyl acetate was used as a substrate, the esterase specific activities of yeast expressing the protein fusions were 103 mU mg(-1) protein for Kre1/EstA/Cwp2p and 72 mU mg(-1) protein for Kre1/EstA/Flo1p. In vivo cell wall targeting was confirmed by esterase solubilization after laminarinase treatment and immunofluorescence microscopy. EstA expression resulted in cell wall-associated esterase activities of 2.72 U mg(-1) protein for Kre1/EstA/Cwp2p and 1.27 U mg(-1) protein for Kre1/EstA/Flo1p. Furthermore, esterase display on the yeast cell surface enabled the cells to effectively grow on the esterase-dependent carbon source glycerol triacetate (Triacetin). In the case of Kre1/EstA/Flo1p, in vivo maturation within the yeast secretory pathway and final incorporation into the wall were further enhanced when there was constitutive activation of the unfolded protein response pathway. Our results demonstrate that esterase cell surface display in yeast, which, as shown here, is remarkably more effective than EstA surface display in Escherichia coli, can be further optimized by activating the protein folding machinery in the eukaryotic secretion pathway. PMID:16980424

Breinig, Frank; Diehl, Björn; Rau, Sabrina; Zimmer, Christian; Schwab, Helmut; Schmitt, Manfred J

2006-11-01

209

Characterization of protein unfolding with solid-state nanopores.  

PubMed

In this work, we review the process of protein unfolding characterized by a solid-state nanopore based device. The occupied or excluded volume of a protein molecule in a nanopore depends on the protein's conformation or shape. A folded protein has a larger excluded volume in a nanopore thus it blocks more ionic current flow than its unfolded form and produces a greater current blockage amplitude. The time duration a protein stays in a pore also depends on the protein's folding state. We use Bovine Serum Albumin (BSA) as a model protein to discuss this current blockage amplitude and the time duration associated with the protein unfolding process. BSA molecules were measured in folded, partially unfolded, and completely unfolded conformations in solid-state nanopores. We discuss experimental results, data analysis, and theoretical considerations of BSA protein unfolding measured with silicon nitride nanopores. We show this nanopore method is capable of characterizing a protein's unfolding process at single molecule level. Problems and future studies in characterization of protein unfolding using a solid-state nanopore device will also be discussed. PMID:24370259

Li, Jiali; Fologea, Daniel; Rollings, Ryan; Ledden, Brad

2014-03-01

210

Solvable model of mechanical unfolding of proteins  

NASA Astrophysics Data System (ADS)

We present exact analytical results describing single-molecule experiments on mechanical unfolding of proteins within a realistic model [1]. The corresponding relation between the extension at a given temperature of the macromolecule and the applied force is derived [2]. The configuration partition function is calculated exactly for a distance constraint protein model that describes the beta-hairpin to coil transition. The resulting extension-force curve is derived, and the results agree with the data from the single-molecule pulling experiments. [1] O.K.Vorov, A.Y.Istomin, D.R.Livesay, D.J.Jacobs, subm. to Phys.Rev.Lett., 2007. [2] O.K.Vorov, D.R.Livesay, D.J.Jacobs, to be subm. to Science, 2007, in preparation.

Vorov, Oleg; Livesay, Dennis; Jacobs, Donald

2008-03-01

211

Comparison of first dimension IPG and NEPHGE techniques in two-dimensional gel electrophoresis experiment with cytosolic unfolded protein response in Saccharomyces cerevisiae  

PubMed Central

Background Two-dimensional gel electrophoresis (2DE) is one of the most popular methods in proteomics. Currently, most 2DE experiments are performed using immobilized pH gradient (IPG) in the first dimension; however, some laboratories still use carrier ampholytes-based isoelectric focusing technique. The aim of this study was to directly compare IPG-based and non-equilibrium pH gradient electrophoresis (NEPHGE)-based 2DE techniques by using the same samples and identical second dimension procedures. We have used commercially available Invitrogen ZOOM IPGRunner and WITAvision systems for IPG and NEPHGE, respectively. The effectiveness of IPG-based and NEPHGE-based 2DE methods was compared by analysing differential protein expression during cytosolic unfolded protein response (UPR-Cyto) in Saccharomyces cerevisiae. Results Protein loss during 2DE procedure was higher in IPG-based method, especially for basic (pI?>?7) proteins. Overall reproducibility of spots was slightly better in NEPHGE-based method; however, there was a marked difference when evaluating basic and acidic protein spots. Using Coomassie staining, about half of detected basic protein spots were not reproducible by IPG-based 2DE, whereas NEPHGE-based method showed excellent reproducibility in the basic gel zone. The reproducibility of acidic proteins was similar in both methods. Absolute and relative volume variability of separate protein spots was comparable in both 2DE techniques. Regarding proteomic analysis of UPR-Cyto, the results exemplified parameters of general comparison of the methods. New highly basic protein Sis1p, overexpressed during UPR-Cyto stress, was identified by NEPHGE-based 2DE method, whereas IPG-based method showed unreliable results in the basic pI range and did not provide any new information on basic UPR-Cyto proteins. In the acidic range, the main UPR-Cyto proteins were detected and quantified by both methods. The drawback of NEPHGE-based 2DE method is its failure to detect some highly acidic proteins. The advantage of NEPHGE is higher protein capacity with good reproducibility and quality of spots at high protein load. Conclusions Comparison of broad range (pH 3–10) gradient-based 2DE methods suggests that NEPHGE-based method is preferable over IPG (Invitrogen) 2DE method for the analysis of basic proteins. Nevertheless, the narrow range (pH 4–7) IPG technique is a method of choice for the analysis of acidic proteins.

2013-01-01

212

TMBIM3/GRINA is a novel unfolded protein response (UPR) target gene that controls apoptosis through the modulation of ER calcium homeostasis  

PubMed Central

Transmembrane BAX inhibitor motif-containing (TMBIM)-6, also known as BAX-inhibitor 1 (BI-1), is an anti-apoptotic protein that belongs to a putative family of highly conserved and poorly characterized genes. Here we report the function of TMBIM3/GRINA in the control of cell death by endoplasmic reticulum (ER) stress. Tmbim3 mRNA levels are strongly upregulated in cellular and animal models of ER stress, controlled by the PERK signaling branch of the unfolded protein response. TMBIM3/GRINA synergies with TMBIM6/BI-1 in the modulation of ER calcium homeostasis and apoptosis, associated with physical interactions with inositol trisphosphate receptors. Loss-of-function studies in D. melanogaster demonstrated that TMBIM3/GRINA and TMBIM6/BI-1 have synergistic activities against ER stress in vivo. Similarly, manipulation of TMBIM3/GRINA levels in zebrafish embryos revealed an essential role in the control of apoptosis during neuronal development and in experimental models of ER stress. These findings suggest the existence of a conserved group of functionally related cell death regulators across species beyond the BCL-2 family of proteins operating at the ER membrane.

Rojas-Rivera, D; Armisen, R; Colombo, A; Martinez, G; Eguiguren, A L; Diaz, A; Kiviluoto, S; Rodriguez, D; Patron, M; Rizzuto, R; Bultynck, G; Concha, M L; Sierralta, J; Stutzin, A; Hetz, C

2012-01-01

213

Deregulated unfolded protein response in chronic wounds of diabetic ob/ob mice: a potential connection to inflammatory and angiogenic disorders in diabetes-impaired wound healing.  

PubMed

Type-2 diabetes mellitus (T2D) represents an important metabolic disorder, firmly connected to obesity and low level of chronic inflammation caused by deregulation of fat metabolism. The convergence of chronic inflammatory signals and nutrient overloading at the endoplasmic reticulum (ER) leads to activation of ER-specific stress responses, the unfolded protein response (UPR). As obesity and T2D are often associated with impaired wound healing, we investigated the role of UPR in the pathologic of diabetic-impaired cutaneuos wound healing. We determined the expression patterns of the three UPR branches during normal and diabetes-impaired skin repair. In healthy and diabetic mice, injury led to a strong induction of BiP (BiP/Grp78), C/EBP homologous protein (CHOP) and splicing of X-box-binding protein (XBP)1. Diabetic-impaired wounds showed gross and sustained induction of UPR associated with increased expression of the pro-inflammatory chemokine macrophage inflammatory protein (MIP)2 as compared to normal healing wounds. In vitro, treatment of RAW264.7 macrophages with tunicamycin, and subsequently stimulation with lipopolysaccharide (LPS) and interferon (IFN)-? enhances MIP2 mRNA und protein expression compared to proinflammatory stimulation alone. However, LPS/IFN? induced vascular endothelial growth factor (VEGF) production was blunted by tunicamycin induced-ER stress. Hence, UPR is activated following skin injury, and functionally connected to the production of proinflammatory mediators. In addition, prolongation of UPR in diabetic non-healing wounds aggravates ER stress and weakens the angiogenic phenotype of wound macrophages. PMID:24583133

Schürmann, Christoph; Goren, Itamar; Linke, Andreas; Pfeilschifter, Josef; Frank, Stefan

2014-03-28

214

Rice black-streaked dwarf virus P10 induces membranous structures at the ER and elicits the unfolded protein response in Nicotiana benthamiana.  

PubMed

Endoplasmic reticular (ER) membrane modifications play an important role in viral RNA replication and virion assembly but little is known about the involvement of ER-membrane remodeling in the infection cycle of fijiviruses in plant cells. The subcellular localization of Rice black-streaked dwarf virus outer capsid P10 was therefore examined using live-cell imaging. P10 fused to eGFP formed vesicular structures associated with ER membranes in Nicotiana benthamiana epidermal cells and in rice protoplasts. Subcellular fractionation experiments confirmed that P10 is an integral membrane protein. Three predicted transmembrane domains and two less-well-defined domains were each able to target eGFP to the ER. Disruption of the actin cytoskeleton with LatB, indicated that the maintenance of P10-induced membrane structures required the intact actin cytoskeleton. P10 induced the expression of ER stress marker genes, including ER stress-related chaperones and transcription factor, indicating that RBSDV P10 triggers ER stress and the unfolded protein response. PMID:24210107

Sun, Zongtao; Yang, Di; Xie, Li; Sun, Liying; Zhang, Shanglin; Zhu, Qisong; Li, Junmin; Wang, Xu; Chen, Jianping

2013-12-01

215

?B-crystallin, a effector of unfolded protein response, confers anti-VEGF resistance to breast cancer via maintenance of intracrine VEGF in endothelial cells  

PubMed Central

Effective inhibition of angiogenesis targeting the tumor endothelial cells requires identification of key cellular and molecular mechanisms associated with survival of vasculatures within the tumor microenvironment. Intracellular autocrine (intracrine) VEGF production by endothelial cells plays a critical role on the vasculature homeostasis. In vitro breast cancer cell-stimulated activation of the unfolded protein response (UPR) of the endothelial cells contributes to maintenance of the intracrine VEGF levels in the endothelial cells through the up-regulation of a previous un-described downstream effector- ?B-crystallin (CRYAB). Short interfering RNA-mediated knockdown of two major UPR proteins-IRE1 and ATF6, led to attenuated CRYAB expression of the endothelial cells. Finally, inhibition of CRYAB blocked the breast cancer cell-stimulated increase in the endogenous VEGF levels of the endothelial cells. A VEGF limited proteolysis assay further revealed that CRYAB protected VEGF for proteolytic degradation. Here we report that the molecular chaperone-CRYAB) was significantly increased and co-localized with tumor vessels in a breast cancer xenograft. Specifically, neutralization of VEGF induced higher levels of CRYAB expression in the endothelial cells co-cultured with MDA-MB-231 or the breast cancer xenograft with a significant survival benefit. However, knockdown of CRYAB had a greater inhibitory effect on endothelial survival. These findings underscore the importance of defining a role for intracrine VEGF signaling in sustaining aberrant tumor angiogenesis and strongly implicate UPR/CRYAB as dichotomous parts of a crucial regulation pathway for maintaining intracrine VEGF signaling.

Ruan, Qing; Han, Song; Jiang, Wen G.; Boulton, Michael E.; Chen, Zhi J.; Law, Brian K.; Cai, Jun

2013-01-01

216

Mutual Cross Talk between the Regulators Hac1 of the Unfolded Protein Response and Gcn4 of the General Amino Acid Control of Saccharomyces cerevisiae  

PubMed Central

Hac1 is the activator of the cellular response to the accumulation of unfolded proteins in the endoplasmic reticulum. Hac1 function requires the activity of Gcn4, which mainly acts as a regulator of the general amino acid control network providing Saccharomyces cerevisiae cells with amino acids. Here, we demonstrate novel functions of Hac1 and describe a mutual connection between Hac1 and Gcn4. Hac1 is required for induction of Gcn4-responsive promoter elements in haploid as well as diploid cells and therefore participates in the cellular amino acid supply. Furthermore, Hac1 and Gcn4 mutually influence their mRNA expression levels. Hac1 is also involved in FLO11 expression and adhesion upon amino acid starvation. Hac1 and Gcn4 act through the same promoter regions of the FLO11 flocculin. The results indicate an indirect effect of both transcription factors on FLO11 expression. Our data suggest a complex mutual cross talk between the Hac1- and Gcn4-controlled networks.

Herzog, Britta; Popova, Blagovesta; Jakobshagen, Antonia; Shahpasandzadeh, Hedieh

2013-01-01

217

Failure of the adaptive unfolded protein response in islets of obese mice is linked with abnormalities in ?-cell gene expression and progression to diabetes.  

PubMed

The normal ?-cell response to obesity-associated insulin resistance is hypersecretion of insulin. Type 2 diabetes develops in subjects with ?-cells that are susceptible to failure. Here, we investigated the time-dependent gene expression changes in islets of diabetes-prone db/db and diabetes-resistant ob/ob mice. The expressions of adaptive unfolded protein response (UPR) genes were progressively induced in islets of ob/ob mice, whereas they declined in diabetic db/db mice. Genes important for ?-cell function and maintenance of the islet phenotype were reduced with time in db/db mice, whereas they were preserved in ob/ob mice. Inflammation and antioxidant genes displayed time-dependent upregulation in db/db islets but were unchanged in ob/ob islets. Treatment of db/db mouse islets with the chemical chaperone 4-phenylbutyric acid partially restored the changes in several ?-cell function genes and transcription factors but did not affect inflammation or antioxidant gene expression. These data suggest that the maintenance (or suppression) of the adaptive UPR is associated with ?-cell compensation (or failure) in obese mice. Inflammation, oxidative stress, and a progressive loss of ?-cell differentiation accompany diabetes progression. The ability to maintain the adaptive UPR in islets may protect against the gene expression changes that underlie diabetes development in obese mice. PMID:23274897

Chan, Jeng Yie; Luzuriaga, Jude; Bensellam, Mohammed; Biden, Trevor J; Laybutt, D Ross

2013-05-01

218

Failure of the Adaptive Unfolded Protein Response in Islets of Obese Mice Is Linked With Abnormalities in ?-Cell Gene Expression and Progression to Diabetes  

PubMed Central

The normal ?-cell response to obesity-associated insulin resistance is hypersecretion of insulin. Type 2 diabetes develops in subjects with ?-cells that are susceptible to failure. Here, we investigated the time-dependent gene expression changes in islets of diabetes-prone db/db and diabetes-resistant ob/ob mice. The expressions of adaptive unfolded protein response (UPR) genes were progressively induced in islets of ob/ob mice, whereas they declined in diabetic db/db mice. Genes important for ?-cell function and maintenance of the islet phenotype were reduced with time in db/db mice, whereas they were preserved in ob/ob mice. Inflammation and antioxidant genes displayed time-dependent upregulation in db/db islets but were unchanged in ob/ob islets. Treatment of db/db mouse islets with the chemical chaperone 4-phenylbutyric acid partially restored the changes in several ?-cell function genes and transcription factors but did not affect inflammation or antioxidant gene expression. These data suggest that the maintenance (or suppression) of the adaptive UPR is associated with ?-cell compensation (or failure) in obese mice. Inflammation, oxidative stress, and a progressive loss of ?-cell differentiation accompany diabetes progression. The ability to maintain the adaptive UPR in islets may protect against the gene expression changes that underlie diabetes development in obese mice.

Chan, Jeng Yie; Luzuriaga, Jude; Bensellam, Mohammed; Biden, Trevor J.; Laybutt, D. Ross

2013-01-01

219

Genome Wide Analysis Reveals Inositol, not Choline, as the Major Effector of Ino2p-Ino4p and Unfolded Protein Response Target Gene Expression in Yeast  

PubMed Central

SUMMARY In the yeast Saccharomyces cerevisiae the transcription of many genes encoding enzymes of phospholipid biosynthesis are repressed in cells grown in the presence of the phospholipid precursors inositol and choline. A genome-wide approach using cDNA microarray technology was utilized to profile the changes in the expression of all genes in yeast that respond to the exogenous presence of inositol and choline. We report that the global response to inositol is completely distinct from the effect of choline. Whereas the effect of inositol on gene expression was primarily repressing, the effect of choline on gene expression was activating. Moreover, the combination inositol and choline increased the number of repressed genes compared to inositol alone and enhanced the repression levels of a subset of genes that responded to inositol. In all, 110 genes were repressed in the presence of inositol and choline. Two distinct sets of genes exhibited differential expression in response to inositol or the combination of inositol and choline in wild type cells. One set of genes contained the UASINO sequence and were bound by Ino2p and Ino4p. Many of these genes were also negatively regulated by OPI1, suggesting a common regulatory mechanism for Ino2p, Ino4p, and Opi1p. Another non-overlapping set of genes were coregulated by the unfolded protein response pathway, an ER-localized stress response pathway, but were not dependent on OPI1 and did not show further repression when choline was present together with inositol. These results suggest that inositol is the major effector of target gene expression, while choline plays a minor role.

Jesch, Stephen A.; Zhao, Xin; Wells, Martin T.; Henry, Susan A.

2005-01-01

220

Unfolding study of a trimeric membrane protein AcrB.  

PubMed

The folding of a multi-domain trimeric ?-helical membrane protein, Escherichia coli inner membrane protein AcrB, was investigated. AcrB contains both a transmembrane domain and a large periplasmic domain. Protein unfolding in sodium dodecyl sulfate (SDS) and urea was monitored using the intrinsic fluorescence and circular dichroism spectroscopy. The SDS denaturation curve displayed a sigmoidal profile, which could be fitted with a two-state unfolding model. To investigate the unfolding of separate domains, a triple mutant was created, in which all three Trp residues in the transmembrane domain were replaced with Phe. The SDS unfolding profile of the mutant was comparable to that of the wild type AcrB, suggesting that the observed signal change was largely originated from the unfolding of the soluble domain. Strengthening of trimer association through the introduction of an inter-subunit disulfide bond had little effect on the unfolding profile, suggesting that trimer dissociation was not the rate-limiting step in unfolding monitored by fluorescence emission. Under our experimental condition, AcrB unfolding was not reversible. Furthermore, we experimented with the refolding of a monomeric mutant, AcrB?loop , from the SDS unfolded state. The CD spectrum of the refolded AcrB?loop superimposed well onto the spectra of the original folded protein, while the fluorescence spectrum was not fully recovered. In summary, our results suggested that the unfolding of the trimeric AcrB started with a local structural rearrangement. While the refolding of secondary structure in individual monomers could be achieved, the re-association of the trimer might be the limiting factor to obtain folded wild-type AcrB. PMID:24715637

Ye, Cui; Wang, Zhaoshuai; Lu, Wei; Wei, Yinan

2014-07-01

221

Thermodynamic cycles as probes of structure in unfolded proteins.  

PubMed

The relationship between structure and stability has been investigated for the folded forms and the unfolded forms of iso-2 cytochrome c and a variant protein with a stability-enhancing mutation, N52I iso-2. Differential scanning calorimetry has been used to measure the reversible unfolding transitions for the proteins in both heme oxidation states. Reduction potentials have been measured as a function of temperature for the folded forms of the proteins. The combination of measurements of thermal stability and reduction potential gives three sides of a thermodynamic cycle and allows prediction of the reduction potential of the thermally unfolded state. The free energies of electron binding for the thermally unfolded proteins differ from those expected for a fully unfolded protein, suggesting that residual structure modulates the reduction potential. At temperatures near 50 degrees C the N52I mutation has a small but significant effect on oxidation state-sensitive structure in the thermally unfolded protein. Inspection of the high-resolution X-ray crystallographic structures of iso-2 and N52I iso-2 shows that the effects of the N52I mutation and oxidation state on native protein stability are correlated with changes in the mobility of specific polypeptide chain segments and with altered hydrogen bonding involving a conserved water molecule. However, there is no clear explanation of oxidation state or mutation-induced differences in stability of the proteins in terms of observed changes in structure and mobility of the folded forms of the proteins alone. PMID:8639684

McGee, W A; Rosell, F I; Liggins, J R; Rodriguez-Ghidarpour, S; Luo, Y; Chen, J; Brayer, G D; Mauk, A G; Nall, B T

1996-02-13

222

Folding of an unfolded protein by macromolecular crowding in vitro.  

PubMed

Protein folding in vivo takes place in a highly crowded environment. The resulting excluded volume forces are thought to stabilize folded forms of proteins. In agreement, many in vitro studies have shown that the presence of macromolecular crowding agents increases the stability of folded proteins but often by only a few kJ per mol. Although it should not matter at what position in the transition between folded and unfolded forms the effect of crowding is employed, there have been no studies assessing whether excluded volume forces alone can correctly fold polypeptides that are mostly unfolded. However, some studies have indicated that the effect of crowding becomes larger the more destabilized the protein is (but still being folded), suggesting that the crowding effect may be exaggerated for unfolded proteins. To address this question directly, we turned to a destabilized mutant of protein L that is mostly unfolded in water but can be folded upon addition of salt. We find that the effect of 200 mg/mL Dextran 20 on the folding equilibrium constant for unfolded protein L (??GU ? 2 kJ mol(-1)) matches the crowding effects found on the folded wild type protein and the mutant when prefolded by salt. This result indicates that the excluded volume effect is independent of starting protein stability and that crowding can shift the reaction toward the folded form when the polypeptide is in the transition region between folded and unfolded states. PMID:24665900

Adén, Jörgen; Wittung-Stafshede, Pernilla

2014-04-15

223

Unfolded protein stress in the endoplasmic reticulum and mitochondria: a role in neurodegeneration  

PubMed Central

Protein-folding occurs in several intracellular locations including the endoplasmic reticulum and mitochondria. In normal conditions there is a balance between the levels of unfolded proteins and protein folding machinery. Disruption of homeostasis and an accumulation of unfolded proteins trigger stress responses, or unfolded protein responses (UPR), in these organelles. These pathways signal to increase the folding capacity, inhibit protein import or expression, increase protein degradation, and potentially trigger cell death. Many aging-related neurodegenerative diseases involve the accumulation of misfolded proteins in both the endoplasmic reticulum and mitochondria. The exact participation of the UPRs in the onset of neurodegeneration is unclear, but there is significant evidence for the alteration of these pathways in the endoplasmic reticulum and mitochondria. Here we will discuss the involvement of endoplasmic reticulum and mitochondrial stress and the possible contributions of the UPR in these organelles to the development of two neurodegenerative diseases, Parkinson's disease (PD) and Alzheimer's disease (AD).

Bernales, Sebastian; Soto, Marisol Morales; McCullagh, Emma

2012-01-01

224

S(+)-ibuprofen destabilizes MYC/MYCN and AKT, increases p53 expression, and induces unfolded protein response and favorable phenotype in neuroblastoma cell lines  

PubMed Central

Neuroblastoma is a common pediatric solid tumor that exhibits a striking clinical bipolarity favorable and unfavorable. The survival rate of children with unfavorable neuroblastoma remains low among all childhood cancers. MYCN and MYC play a crucial role in determining the malignancy of unfavorable neuroblastomas, whereas high-level expression of the favorable neuroblastoma genes is associated with a good disease outcome and confers growth suppression of neuroblastoma cells. A small fraction of neuroblastomas harbors TP53 mutations at diagnosis, but a higher proportion of the relapse cases acquire TP53 mutations. In this study, we investigated the effect of S(+)-ibuprofen on neuroblastoma cell lines, focusing on the expression of the MYCN, MYC, AKT, p53 proteins and the favorable neuroblastoma genes in vitro as biomarkers of malignancy. Treatment of neuroblastoma cell lines with S(+)-ibuprofen resulted in a significant growth suppression. This growth effect was accompanied by a marked decrease in the expression of MYC, MYCN, AKT and an increase in p53 expression in neuroblastoma cell lines without TP53 mutation. In addition, S(+)-ibuprofen enhanced the expression of some favorable neuroblastoma genes (EPHB6, CD44) and genes involved in growth suppression and differentiation (EGR1, EPHA2, NRG1 and SEL1L). Gene expression profile and Ingenuity pathway analyses using TP53-mutated SKNAS cells further revealed that S(+)-ibuprofen suppressed molecular pathways associated with cell growth and conversely enhanced those of cell cycle arrest and the unfolded protein response. Collectively, these results suggest that S(+)-ibuprofen or its related compounds may have the potential for therapeutic and/or palliative use for unfavorable neuroblastoma.

IKEGAKI, NAOHIKO; HICKS, SAKEENAH L.; REGAN, PAUL L.; JACOBS, JOSHUA; JUMBO, AMINA S.; LEONHARDT, PAYTON; RAPPAPORT, ERIC F.; TANG, XAO X.

2014-01-01

225

S(+)-ibuprofen destabilizes MYC/MYCN and AKT, increases p53 expression, and induces unfolded protein response and favorable phenotype in neuroblastoma cell lines.  

PubMed

Neuroblastoma is a common pediatric solid tumor that exhibits a striking clinical bipolarity: favorable and unfavorable. The survival rate of children with unfavorable neuroblastoma remains low among all childhood cancers. MYCN and MYC play a crucial role in determining the malignancy of unfavorable neuroblastomas, whereas high-level expression of the favorable neuroblastoma genes is associated with a good disease outcome and confers growth suppression of neuroblastoma cells. A small fraction of neuroblastomas harbors TP53 mutations at diagnosis, but a higher proportion of the relapse cases acquire TP53 mutations. In this study, we investigated the effect of S(+)-ibuprofen on neuroblastoma cell lines, focusing on the expression of the MYCN, MYC, AKT, p53 proteins and the favorable neuroblastoma genes in vitro as biomarkers of malignancy. Treatment of neuroblastoma cell lines with S(+)-ibuprofen resulted in a significant growth suppression. This growth effect was accompanied by a marked decrease in the expression of MYC, MYCN, AKT and an increase in p53 expression in neuroblastoma cell lines without TP53 mutation. In addition, S(+)-ibuprofen enhanced the expression of some favorable neuroblastoma genes (EPHB6, CD44) and genes involved in growth suppression and differentiation (EGR1, EPHA2, NRG1 and SEL1L). Gene expression profile and Ingenuity pathway analyses using TP53-mutated SKNAS cells further revealed that S(+)-ibuprofen suppressed molecular pathways associated with cell growth and conversely enhanced those of cell cycle arrest and the unfolded protein response. Collectively, these results suggest that S(+)-ibuprofen or its related compounds may have the potential for therapeutic and/or palliative use for unfavorable neuroblastoma. PMID:24173829

Ikegaki, Naohiko; Hicks, Sakeenah L; Regan, Paul L; Jacobs, Joshua; Jumbo, Amina S; Leonhardt, Payton; Rappaport, Eric F; Tang, Xao X

2014-01-01

226

Modulation of the rate of retinal degeneration in T17M RHO mice by reprogramming the Unfolded Protein Response  

PubMed Central

The goal of this study is to validate whether reprogramming of the UPR via modulation of pro-apoptotic caspase-7 and CHOP proteins could be an effective approach to slow down the rate of retinal degeneration in ADRP mice. In order to pursue our goal we created the T17M RHO CASP7 and T17M RHO CHOP mice to study the impact of the CASP7 or CHOP ablations in T17M RHO retina by ERG, SD-OCT, histology and western blot analysis. The scotopic ERG demonstrated that the ablation of the CASP7 in T17M RHO retina leads to significant preservation of the function of photoreceptors compared to control. Surprisingly, the ablation of pro-apoptotic CHOP protein in T17M RHO mice led to a more severe form of retinal degeneration. Results of the SD-OCT and histology were in agreement with the ERG data. The further analysis demonstrated that the preservation of the structure and function or the acceleration of the onset of the T17M RHO photoreceptor degeneration occurred via reprogramming of the UPR. In addition, the CASP7 ablation leads to the inhibition of cJUN mediated apoptosis, while the ablation of CHOP induces an increase in the HDAC. Thus, manipulation with the UPR requires careful examination in order to achieve a therapeutic effect.

Choudhury, Shreyasi; Nashine, Sonali; Bhootada, Yogesh; Kunte, Mansi Motiwale; Gorbatyuk, Oleg; Lewin, Alfred S.; Gorbatyuk, Marina

2014-01-01

227

Unfolded protein ensembles, folding trajectories, and refolding rate prediction  

NASA Astrophysics Data System (ADS)

Computer simulations can provide critical information on the unfolded ensemble of proteins under physiological conditions, by explicitly characterizing the geometrical properties of the diverse conformations that are sampled in the unfolded state. A general computational analysis across many proteins has not been implemented however. Here, we develop a method for generating a diverse conformational ensemble, to characterize properties of the unfolded states of intrinsically disordered or intrinsically folded proteins. The method allows unfolded proteins to retain disulfide bonds. We examined physical properties of the unfolded ensembles of several proteins, including chemical shifts, clustering properties, and scaling exponents for the radius of gyration with polymer length. A problem relating simulated and experimental residual dipolar couplings is discussed. We apply our generated ensembles to the problem of folding kinetics, by examining whether the ensembles of some proteins are closer geometrically to their folded structures than others. We find that for a randomly selected dataset of 15 non-homologous 2- and 3-state proteins, quantities such as the average root mean squared deviation between the folded structure and unfolded ensemble correlate with folding rates as strongly as absolute contact order. We introduce a new order parameter that measures the distance travelled per residue, which naturally partitions into a smooth ``laminar'' and subsequent ``turbulent'' part of the trajectory. This latter conceptually simple measure with no fitting parameters predicts folding rates in 0 M denaturant with remarkable accuracy (r = -0.95, p = 1 × 10-7). The high correlation between folding times and sterically modulated, reconfigurational motion supports the rapid collapse of proteins prior to the transition state as a generic feature in the folding of both two-state and multi-state proteins. This method for generating unfolded ensembles provides a powerful approach to address various questions in protein evolution, misfolding and aggregation, transient structures, and molten globule and disordered protein phases.

Das, A.; Sin, B. K.; Mohazab, A. R.; Plotkin, S. S.

2013-09-01

228

Increasing Temperature Accelerates Protein Unfolding Without Changing the Pathway of Unfolding  

Microsoft Academic Search

We have traditionally relied on extremely elevated temperatures (498K, 225°C) to investigate the unfolding process of proteins within the timescale available to molecular dynamics simulations with explicit solvent. However, recent advances in computer hardware have allowed us to extend our thermal denaturation studies to much lower temperatures. Here we describe the results of simulations of chymotrypsin inhibitor 2 at seven

Ryan Day; Brian J Bennion; Sihyun Ham; Valerie Daggett

2002-01-01

229

HLA-B27 Misfolding and the Unfolded Protein Response Augment IL-23 Production and are Associated with Th17 Activation in Transgenic Rats1  

PubMed Central

Objective To determine whether HLA-B27 misfolding and the unfolded protein response (UPR) results in cytokine dysregulation, and whether this is associated with Th1 and/or Th17 activation in HLA-B27/human beta-2-microglobulin (h?2m) transgenic rats (HLA-B27 transgenic), an animal model of spondyloarthropathy. Methods Cytokine expression in LPS-stimulated macrophages was analyzed in the presence and absence of a UPR induced by chemical agents or HLA-B27 upregulation. Cytokine expression in colon tissue and in cells purified from the lamina propria was determined by real time reverse-transcriptase polymerase chain reaction, and differences in Th1 and Th17 CD4+ T cell populations were quantified with intracellular cytokine staining. Results IL-23 was found to be synergistically upregulated by LPS in macrophages undergoing a UPR induced either by pharmacologic agents or HLA-B27 misfolding. IL-23 was also increased in the colon of HLA-B27 transgenic rats concurrent with the development of intestinal inflammation, and IL-17, a downstream target of IL-23, exhibited robust upregulation in a similar temporal pattern. IL-23 and IL-17 transcripts were localized to CD11+ antigen presenting cells and CD4+ T cells, respectively, from the colonic lamina propria. Colitis was associated with a 6-fold expansion of CD4+ IL-17-expressing T cells. Conclusion The IL-23/IL-17 axis is strongly activated in the colon of HLA-B27 transgenic rats with spondyloarthropathy-like disease. HLA-B27 misfolding and UPR activation in macrophages can result in enhanced induction of the pro-Th17 cytokine, IL-23. These results suggest a possible link between HLA-B27 misfolding and immune dysregulation in this animal model with implications for human disease.

DeLay, Monica L.; Turner, Matthew J.; Klenk, Erin I.; Smith, Judith A.; Sowders, Dawn P.; Colbert, Robert A.

2010-01-01

230

D-Penicillamine targets metastatic melanoma cells with induction of the unfolded protein response (UPR) and Noxa (PMAIP1)-dependent mitochondrial apoptosis  

PubMed Central

D-penicillamine (3,3-Dimethyl-D-cysteine; DP) is an FDA-approved redox-active D-cysteine-derivative with antioxidant, disulfide-reducing, and metal chelating properties used therapeutically for the control of copper-related pathology in Wilson’s disease and reductive cystine-solubilization in cystinuria. Based on the established sensitivity of metastatic melanoma cells to pharmacological modulation of cellular oxidative stress, we tested feasibility of using DP for chemotherapeutic intervention targeting human A375 melanoma cells in vitro and in vivo. DP treatment induced caspase-dependent cell death in cultured human metastatic melanoma cells (A375, G361) without compromising viability of primary epidermal melanocytes, an effect not observed with the thiol-antioxidants N-acetyl-L-cysteine (NAC) and dithiothreitol. Focused gene expression array analysis followed by immunoblot detection revealed that DP rapidly activates the cytotoxic unfolded protein response (UPR; involving phospho-PERK, phospho-eIF2?, Grp78, CHOP, and Hsp70) and the mitochondrial pathway of apoptosis with p53 upregulation and modulation of Bcl-2 family members (involving Noxa, Mcl-1, and Bcl-2). DP (but not NAC) induced oxidative stress with early impairment of glutathione homeostasis and mitochondrial transmembrane potential. SiRNA-based antagonism of PMAIP1 expression blocked DP-induced upregulation of the proapoptotic BH3-only effector Noxa and prevented downregulation of the Noxa-antagonist Mcl-1, rescuing melanoma cells from DP-induced apoptosis. Intraperitoneal administration of DP displayed significant antimelanoma activity in a murine A375 xenograft model. It remains to be seen if melanoma cell-directed induction of UPR and apoptosis using DP or improved DP-derivatives can be harnessed for future chemotherapeutic intervention.

Qiao, Shuxi; Cabello, Christopher M.; Lamore, Sarah D.; Lesson, Jessica L.; Wondrak, Georg T.

2013-01-01

231

N-Octanoyl Dopamine Treatment of Endothelial Cells Induces the Unfolded Protein Response and Results in Hypometabolism and Tolerance to Hypothermia  

PubMed Central

Aim N-acyl dopamines (NADD) are gaining attention in the field of inflammatory and neurological disorders. Due to their hydrophobicity, NADD may have access to the endoplasmic reticulum (ER). We therefore investigated if NADD induce the unfolded protein response (UPR) and if this in turn influences cell behaviour. Methods Genome wide gene expression profiling, confirmatory qPCR and reporter assays were employed on human umbilical vein endothelial cells (HUVEC) to validate induction of UPR target genes and UPR sensor activation by N-octanoyl dopamine (NOD). Intracellular ATP, apoptosis and induction of thermotolerance were used as functional parameters to assess adaptation of HUVEC. Results NOD, but not dopamine dose dependently induces the UPR. This was also found for other synthetic NADD. Induction of the UPR was dependent on the redox activity of NADD and was not caused by selective activation of a particular UPR sensor. UPR induction did not result in cell apoptosis, yet NOD strongly impaired cell proliferation by attenuation of cells in the S-G2/M phase. Long-term treatment of HUVEC with low NOD concentration showed decreased intracellular ATP concentration paralleled with activation of AMPK. These cells were significantly more resistant to cold inflicted injury. Conclusions We provide for the first time evidence that NADD induce the UPR in vitro. It remains to be assessed if UPR induction is causally associated with hypometabolism and thermotolerance. Further pharmacokinetic studies are warranted to address if the NADD concentrations used in vitro can be obtained in vivo and if this in turn shows therapeutic efficacy.

Stamellou, Eleni; Fontana, Johann; Wedel, Johannes; Ntasis, Emmanouil; Sticht, Carsten; Becker, Anja; Pallavi, Prama; Wolf, Kerstin; Kramer, Bernhard K.; Hafner, Mathias; van Son, Willem J.; Yard, Benito A.

2014-01-01

232

D-Penicillamine targets metastatic melanoma cells with induction of the unfolded protein response (UPR) and Noxa (PMAIP1)-dependent mitochondrial apoptosis.  

PubMed

D-Penicillamine (3,3-dimethyl-D-cysteine; DP) is an FDA-approved redox-active D-cysteine-derivative with antioxidant, disulfide-reducing, and metal chelating properties used therapeutically for the control of copper-related pathology in Wilson's disease and reductive cystine-solubilization in cystinuria. Based on the established sensitivity of metastatic melanoma cells to pharmacological modulation of cellular oxidative stress, we tested feasibility of using DP for chemotherapeutic intervention targeting human A375 melanoma cells in vitro and in vivo. DP treatment induced caspase-dependent cell death in cultured human metastatic melanoma cells (A375, G361) without compromising viability of primary epidermal melanocytes, an effect not observed with the thiol-antioxidants N-acetyl-L-cysteine (NAC) and dithiothreitol. Focused gene expression array analysis followed by immunoblot detection revealed that DP rapidly activates the cytotoxic unfolded protein response (UPR; involving phospho-PERK, phospho-eIF2?, Grp78, CHOP, and Hsp70) and the mitochondrial pathway of apoptosis with p53 upregulation and modulation of Bcl-2 family members (involving Noxa, Mcl-1, and Bcl-2). DP (but not NAC) induced oxidative stress with early impairment of glutathione homeostasis and mitochondrial transmembrane potential. SiRNA-based antagonism of PMAIP1 expression blocked DP-induced upregulation of the proapoptotic BH3-only effector Noxa and prevented downregulation of the Noxa-antagonist Mcl-1, rescuing melanoma cells from DP-induced apoptosis. Intraperitoneal administration of DP displayed significant antimelanoma activity in a murine A375 xenograft model. It remains to be seen if melanoma cell-directed induction of UPR and apoptosis using DP or improved DP-derivatives can be harnessed for future chemotherapeutic intervention. PMID:22843330

Qiao, Shuxi; Cabello, Christopher M; Lamore, Sarah D; Lesson, Jessica L; Wondrak, Georg T

2012-10-01

233

Dual Regulation of Cadmium-Induced Apoptosis by mTORC1 through Selective Induction of IRE1 Branches in Unfolded Protein Response  

PubMed Central

Cadmium (Cd) causes generation of reactive oxygen species (ROS) that trigger renal tubular injury. We found that rapamycin, an inhibitor of mTORC1, attenuated Cd-induced apoptosis in renal tubular cells. Knockdown of Raptor, a positive regulator of mTORC1, also had the similar effect. However, rapamycin did not alter generation of ROS, suggesting that mTORC1 is a target downstream of ROS. Indeed, ROS caused activation of mTORC1, which contributed to induction of a selective branch of the unfolded protein response (UPR); i.e., the IRE1 pathway. Although Cd triggered three major UPR pathways, activation of mTORC1 by Cd did not contribute to induction of the PERK–eIF2? and ATF6 pathways. Consistently, knockdown of Raptor caused suppression of JNK without affecting the PERK–eIF2? pathway in Cd-exposed cells. Knockdown of TSC2, a negative regulator of mTORC1, caused activation of mTORC1 and enhanced Cd induction of the IRE1–JNK pathway and apoptosis without affecting other UPR branches. Inhibition of IRE1? kinase led to suppression of JNK activity and apoptosis in Cd-treated cells. Dominant-negative inhibition of JNK also suppressed Cd-induced apoptosis. In contrast, inhibition of IRE1? endoribonuclease activity or downstream XBP1 modestly enhanced Cd-induced apoptosis. In vivo, administration with rapamycin suppressed activation of mTORC1 and JNK, but not eIF2?, in the kidney of Cd-treated mice. It was correlated with attenuation of tubular injury and apoptotic cell death in the tubules. These results elucidate dual regulation of Cd-induced renal injury by mTORC1 through selective induction of IRE1 signaling.

Kato, Hironori; Katoh, Ryohei; Kitamura, Masanori

2013-01-01

234

Quality control for unfolded proteins at the plasma membrane  

PubMed Central

Cellular protein homeostasis profoundly depends on the disposal of terminally damaged polypeptides. To demonstrate the operation and elucidate the molecular basis of quality control of conformationally impaired plasma membrane (PM) proteins, we constructed CD4 chimeras containing the wild type or a temperature-sensitive bacteriophage ? domain in their cytoplasmic region. Using proteomic, biochemical, and genetic approaches, we showed that thermal unfolding of the ? domain at the PM provoked the recruitment of Hsp40/Hsc70/Hsp90 chaperones and the E2–E3 complex. Mixed-chain polyubiquitination, monitored by bioluminescence resonance energy transfer and immunoblotting, is responsible for the nonnative chimera–accelerated internalization, impaired recycling, and endosomal sorting complex required for transport–dependent lysosomal degradation. A similar paradigm prevails for mutant dopamine D4.4 and vasopressin V2 receptor removal from the PM. These results outline a peripheral proteostatic mechanism in higher eukaryotes and its potential contribution to the pathogenesis of a subset of conformational diseases.

Apaja, Pirjo M.; Xu, Haijin

2010-01-01

235

Studying the unfolding process of protein G and protein L under physical property space  

PubMed Central

Background The studies on protein folding/unfolding indicate that the native state topology is an important determinant of protein folding mechanism. The folding/unfolding behaviors of proteins which have similar topologies have been studied under Cartesian space and the results indicate that some proteins share the similar folding/unfolding characters. Results We construct physical property space with twelve different physical properties. By studying the unfolding process of the protein G and protein L under the property space, we find that the two proteins have the similar unfolding pathways that can be divided into three types and the one which with the umbrella-shape represents the preferred pathway. Moreover, the unfolding simulation time of the two proteins is different and protein L unfolding faster than protein G. Additionally, the distributing area of unfolded state ensemble of protein L is larger than that of protein G. Conclusion Under the physical property space, the protein G and protein L have the similar folding/unfolding behaviors, which agree with the previous results obtained from the studies under Cartesian coordinate space. At the same time, some different unfolding properties can be detected easily, which can not be analyzed under Cartesian coordinate space.

Zhao, Liling; Wang, Jihua; Dou, Xianghua; Cao, Zanxia

2009-01-01

236

GroEL stimulates protein folding through forced unfolding  

PubMed Central

Many proteins cannot fold without the assistance of chaperonin machines like GroEL and GroES. The nature of this assistance, however, remains poorly understood. Here we demonstrate that unfolding of a substrate protein by GroEL enhances protein folding. We first show that capture of a protein on the open ring of a GroEL–ADP–GroES complex, GroEL’s physiological acceptor state for non-native proteins in vivo, leaves the substrate protein in an unexpectedly compact state. Subsequent binding of ATP to the same GroEL ring causes rapid, forced unfolding of the substrate protein. Notably, the fraction of the substrate protein that commits to the native state following GroES binding and protein release into the GroEL–GroES cavity is proportional to the extent of substrate-protein unfolding. Forced protein unfolding is thus a central component of the multilayered stimulatory mechanism used by GroEL to drive protein folding.

Lin, Zong; Madan, Damian; Rye, Hays S

2013-01-01

237

Forced unfolding of protein domains determines cytoskeletal rheology  

NASA Astrophysics Data System (ADS)

Cells have recently been shown to have a power-law dynamic shear modulus over wide frequency range; the value of the exponent being non-universal, varying from 0.1-0.25 depending on cell type. This observation has been interpreted as evidence for the Soft Glassy Rheology (SGR) model, a trap-type glass model with an effective granular temperature. We propose a simple, alternative model of cytoskeletal mechanics based on the thermally activated, forced unfolding of domains in proteins cross-linking a stressed semi-flexible polymer gel. It directly relates a cell’s mechanical response to biophysical parameters of the cytoskeleton’s molecular constituents. Simulations indicate that unfolding events in a random network display a collective self-organization, giving rise to an exponential distribution of crosslink stress that can reproduce cell viscoelasticity. The model suggests natural explanations for the observed correlation between cell rheology and intracellular static stress, including those previously explained using the tensegrity concept. Moreover, our model provides insight into potential mechanisms of mechanotransduction as well as cell shape sensing and maintenance.

Crocker, John

2005-03-01

238

Effect of antimicrobial preservatives on partial protein unfolding and aggregation.  

PubMed

One-third of protein formulations are multi-dose. These require antimicrobial preservatives (APs); however, some APs have been shown to cause protein aggregation. Our previous work on a model protein cytochrome c indicated that partial protein unfolding, rather than complete unfolding, triggers aggregation. Here, we examined the relative strength of five commonly used APs on such unfolding and aggregation, and explored whether stabilizing the aggregation 'hot-spot' reduces such aggregation. All APs induced protein aggregation in the order m-cresol > phenol > benzyl alcohol > phenoxyethanol > chlorobutanol. All these enhanced the partial protein unfolding that includes a local region which was predicted to be the aggregation 'hot-spot'. The extent of destabilization correlated with the extent of aggregation. Further, we show that stabilizing the 'hot-spot' reduces aggregation induced by all five APs. These results indicate that m-cresol causes the most protein aggregation, whereas chlorobutanol causes the least protein aggregation. The same protein region acts as the 'hot-spot' for aggregation induced by different APs, implying that developing strategies to prevent protein aggregation induced by one AP will also work for others. PMID:23169345

Hutchings, Regina L; M Singh, Surinder; Cabello-Villegas, Javier; Mallela, Krishna M G

2013-02-01

239

Mechanically Unfolding the Small, Topologically Simple Protein L  

PubMed Central

?-sheet proteins are generally more able to resist mechanical deformation than ?-helical proteins. Experiments measuring the mechanical resistance of ?-sheet proteins extended by their termini led to the hypothesis that parallel, directly hydrogen-bonded terminal ?-strands provide the greatest mechanical strength. Here we test this hypothesis by measuring the mechanical properties of protein L, a domain with a topology predicted to be mechanically strong, but with no known mechanical function. A pentamer of this small, topologically simple protein is resistant to mechanical deformation over a wide range of extension rates. Molecular dynamics simulations show the energy landscape for protein L is highly restricted for mechanical unfolding and that this protein unfolds by the shearing apart of two structural units in a mechanism similar to that proposed for ubiquitin, which belongs to the same structural class as protein L, but unfolds at a significantly higher force. These data suggest that the mechanism of mechanical unfolding is conserved in proteins within the same fold family and demonstrate that although the topology and presence of a hydrogen-bonded clamp are of central importance in determining mechanical strength, hydrophobic interactions also play an important role in modulating the mechanical resistance of these similar proteins.

Brockwell, David J.; Beddard, Godfrey S.; Paci, Emanuele; West, Dan K.; Olmsted, Peter D.; Smith, D. Alastair; Radford, Sheena E.

2005-01-01

240

Protein Unfolding under Force: Crack Propagation in a Network  

PubMed Central

The mechanical unfolding of a set of 12 proteins with diverse topologies is investigated using an all-atom constraint-based model. Proteins are represented as polypeptides cross-linked by hydrogen bonds, salt bridges, and hydrophobic contacts, each modeled as a harmonic inequality constraint capable of supporting a finite load before breaking. Stereochemically acceptable unfolding pathways are generated by minimally overloading the network in an iterative fashion, analogous to crack propagation in solids. By comparing the pathways to those from molecular dynamics simulations and intermediates identified from experiment, it is demonstrated that the dominant unfolding pathways for 9 of the 12 proteins studied are well described by crack propagation in a network.

de Graff, Adam M.R.; Shannon, Gareth; Farrell, Daniel W.; Williams, Philip M.; Thorpe, M.F.

2011-01-01

241

Sequential Unfolding of Beta Helical Protein by Single-Molecule Atomic Force Microscopy  

PubMed Central

The parallel ?helix is a common fold among extracellular proteins, however its mechanical properties remain unexplored. In Gram-negative bacteria, extracellular proteins of diverse functions of the large ‘TpsA’ family all fold into long ?helices. Here, single-molecule atomic force microscopy and steered molecular dynamics simulations were combined to investigate the mechanical properties of a prototypic TpsA protein, FHA, the major adhesin of Bordetella pertussis. Strong extension forces were required to fully unfold this highly repetitive protein, and unfolding occurred along a stepwise, hierarchical process. Our analyses showed that the extremities of the ?helix unfold early, while central regions of the helix are more resistant to mechanical unfolding. In particular, a mechanically resistant subdomain conserved among TpsA proteins and critical for secretion was identified. This nucleus harbors structural elements packed against the ?helix that might contribute to stabilizing the N-terminal region of FHA. Hierarchical unfolding of the ?helix in response to a mechanical stress may maintain ?-helical portions that can serve as templates for regaining the native structure after stress. The mechanical properties uncovered here might apply to many proteins with ?-helical or related folds, both in prokaryotes and in eukaryotes, and play key roles in their structural integrity and functions.

Jamin, Marc; Jacob-Dubuisson, Francoise

2013-01-01

242

An Item Response Unfolding Model for Graphic Rating Scales  

ERIC Educational Resources Information Center

The graphic rating scale, a measurement tool used in many areas of psychology, usually takes a form of a fixed-length line segment, with both ends bounded and labeled as extreme responses. The raters mark somewhere on the line, and the length of the line segment from one endpoint to the mark is taken as the measure. An item response unfolding

Liu, Ying

2009-01-01

243

Down-modulation of SEL1L, an unfolded protein response and endoplasmic reticulum-associated degradation protein, sensitizes glioma stem cells to the cytotoxic effect of valproic acid.  

PubMed

Valproic acid (VPA), an histone deacetylase inhibitor, is emerging as a promising therapeutic agent for the treatments of gliomas by virtue of its ability to reactivate the expression of epigenetically silenced genes. VPA induces the unfolded protein response (UPR), an adaptive pathway displaying a dichotomic yin yang characteristic; it initially contributes in safeguarding the malignant cell survival, whereas long-lasting activation favors a proapoptotic response. By triggering UPR, VPA might tip the balance between cellular adaptation and programmed cell death via the deregulation of protein homeostasis and induction of proteotoxicity. Here we aimed to investigate the impact of proteostasis on glioma stem cells (GSC) using VPA treatment combined with subversion of SEL1L, a crucial protein involved in homeostatic pathways, cancer aggressiveness, and stem cell state maintenance. We investigated the global expression of GSC lines untreated and treated with VPA, SEL1L interference, and GSC line response to VPA treatment by analyzing cell viability via MTT assay, neurosphere formation, and endoplasmic reticulum stress/UPR-responsive proteins. Moreover, SEL1L immunohistochemistry was performed on primary glial tumors. The results show that (i) VPA affects GSC lines viability and anchorage-dependent growth by inducing differentiative programs and cell cycle progression, (ii) SEL1L down-modulation synergy enhances VPA cytotoxic effects by influencing GSCs proliferation and self-renewal properties, and (iii) SEL1L expression is indicative of glioma proliferation rate, malignancy, and endoplasmic reticulum stress statuses. Targeting the proteostasis network in association to VPA treatment may provide an alternative approach to deplete GSC and improve glioma treatments. PMID:24311781

Cattaneo, Monica; Baronchelli, Simona; Schiffer, Davide; Mellai, Marta; Caldera, Valentina; Saccani, Gloria Jotti; Dalpra, Leda; Daga, Antonio; Orlandi, Rosaria; DeBlasio, Pasquale; Biunno, Ida

2014-01-31

244

Molecular Dynamics Simulation of protein unfolding on lipid nanodomains  

NASA Astrophysics Data System (ADS)

Beta amyloid (beta-40), a short peptide associated with Alzheimer disease, can exist in the alpha-helix, beta-sheet or global conformation depending on its environment. The role of self-assembling lipid nanodomains on the conformational transition of this peptide was examined using Coarse-grained MD simulations. A stable cholesterol superlattice consisting of phosphatidylcholine and cholesterol was constructed to mimic the neuronal membrane. The unfolding kinetics of beta-40 on this superlattice surface as compared with that in solution was determined. Our results suggest a critical role of the nanostructure of lipid surface on protein unfolding at a biologically relevant time scale of hundreds of nanoseconds.

Qiu, Liming; Vaughn, Mark; Cheng, Kwan

2008-10-01

245

Amyloid protein unfolding and insertion kinetics on neuronal membrane mimics  

NASA Astrophysics Data System (ADS)

Atomistic details of beta-amyloid (A? ) protein unfolding and lipid interaction kinetics mediated by the neuronal membrane surface are important for developing new therapeutic strategies to prevent and cure Alzheimer's disease. Using all-atom MD simulations, we explored the early unfolding and insertion kinetics of 40 and 42 residue long A? in binary lipid mixtures with and without cholesterol that mimic the cholesterol-depleted and cholesterol-enriched lipid nanodomains of neurons. The protein conformational transition kinetics was evaluated from the secondary structure profile versus simulation time plot. The extent of membrane disruption was examined by the calculated order parameters of lipid acyl chains and cholesterol fused rings as well as the density profiles of water and lipid headgroups at defined regions across the lipid bilayer from our simulations. Our results revealed that both the cholesterol content and the length of the protein affect the protein-insertion and membrane stability in our model lipid bilayer systems.

Qiu, Liming; Buie, Creighton; Vaughn, Mark; Cheng, Kwan

2010-03-01

246

Thermodynamic analysis of unfolding and dissociation in lactose repressor protein.  

PubMed

Lactose repressor protein, regulator of lac enzyme expression in Escherichia coli, maintains its structure and function at extremely low protein concentrations (<10(-)12 M). To examine the unfolding and dissociation of this tetrameric protein, structural transitions in the presence of varying concentrations of urea were monitored by fluorescence and circular dichroism spectroscopy, analytical ultracentrifugation, and functional activities. The spectroscopic data demonstrated a single cooperative transition with no evidence of folded dimeric or monomeric species of this protein. These spectroscopic transitions were reversible provided a long incubation step was employed in the refolding reaction at approximately 3 M urea. The refolded repressor protein possessed the same functional and structural properties as wild-type repressor protein. The absence of concentration dependence expected for tetramer dissociation to unfolded monomer (M4 <--> 4U) in the spectral transitions indicates that the disruption of the monomer-monomer interface and monomer unfolding are a concerted reaction (M4 <--> U4) that may occur prior to the dissociation of the dimer-dimer interface. Thus, we propose that the unfolded monomers remain associated at the C-terminus by the 4-helical coiled-coil structure that forms the dimer-dimer interface and that this intermediate is the end point detected in the spectral transitions. Efforts to confirm the existence of this species by ultracentrifugation were inhibited by the aggregation of this intermediate. Based upon these observations, the wild-type fluorescence and CD data were fit to a model, M4 <--> U4, which resulted in an overall DeltaG degrees for unfolding of 40 kcal/mol. Using a mutant protein, K84L, in which the monomer-monomer interface is stabilized, sedimentation equilibrium results demonstrated that the dimer-dimer interface of lac repressor could persist at higher levels of urea than the monomer-monomer interface. The tetramer-dimer transition monitored using this mutant repressor yields a DeltaG degrees of 20.4 kcal/mol. Using this free energy value for the dissociation process of U4 <--> 4U, an overall free energy change of approximately 60 kcal/mol was calculated for dissociation of all interfaces and unfolding of the tetrameric lac repressor, reflecting the exceptional stability of this protein. PMID:10350470

Barry, J K; Matthews, K S

1999-05-18

247

Single-shot NMR measurement of protein unfolding landscapes.  

PubMed

The transient unfolding events from the native state of a protein towards higher energy states can be closely investigated by studying the process of hydrogen exchange. Here, we present BLUU-Tramp (Biophysics Laboratory University of Udine-Temperature ramp), a new method to measure the rates for the exchange process and the underlying equilibrium thermodynamic parameters, using just a single sample preparation, in a single experiment that lasts some 20 to 60h depending on the protein thermal stability, to record hundreds of points over a virtually continuous temperature window. The method is suitable also in presence of other proteins in the sample, if only the target protein is (15)N-labelled. This allows the complete thermodynamic description of the unfolding landscape at an atomic level in the presence of small or macromolecular ligands or cosolutes, or in physiological environments. The method was successfully tested with human ubiquitin. Then the unfolding thermodynamic parameters were satisfactorily determined for the amyloidogenic protein ?(2)-microglobulin, in aqueous buffer and in synovial liquid, that is the natural medium of amyloid deposition in joints. PMID:22522028

Rennella, Enrico; Corazza, Alessandra; Codutti, Luca; Causero, Araldo; Bellotti, Vittorio; Stoppini, Monica; Viglino, Paolo; Fogolari, Federico; Esposito, Gennaro

2012-06-01

248

Usage of Simulated Response Matrices for the Scintillation Spectra Unfolding  

NASA Astrophysics Data System (ADS)

Response of the scintillation spectrometer to incident photons has complex character due to complex character of photon interaction processes. Experimental spectrum thus provide qualitative information given by the identifiable peaks positions, while extraction of the full, quantitative information (i.e. photon flux energy distribution) require spectra unfolding based on the spectrometer response (represented by the response matrix) knowledge. As experimental determination of response matrix is difficult or impossible in most cases, the Monte Carlo simulation is the proved solution. Usage of the unfolding for experimental spectra processing enable to determine dosimetric characteristics of the incident photon fields and/or characteristics of the sources, creating those photon fields. The response matrices for scintillation spectrometers with different detectors (commonly used NaI, BGO and new LaBr) were calculated using Monte Carlo method. The response to the internal activity of the LaBr detector was also simulated and considered in spectra processing. Calculated matrices were used for unfolding of the experimental spectra from different applications to determine desired dosimetric quantities. Typical applications are environmental monitoring, monitoring of the working environment, accidental monitoring and contamination measurement, etc. Method is suitable also for the airborne monitoring or security applications. Results for individual detection systems are compared and discussed with aim to analyze potential advantages of the LaBr detector for considered applications.

Kluso?, J.; Urban, T.

2014-06-01

249

Protein Unfolding Coupled to Ligand Binding: Differential Scanning Calorimetry Simulation Approach  

ERIC Educational Resources Information Center

A comprehensive theoretical description of thermal protein unfolding coupled to ligand binding is presented. The thermodynamic concepts are independent of the method used to monitor protein unfolding but a differential scanning calorimetry is being used as a tool for examining the unfolding process.

Celej, Maria Soledad; Fidelio, Gerardo Daniel; Dassie, Sergio Alberto

2005-01-01

250

Protein under tension and mechanical unfolding  

NASA Astrophysics Data System (ADS)

The mechanical properties of proteins are important for a wide variety of functions ranging from stabilizing cellular structures to the transduction of signals across the membrane. We examined changes in protein conformation under external force fields by simple theoretical methods and new simulation techniques. The theoretical model solved a Gaussian chain plus native contact residue-level model under approximations. The simulations used the force ensemble replica exchange method and all-atom stochastic dynamics with a generalized Born plus solvent accessible surface as the solvation model. We applied these methods to study the protein spectrin as well as the domains of titin. Both global properties (such as energy and extension) and local roperties (especially, the specific contacts maintained and the secondary structure) are shown as functions of external force.

Shen, Tongye; Canino, Larry; Wolynes, Peter G.; McCammon, J. Andrew

2003-03-01

251

OASIS/CREB3L1 is induced by endoplasmic reticulum stress in human glioma cell lines and contributes to the unfolded protein response, extracellular matrix production and cell migration.  

PubMed

OASIS is a transcription factor similar to ATF6 that is activated by endoplasmic reticulum stress. In this study we investigated the expression of OASIS in human glioma cell lines and the effect of OASIS knock-down on the ER stress response and cell migration. OASIS mRNA was detected in three distinct glioma cell lines (U373, A172 and U87) and expression levels were increased upon treatment with ER stress-inducing compounds in the U373 and U87 lines. OASIS protein, which is glycosylated on Asn-513, was detected in the U373 and U87 glioma lines at low levels in control cells and protein expression was induced by ER stress. Knock-down of OASIS in human glioma cell lines resulted in an attenuated unfolded protein response to ER stress (reduced GRP78/BiP and GRP94 induction) and decreased expression of chondroitin sulfate proteoglycan extracellular matrix proteins, but induction of the collagen gene Col1a1 was unaffected. Cells in which OASIS was knocked-down exhibited altered cell morphology and reduced cell migration. These results suggest that OASIS is important for the ER stress response and maintenance of some extracellular matrix proteins in human glioma cells. PMID:23335989

Vellanki, Ravi N; Zhang, Liling; Volchuk, Allen

2013-01-01

252

Exploring early stages of the chemical unfolding of proteins at the proteome scale.  

PubMed

After decades of using urea as denaturant, the kinetic role of this molecule in the unfolding process is still undefined: does urea actively induce protein unfolding or passively stabilize the unfolded state? By analyzing a set of 30 proteins (representative of all native folds) through extensive molecular dynamics simulations in denaturant (using a range of force-fields), we derived robust rules for urea unfolding that are valid at the proteome level. Irrespective of the protein fold, presence or absence of disulphide bridges, and secondary structure composition, urea concentrates in the first solvation shell of quasi-native proteins, but with a density lower than that of the fully unfolded state. The presence of urea does not alter the spontaneous vibration pattern of proteins. In fact, it reduces the magnitude of such vibrations, leading to a counterintuitive slow down of the atomic-motions that opposes unfolding. Urea stickiness and slow diffusion is, however, crucial for unfolding. Long residence urea molecules placed around the hydrophobic core are crucial to stabilize partially open structures generated by thermal fluctuations. Our simulations indicate that although urea does not favor the formation of partially open microstates, it is not a mere spectator of unfolding that simply displaces to the right of the folded ?? unfolded equilibrium. On the contrary, urea actively favors unfolding: it selects and stabilizes partially unfolded microstates, slowly driving the protein conformational ensemble far from the native one and also from the conformations sampled during thermal unfolding. PMID:24348236

Candotti, Michela; Pérez, Alberto; Ferrer-Costa, Carles; Rueda, Manuel; Meyer, Tim; Gelpí, Josep Lluís; Orozco, Modesto

2013-12-01

253

Exploring Early Stages of the Chemical Unfolding of Proteins at the Proteome Scale  

PubMed Central

After decades of using urea as denaturant, the kinetic role of this molecule in the unfolding process is still undefined: does urea actively induce protein unfolding or passively stabilize the unfolded state? By analyzing a set of 30 proteins (representative of all native folds) through extensive molecular dynamics simulations in denaturant (using a range of force-fields), we derived robust rules for urea unfolding that are valid at the proteome level. Irrespective of the protein fold, presence or absence of disulphide bridges, and secondary structure composition, urea concentrates in the first solvation shell of quasi-native proteins, but with a density lower than that of the fully unfolded state. The presence of urea does not alter the spontaneous vibration pattern of proteins. In fact, it reduces the magnitude of such vibrations, leading to a counterintuitive slow down of the atomic-motions that opposes unfolding. Urea stickiness and slow diffusion is, however, crucial for unfolding. Long residence urea molecules placed around the hydrophobic core are crucial to stabilize partially open structures generated by thermal fluctuations. Our simulations indicate that although urea does not favor the formation of partially open microstates, it is not a mere spectator of unfolding that simply displaces to the right of the folded??unfolded equilibrium. On the contrary, urea actively favors unfolding: it selects and stabilizes partially unfolded microstates, slowly driving the protein conformational ensemble far from the native one and also from the conformations sampled during thermal unfolding.

Candotti, Michela; Perez, Alberto; Ferrer-Costa, Carles; Rueda, Manuel; Meyer, Tim; Gelpi, Josep Lluis; Orozco, Modesto

2013-01-01

254

Differential regulation of adaptive and apoptotic unfolded protein response signalling by cytokine-induced nitric oxide production in mouse pancreatic beta cells  

Microsoft Academic Search

Aims\\/hypothesis  Pro-inflammatory cytokines such as IL-1?, IFN-? and TNF-? may contribute to pancreatic beta cell destruction in type 1 diabetes.\\u000a A mechanism requiring nitric oxide, which is generated by inducible nitric oxide synthase (iNOS), in cytokine-induced endoplasmic\\u000a reticulum (ER) stress and apoptosis has been proposed. Here, we tested the role of nitric oxide in cytokine-induced ER stress\\u000a and the subsequent unfolded

J. Y. Chan; G. J. Cooney; T. J. Biden; D. R. Laybutt

2011-01-01

255

Tannin-assisted aggregation of natively unfolded proteins  

NASA Astrophysics Data System (ADS)

Tannin-protein interactions are essentially physical: hydrophobic and hydrogen-bond-mediated. We explored the tannin-assisted protein aggregation on the case of ?-casein, which is a natively unfolded protein known for its ability to form micellar aggregates. We used several tannins with specified length. Our SAXS results show that small tannins increase the number of proteins per micelle, but keeping their size constant. It leads to a tannin-assisted compactization of micelles. Larger tannins, with linear dimensions greater than the crown width of micelles, lead to the aggregation of micelles by a bridging effect. Experimental results can be understood within a model where tannins are treated as effective enhancers of hydrophobic attraction between specific sites in proteins.

Zanchi, D.; Narayanan, T.; Hagenmuller, D.; Baron, A.; Guyot, S.; Cabane, B.; Bouhallab, S.

2008-06-01

256

Counting peptide-water hydrogen bonds in unfolded proteins  

PubMed Central

It is often assumed that the peptide backbone forms a substantial number of additional hydrogen bonds when a protein unfolds. We challenge that assumption in this article. Early surveys of hydrogen bonding in proteins of known structure typically found that most, but not all, backbone polar groups are satisfied, either by intramolecular partners or by water. When the protein is folded, these groups form approximately two hydrogen bonds per peptide unit, one donor or acceptor for each carbonyl oxygen or amide hydrogen, respectively. But when unfolded, the backbone chain is often believed to form three hydrogen bonds per peptide unit, one partner for each oxygen lone pair or amide hydrogen. This assumption is based on the properties of small model compounds, like N-methylacetamide, or simply accepted as self-evident fact. If valid, a chain of N residues would have approximately 2N backbone hydrogen bonds when folded but 3N backbone hydrogen bonds when unfolded, a sufficient difference to overshadow any uncertainties involved in calculating these per-residue averages. Here, we use exhaustive conformational sampling to monitor the number of H-bonds in a statistically adequate population of blocked polyalanyl-six-mers as the solvent quality ranges from good to poor. Solvent quality is represented by a scalar parameter used to Boltzmann-weight the population energy. Recent experimental studies show that a repeating (Gly-Ser) polypeptide undergoes a denaturant-induced expansion accompanied by breaking intramolecular peptide H-bonds. Results from our simulations augment this experimental finding by showing that the number of H-bonds is approximately conserved during such expansion?compaction transitions.

Gong, Haipeng; Porter, Lauren L; Rose, George D

2011-01-01

257

Using Data Augmentation and Markov Chain Monte Carlo for the Estimation of Unfolding Response Models  

ERIC Educational Resources Information Center

Unfolding response models, a class of item response theory (IRT) models that assume a unimodal item response function (IRF), are often used for the measurement of attitudes. Verhelst and Verstralen (1993)and Andrich and Luo (1993) independently developed unfolding response models by relating the observed responses to a more common monotone IRT…

Johnson, Matthew S.; Junker, Brian W.

2003-01-01

258

Global analysis of three-state protein unfolding data  

PubMed Central

A new method for analyzing three-state protein unfolding equilibria is described that overcomes the difficulties created by direct effects of denaturants on circular dichroism (CD) and fluorescence spectra of the intermediate state. The procedure begins with a singular value analysis of the data matrix to determine the number of contributing species and perturbations. This result is used to choose a fitting model and remove all spectra from the fitting equation. Because the fitting model is a product of a matrix function which is nonlinear in the thermodynamic parameters and a matrix that is linear in the parameters that specify component spectra, the problem is solved with a variable projection algorithm. Advantages of this procedure are perturbation spectra do not have to be estimated before fitting, arbitrary assumptions about magnitudes of parameters that describe the intermediate state are not required, and multiple experiments involving different spectroscopic techniques can be simultaneously analyzed. Two tests of this method were performed: First, simulated three-state data were analyzed, and the original and recovered thermodynamic parameters agreed within one standard error, whereas recovered and original component spectra agreed within 0.5%. Second, guanidine-induced unfolding titrations of the human retinoid-X-receptor ligand-binding domain were analyzed according to a three-state model. The standard unfolding free energy changes in the absence of guanidine and the guanidine concentrations at zero free-energy change for both transitions were determined from a joint analysis of fluorescence and CD spectra. Realistic spectra of the three protein states were also obtained.

Harder, Mark E.; Deinzer, Max L.; Leid, Mark E.; Schimerlik, Michael I.

2004-01-01

259

Changes in solvent exposure reveal the kinetics and equilibria of adsorbed protein unfolding in hydrophobic interaction chromatography  

PubMed Central

Hydrogen exchange has been a useful technique for studying the conformational state of proteins, both in bulk solution and at interfaces, for several decades. Here, we propose a physically-based model of simultaneous protein adsorption, unfolding and hydrogen exchange in HIC. An accompanying experimental protocol, utilizing mass spectrometry to quantify deuterium labeling, enables the determination of both the equilibrium partitioning between conformational states and pseudo-first order rate constants for folding and unfolding of adsorbed protein. Unlike chromatographic techniques, which rely on the interpretation of bulk phase behavior, this methodology utilizes the measurement of a molecular property (solvent exposure) and provides insight into the nature of the unfolded conformation in the adsorbed phase. Three model proteins of varying conformational stability, ?-chymotrypsinogen A, ?-lactoglobulin B, and holo ?-lactalbumin, are studied on Sepharose™ HIC resins possessing assorted ligand chemistries and densities. ?-Chymotrypsinogen, the most conformationally stable protein in the set, exhibits no change in solvent exposure at all of the conditions studied, even when isocratic pulse-response chromatography suggests nearly irreversible adsorption. Apparent unfolding energies of adsorbed ?-lactoglobulin B and holo ?-lactalbumin range from ?4 to 3 kJ/mol and are dependent on resin properties and salt concentration. Characteristic pseudo-first order rate constants for surface-induced unfolding are 0.2 to 0.9 min?1. While poor protein recovery in HIC is often associated with irreversible unfolding, this study documents that non-eluting behavior can occur when surface unfolding is reversible or does not occur at all. Further, this hydrogen exchange technique can be used to assess the conformation of adsorbed protein under conditions where the protein is non-eluting and chromatographic methods are not applicable.

Deitcher, R.W.; O'Connell, J.P.; Fernandez, E.J.

2010-01-01

260

Unfolding of DNA quadruplexes induced by HIV-1 nucleocapsid protein  

PubMed Central

The human immunodeficiency virus type 1 nucleocapsid protein (NC) is a nucleic acid chaperone that catalyzes the rearrangement of nucleic acids into their thermodynamically most stable structures. In the present study, a combination of optical and thermodynamic techniques were used to characterize the influence of NC on the secondary structure, thermal stability and energetics of monomolecular DNA quadruplexes formed by the sequence d(GGTTGGTGTGGTTGG) in the presence of K+ or Sr2+. Circular dichroism studies demonstrate that NC effectively unfolds the quadruplexes. Studies carried out with NC variants suggest that destabilization is mediated by the zinc fingers of NC. Calorimetric studies reveal that NC destabilization is enthalpic in origin, probably owing to unstacking of the G-quartets upon protein binding. In contrast, parallel studies performed on a related DNA duplex reveal that under conditions where NC readily destabilizes and unfolds the quadruplexes, its effect on the DNA duplex is much less pronounced. The differences in NC's ability to destabilize quadruplex versus duplex is in accordance with the higher ?G of melting for the latter, and with the inverse correlation between nucleic acid stability and the destabilizing activity of NC.

Kankia, Besik I.; Barany, George; Musier-Forsyth, Karin

2005-01-01

261

The effect of pulling geometry and concatamer architecture on the mechanical unfolding force of ?-sheet proteins  

NASA Astrophysics Data System (ADS)

The molecular origins of mechanical resistance in proteins are still unclear, although the orientation of secondary structural elements relative to the applied force vector is thought to play an important role. By using a novel method of protein immobilisation that allows force to be applied to the same all ?-protein, E2lip3, in two different directions, we show that the energy landscape for mechanical unfolding is remarkably anisotropic. These results, in combination with molecular dynamics simulations, reveal that the unfolding pathway depends on pulling geometry, leading to unfolding forces that differ by an order of magnitude. We also demonstrate how, for a given pulling direction, kinetic effects lead to apparent unfolding forces that depend on the number of domains being unfolded and the overall compliance. Hence, the mechanical resistance of a protein is not solely endowed by sequence, topology or unfolding rate constant, but depends critically on the direction of the applied extension.

Olmsted, Pd; Zinober, Rc; Smith, Da; Blake, Aw; Brockwell, Dj; Radford, Se; Beddard, Gs; Perham, Rn; Paci, E.

2004-03-01

262

Ligand-modulated Parallel Mechanical Unfolding Pathways of Maltose-binding Proteins*  

PubMed Central

Protein folding and unfolding are complex phenomena, and it is accepted that multidomain proteins generally follow multiple pathways. Maltose-binding protein (MBP) is a large (a two-domain, 370-amino acid residue) bacterial periplasmic protein involved in maltose uptake. Despite the large size, it has been shown to exhibit an apparent two-state equilibrium unfolding in bulk experiments. Single-molecule studies can uncover rare events that are masked by averaging in bulk studies. Here, we use single-molecule force spectroscopy to study the mechanical unfolding pathways of MBP and its precursor protein (preMBP) in the presence and absence of ligands. Our results show that MBP exhibits kinetic partitioning on mechanical stretching and unfolds via two parallel pathways: one of them involves a mechanically stable intermediate (path I) whereas the other is devoid of it (path II). The apoMBP unfolds via path I in 62% of the mechanical unfolding events, and the remaining 38% follow path II. In the case of maltose-bound MBP, the protein unfolds via the intermediate in 79% of the cases, the remaining 21% via path II. Similarly, on binding to maltotriose, a ligand whose binding strength with the polyprotein is similar to that of maltose, the occurrence of the intermediate is comparable (82% via path I) with that of maltose. The precursor protein preMBP also shows a similar behavior upon mechanical unfolding. The percentages of molecules unfolding via path I are 53% in the apo form and 68% and 72% upon binding to maltose and maltotriose, respectively, for preMBP. These observations demonstrate that ligand binding can modulate the mechanical unfolding pathways of proteins by a kinetic partitioning mechanism. This could be a general mechanism in the unfolding of other large two-domain ligand-binding proteins of the bacterial periplasmic space.

Aggarwal, Vasudha; Kulothungan, S. Rajendra; Balamurali, M. M.; Saranya, S. R.; Varadarajan, Raghavan; Ainavarapu, Sri Rama Koti

2011-01-01

263

Enhancing the Activity of a Protein by Stereospecific Unfolding  

PubMed Central

A central tenet of molecular biology holds that the function of a protein is mediated by its structure. An inactive ground-state conformation may nonetheless be enjoined by the interplay of competing biological constraints. A model is provided by insulin, well characterized at atomic resolution by x-ray crystallography. Here, we demonstrate that the activity of the hormone is enhanced by stereospecific unfolding of a conserved structural element. A bifunctional ?-strand mediates both self-assembly (within ?-cell storage vesicles) and receptor binding (in the bloodstream). This strand is anchored by an invariant side chain (PheB24); its substitution by Ala leads to an unstable but native-like analog of low activity. Substitution by d-Ala is equally destabilizing, and yet the protein diastereomer exhibits enhanced activity with segmental unfolding of the ?-strand. Corresponding photoactivable derivatives (containing l- or d-para-azido-Phe) cross-link to the insulin receptor with higher d-specific efficiency. Aberrant exposure of hydrophobic surfaces in the analogs is associated with accelerated fibrillation, a form of aggregation-coupled misfolding associated with cellular toxicity. Conservation of PheB24, enforced by its dual role in native self-assembly and induced fit, thus highlights the implicit role of misfolding as an evolutionary constraint. Whereas classical crystal structures of insulin depict its storage form, signaling requires engagement of a detachable arm at an extended receptor interface. Because this active conformation resembles an amyloidogenic intermediate, we envisage that induced fit and self-assembly represent complementary molecular adaptations to potential proteotoxicity. The cryptic threat of misfolding poses a universal constraint in the evolution of polypeptide sequences.

Hua, Qing-xin; Xu, Bin; Huang, Kun; Hu, Shi-Quan; Nakagawa, Satoe; Jia, Wenhua; Wang, Shuhua; Whittaker, Jonathan; Katsoyannis, Panayotis G.; Weiss, Michael A.

2009-01-01

264

Experiments & Simulations of Pathway Shifts & T-dependent Forced Unfolding of 3-helix Proteins  

NASA Astrophysics Data System (ADS)

Pathways of unfolding a protein depend in principle on the perturbation - whether it is temperature, denaturant, or even forced extension. Widely-shared, helical-bundle spectrin repeats are known to melt at temperatures as low as 4045 C and are also known to unfold via multiple pathways as single molecules in atomic force microscopy. We sought to determine the coupled effects of temperature on forced unfolding by AFM as well as by Molecular Dynamics. Bimodal distributions of unfolding intervals are seen at all temperatures for the four-repeat b14 spectrinan a-actinin homolog. The major unfolding length corresponds to unfolding of a single repeat, and a minor peak at twice the length (or longer) corresponds to tandem repeats. Increasing temperature shows fewer tandem events but has no effect on unfolding intervals. As T approaches Tm, however, mean unfolding forces in atomic force microscopy also decrease; and circular dichroism studies demonstrate a nearly proportional decrease of helical content in solution. The results imply a thermal softening of a helical linker between repeats which otherwise propagates a helix-to-coil transition to adjacent repeats. Structural changes with T correlate with both unfolding forces and shifts in unfolding pathways.

Discher, Dennis; Law, Richard; Klein, Mike

2004-03-01

265

Conservation between animals and plants of the cis-acting element involved in the unfolded protein responseq  

Microsoft Academic Search

Using Arabidopsis thaliana, we identified the cis-element involved in the plant unfolded protein response (UPR). In transgenic plants, tunicamycin stimulated expression of a reporter gene under the control of the BiP promoter and promoter analysis identified a 24 bp sequence crucial to this induction. When fused with a minimal promoter, a hexamer of this sequence was sufficient for induction of

Dong-Ha Oh; Chang-Seob Kwon; Hiroshi Sano; Won-Il Chung; Nozomu Koizumib

266

Theoretical models for electrochemical impedance spectroscopy and local ?-potential of unfolded proteins in nanopores  

NASA Astrophysics Data System (ADS)

Single solid-state nanopores find increasing use for electrical detection and/or manipulation of macromolecules. These applications exploit the changes in signals due to the geometry and electrical properties of the molecular species found within the nanopore. The sensitivity and resolution of such measurements are also influenced by the geometric and electrical properties of the nanopore. This paper continues the development of an analytical theory to predict the electrochemical impedance spectra of nanopores by including the influence of the presence of an unfolded protein using the variable topology finite Warburg impedance model previously published by the authors. The local excluded volume of, and charges present on, the segment of protein sampled by the nanopore are shown to influence the shape and peak frequency of the electrochemical impedance spectrum. An analytical theory is used to relate the capacitive response of the electrical double layer at the surface of the protein to both the charge density at the protein surface and the more commonly measured zeta potential. Illustrative examples show how the theory predicts that the varying sequential regions of surface charge density and excluded volume dictated by the protein primary structure may allow for an impedance-based approach to identifying unfolded proteins.

Vitarelli, Michael J.; Talaga, David S.

2013-09-01

267

Dual Functions of Yeast tRNA Ligase in the Unfolded Protein Response: Unconventional Cytoplasmic Splicing of HAC1 Pre-mRNA Is Not Sufficient to Release Translational Attenuation  

PubMed Central

The unfolded protein response (UPR) is an essential signal transduction to cope with protein-folding stress in the endoplasmic reticulum. In the yeast UPR, the unconventional splicing of HAC1 mRNA is a key step. Translation of HAC1 pre-mRNA (HAC1u mRNA) is attenuated on polysomes and restarted only after splicing upon the UPR. However, the precise mechanism of this restart remained unclear. Here we show that yeast tRNA ligase (Rlg1p/Trl1p) acting on HAC1 ligation has an unexpected role in HAC1 translation. An RLG1 homologue from Arabidopsis thaliana (AtRLG1) substitutes for yeast RLG1 in tRNA splicing but not in the UPR. Surprisingly, AtRlg1p ligates HAC1 exons, but the spliced mRNA (HAC1i mRNA) is not translated efficiently. In the AtRLG1 cells, the HAC1 intron is circularized after splicing and remains associated on polysomes, impairing relief of the translational repression of HAC1i mRNA. Furthermore, the HAC1 5? UTR itself enables yeast Rlg1p to regulate translation of the following ORF. RNA IP revealed that yeast Rlg1p is integrated in HAC1 mRNP, before Ire1p cleaves HAC1u mRNA. These results indicate that the splicing and the release of translational attenuation of HAC1 mRNA are separable steps and that Rlg1p has pivotal roles in both of these steps.

Mori, Takao; Ogasawara, Chiharu; Inada, Toshifumi; Englert, Markus; Beier, Hildburg; Takezawa, Mine; Endo, Toshiya

2010-01-01

268

Mammalian Target of Rapamycin Complex 1 (mTORC1) Enhances Bortezomib-induced Death in Tuberous Sclerosis Complex (TSC)-null Cells by a c-MYC-dependent Induction of the Unfolded Protein Response*  

PubMed Central

Many factors, including duration and intensity of the unfolded protein response (UPR), dictate whether cells will adapt to endoplasmic reticulum stress or undergo apoptosis. In tuberous sclerosis (TSC), elevation of mammalian target of rapamycin complex 1 (mTORC1) activity has been proposed to compound the induction of UPR transcription factors ATF4 and CHOP, suggesting that the UPR could be targeted to eradicate TSC1/2-null cells during patient therapy. Here we report that control of c-MYC translation by mTORC1 plays a key role in determining whether TSC2-null Elt3 rat leiomyoma cells apoptose in response to UPR induction by the proteasome inhibitor bortezomib. Although bortezomib induces eukaryotic initiating factor 2? phosphorylation, mTORC1 activity was also required for downstream induction of the UPR transcription factors ATF4 and CHOP by a mechanism involving increased expression of c-MYC. Although bortezomib-induced c-MYC transcription was resistant to rapamycin treatment, mTORC1 activity was required for efficient c-MYC translation. c-MYC subsequently bound to the ATF4 promoter, suggesting direct involvement of an mTORC1/c-MYC-driven signaling pathway in the activation of the UPR. Consistent with this notion, exogenously expressed c-MYC reversed the ability of rapamycin to prevent bortezomib-induced CHOP and ATF4 expression as well as apoptosis. These findings indicate that the induction of ATF4/CHOP expression occurs via mTORC1 regulation of c-MYC and that this signaling pathway is a major determinant in the ability of bortezomib to induce apoptosis.

Babcock, Justin T.; Nguyen, Hoa B.; He, Yujun; Hendricks, Jeremiah W.; Wek, Ronald C.; Quilliam, Lawrence A.

2013-01-01

269

Sequence-Specific Solvent Accessibilities of Protein Residues in Unfolded Protein Ensembles  

PubMed Central

Protein stability cannot be understood without the correct description of the unfolded state. We present here an efficient method for accurate calculation of atomic solvent exposures for denatured protein ensembles. The method used to generate the ensembles has been shown to reproduce diverse biophysical experimental data corresponding to natively and chemically unfolded proteins. Using a data set of 19 nonhomologous proteins containing from 98 to 579 residues, we report average accessibilities for all residue types. These averaged accessibilities are considerably lower than those previously reported for tripeptides and close to the lower limit reported by Creamer and co-workers. Of importance, we observe remarkable sequence dependence for the exposure to solvent of all residue types, which indicates that average residue solvent exposures can be inappropriate to interpret mutational studies. In addition, we observe smaller influences of both protein size and protein amino acid composition in the averaged residue solvent exposures for individual proteins. Calculating residue-specific solvent accessibilities within the context of real sequences is thus necessary and feasible. The approach presented here may allow a more precise parameterization of protein energetics as a function of polar- and apolar-area burial and opens new ways to investigate the energetics of the unfolded state of proteins.

Bernado, Pau; Blackledge, Martin; Sancho, Javier

2006-01-01

270

Measurement of the Kinetics of Protein Unfolding in Viscous Systems and Implications for Protein Stability in Freeze-Drying  

Microsoft Academic Search

Purpose. The aim of the study is to determine the degree of coupling between protein unfolding rate and system viscosity at low temperatures in systems relevant to freeze-drying. Methods. The cold denaturation of both phosphoglycerate kinase (PGK) and b-lactoglobulin were chosen as models for the protein unfolding kinetics study. The system viscosity was enhanced by adding stabilizers (such as sucrose),

Michael J. Pikal

2005-01-01

271

Mitochondrial disease activates transcripts of the unfolded protein response and cell cycle and inhibits vesicular secretion and oligodendrocyte-specific transcripts  

Microsoft Academic Search

Mutations in gene products expressed in the mitochondrion cause a nuclear transcriptional response that leads to neurological disease. To examine the extent to which the transcriptional profile was shared among 5 mitochondrial diseases (LHON, FRDA, MELAS, KSS, and NARP), we microarrayed mutant and control groups in N-tera2, SH-SY5Y, lymphoblasts, fibroblasts, myoblasts, muscle, and osteosarcoma cybrids. Many more transcripts were observed

Gino Cortopassi; Steven Danielson; Mansour Alemi; Shan Shan Zhan; Warren Tong; Valerio Carelli; Andrea Martinuzzi; Sangot Marzuki; Kari Majamaa; Alice Wong

2006-01-01

272

Unfolding of a small protein proceeds via dry and wet globules and a solvated transition state.  

PubMed

Dissecting a protein unfolding process into individual steps can provide valuable information on the forces that maintain the integrity of the folded structure. Solvation of the protein core determines stability, but it is not clear when such solvation occurs during unfolding. In this study, far-UV circular dichroism measurements suggest a simplistic two-state view of the unfolding of barstar, but the use of multiple other probes brings out the complexity of the unfolding reaction. Near-UV circular dichroism measurements show that unfolding commences with the loosening of tertiary interactions in a native-like intermediate, N(?). Fluorescence resonance energy transfer measurements show that N(?) then expands rapidly but partially to form an early unfolding intermediate IE. Fluorescence spectral measurements indicate that both N(?) and IE have retained native-like solvent accessibility of the core, suggesting that they are dry molten globules. Dynamic quenching measurements at the single tryptophan buried in the core suggest that the core becomes solvated only later in a late wet molten globule, IL, which precedes the unfolded form. Fluorescence anisotropy decay measurements show that tight packing around the core tryptophan is lost when IL forms. Of importance, the slowest step is unfolding of the wet molten globule and involves a solvated transition state. PMID:24268151

Sarkar, Saswata Sankar; Udgaonkar, Jayant B; Krishnamoorthy, Guruswamy

2013-11-19

273

Using circular dichroism collected as a function of temperature to determine the thermodynamics of protein unfolding and binding interactions  

PubMed Central

Circular dichroism (CD) is an excellent spectroscopic technique for following the unfolding and folding of proteins as a function of temperature. One of its principal applications is to determine the effects of mutations and ligands on protein and polypeptide stability If the change in CD as a function of temperature is reversible, analysis of the data may be used to determined the van't Hoff enthalpy (?H) and entropy (?S) of unfolding, the midpoint of the unfolding transition (TM) and the free energy (?G) of unfolding. Binding constants of protein-protein and protein-ligand interactions may also be estimated from the unfolding curves. Analysis of CD spectra obtained as a function of temperature is also useful to determine whether a protein has unfolding intermediates. Measurement of the spectra of five folded proteins and their unfolding curves at a single wavelength takes approximately eight hours.

Greenfield, Norma J.

2009-01-01

274

An Application of Unfolding and Cumulative Item Response Theory Models for Noncognitive Scaling: Examining the Assumptions and Applicability of the Generalized Graded Unfolding Model  

ERIC Educational Resources Information Center

This study examined the applicability of a relatively new unidimensional, unfolding item response theory (IRT) model called the generalized graded unfolding model (GGUM; Roberts, Donoghue, & Laughlin, 2000). A total of four scaling methods were applied. Two commonly used cumulative IRT models for polytomous data, the Partial Credit Model and the…

Sgammato, Adrienne N.

2009-01-01

275

Protein denaturation in vacuo: Mechanism for centrifugal unfolding of neutral lysozyme  

NASA Astrophysics Data System (ADS)

Experiments on anhydrous proteins diffusing in a low-pressure gas reveal transitions that share common features with the unfolding and refolding processes observed in solution. These phenomena force us to re-evaluate the specific role played by solvent on large-scale protein rearrangements. Computer simulations, in combination with tools for molecular shape analysis, provide insights into in vacuo processes. In this work, we deal with one particular aspect of this problem: the conditions and the mechanism for spontaneous unfolding in a globally neutral protein. When coupling the protein to a simulated thermal bath, the flow of energy between rotational modes and internal modes can produce a centrifugal effect leading to unfolding. Using hen lysozyme as a system, we study the reproducibility of the unfolding transition and its dependence on the bath relaxation constant. In addition, we monitor the evolution of large-scale molecular shape features (e.g., chain entanglements) that take place during unfolding. Our results show that a change in bath relaxation constant affects the time scale of the transition and the population of intermediates, without changing the basic unfolding mechanism. We also discuss possible cycles of unfolding-refolding transitions, and their implications for our understanding of the denatured state.

Arteca, Gustavo A.; Tapia, O.

2001-12-01

276

Replication Protein A Unfolds G-Quadruplex Structures with a Varying Degree of Efficiency  

PubMed Central

Replication Protein A (RPA) is known to interact with G-rich sequences that adopt G-quadruplex (GQ) structures. Most studies in the literature have been performed on GQ formed by homogenous sequences, such as the human telomeric repeat, and RPA’s ability to unfold GQ structures of differing stability is not known. We compared the thermal stability of three potential GQ forming DNA sequences (PQS) to their stability against RPA mediated unfolding using single molecule FRET and bulk biophysical and biochemical experiments. One of these sequences is the human telomeric repeat and the other two located in the promoter region of tyrosine hydroxylase gene are highly heterogeneous sequences, which better represent PQS in the genome. The three GQ constructs have thermal stabilities that are significantly different from each other. Our measurements showed that the most thermally stable structure (Tm= 86 °C) was also the most stable against RPA mediated unfolding, although the least thermally stable structure (Tm= 69 °C) had at least an order of magnitude higher stability against RPA mediated unfolding compared to the structure with intermediate thermal stability (Tm= 78 °C). The significance of this observation becomes more evident when considered within the context of cellular environment where protein-DNA interactions can be an important determinant of GQ viability. Considering these, we conclude that thermal stability is not necessarily an adequate criterion for predicting physiological viability of GQ structures. Finally, we measured the time it takes for an RPA molecule to unfold a GQ from a fully folded to a fully unfolded conformation using a single molecule stopped-flow type method. All three GQ structures were unfolded within ?t?0.30±0.10 sec, a surprising result as the unfolding time does not correlate with thermal stability or stability against RPA mediated unfolding. These results suggest that the limiting step in G-quadruplex unfolding by RPA is simply the accessibility of the structure to the RPA protein.

Qureshi, Mohammad H.; Ray, Sujay; Sewell, Abby L.; Basu, Soumitra; Balci, Hamza

2012-01-01

277

Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain  

Microsoft Academic Search

Site-directed spin labeling in combination with paramagnetic relaxation enhancement (PRE) measurements is one of the most promising techniques for studying unfolded proteins. Since the pioneering work of Gillespie and Shortle (J Mol Biol 1997;268:158), PRE data from unfolded proteins have been interpreted using the theory that was originally developed for rotational spin relaxation. At the same time, it can be

Yi Xue; Ivan S. Podkorytov; D. Krishna Rao; Nathan Benjamin; Honglei Sun; Nikolai R. Skrynnikov

2009-01-01

278

Insulin relaxes bladder via PI3K/AKT/eNOS pathway activation in mucosa: unfolded protein response-dependent insulin resistance as a cause of obesity-associated overactive bladder  

PubMed Central

We aimed to investigate the role of insulin in the bladder and its relevance for the development of overactive bladder (OAB) in insulin-resistant obese mice. Bladders from male individuals who were involved in multiple organ donations were used. C57BL6/J mice were fed with a high-fat diet for 10 weeks to induce insulin-resistant obesity. Concentration–response curves to insulin were performed in human and mouse isolated mucosa-intact and mucosa-denuded bladders. Cystometric study was performed in terminally anaesthetized mice. Western blot was performed in bladders to detect phosphorylated endothelial NO synthase (eNOS) (Ser1177) and the phosphorylated protein kinase AKT (Ser473), as well as the unfolded protein response (UPR) markers TRIB3, CHOP and ATF4. Insulin (1–100 nm) produced concentration-dependent mouse and human bladder relaxations that were markedly reduced by mucosal removal or inhibition of the PI3K/AKT/eNOS pathway. In mouse bladders, insulin produced a 3.0-fold increase in cGMP levels (P < 0.05) that was prevented by PI3K/AKT/eNOS pathway inhibition. Phosphoinositide 3-kinase (PI3K) inhibition abolished insulin-induced phosphorylation of AKT and eNOS in bladder mucosa. Obese mice showed greater voiding frequency and non-voiding contractions, indicating overactive detrusor smooth muscle. Insulin failed to relax the bladder or to increase cGMP in the obese group. Insulin-stimulated AKT and eNOS phosphorylation in mucosa was also impaired in obese mice. The UPR markers TRIB3, CHOP and ATF4 were increased in the mucosa of obese mice. The UPR inhibitor 4-phenyl butyric acid normalized all the functional and molecular parameters in obese mice. Our data show that insulin relaxes human and mouse bladder via activation of the PI3K/AKT/eNOS pathway in the bladder mucosa. Endoplasmic reticulum stress-dependent insulin resistance in bladder contributes to OAB in obese mice.

Leiria, Luiz O; Sollon, Carolina; Bau, Fernando R; Monica, Fabiola Z; D'Ancona, Carlos L; De Nucci, Gilberto; Grant, Andrew D; Anhe, Gabriel F; Antunes, Edson

2013-01-01

279

Unfolded state dynamics and structure of protein L characterized by simulation and experiment  

PubMed Central

While several experimental techniques now exist for characterizing protein unfolded states, all-atom simulation of unfolded states has been challenging due to the long time scales and conformational sampling required. We address this problem by using a combination of accelerated calculations on graphics processor units and distributed computing to simulate tens of thousands of molecular dynamics trajectories each up to ~10 ?s (for a total aggregate simulation time of 127 milliseconds). We used this approach in conjunction with Trp-Cys contact quenching experiments to characterize the unfolded structure and dynamics of protein L. We employed a polymer theory method to make quantitative comparisons between high temperature simulated and chemically denatured experimental ensembles, and find that reaction-limited quenching rates calculated from simulation agree remarkably well with experiment. In both experiment and simulation, we find that unfolded state intramolecular diffusion rates are very slow compared to highly denatured chains, and that a single-residue mutation can significantly alter unfolded state dynamics and structure. This work suggests a view of the unfolded state in which surprisingly low diffusion rates could limit folding, and opens the door for all-atom molecular simulation to be a useful predictive tool for characterizing protein unfolded states along with experiments that directly measure intramolecular diffusion.

Voelz, Vincent A.; Singh, Vijay R.; Wedemeyer, William J.; Lapidus, Lisa J.; Pande, Vijay S.

2010-01-01

280

Thermodynamic Analysis of a Molecular Chaperone Binding to Unfolded Protein Substrates  

PubMed Central

Molecular chaperones are a highly diverse group of proteins that recognize and bind unfolded proteins in order to facilitate protein folding and prevent non-specific protein aggregation. The mechanisms by which chaperones bind their protein substrates have been studied for decades. However, there are few reports on the affinity of molecular chaperones for their unfolded protein substrates. Thus, little is known about the relative binding affinities of different chaperones and about the relative binding affinities of chaperones for different unfolded protein substrates. Here we describe the application of SUPREX (stability of unpurified proteins from rates of H/D exchange), an H/D exchange and MALDI-based technique, to study the binding interaction between the molecular chaperone Hsp33 and four different unfolded protein substrates including citrate synthase, lactate dehydrogenase, malate dehydrogenase, and aldolase. The results of our studies suggest that the cooperativity of the Hsp33 folding/unfolding reaction increases upon binding with denatured protein substrates. This is consistent with the burial of significant hydrophobic surface area in Hsp33 when it interacts with its substrate proteins. The SUPREX derived Kd-values for Hsp33 complexes with four different substrates were found to be all within a range of 3-300 nM.

Xu, Ying; Schmitt, Sebastian; Tang, Liangjie; Jakob, Ursula; Fitzgerald, Michael C.

2010-01-01

281

Native topology determines force-induced unfolding pathways in globular proteins  

NASA Astrophysics Data System (ADS)

Single-molecule manipulation techniques reveal that stretching unravels individually folded domains in the muscle protein titin and the extracellular matrix protein tenascin. These elastic proteins contain tandem repeats of folded domains with -sandwich architecture. Herein, we propose by stretching two model sequences (S1 and S2) with four-stranded -barrel topology that unfolding forces and pathways in folded domains can be predicted by using only the structure of the native state. Thermal refolding of S1 and S2 in the absence of force proceeds in an all-or-none fashion. In contrast, phase diagrams in the force-temperature (f,T) plane and steered Langevin dynamics studies of these sequences, which differ in the native registry of the strands, show that S1 unfolds in an allor-none fashion, whereas unfolding of S2 occurs via an obligatory intermediate. Force-induced unfolding is determined by the native topology. After proving that the simulation results for S1 and S2 can be calculated by using native topology alone, we predict the order of unfolding events in Ig domain (Ig27) and two fibronectin III type domains (9FnIII and 10FnIII). The calculated unfolding pathways for these proteins, the location of the transition states, and the pulling speed dependence of the unfolding forces reflect the differences in the way the strands are arranged in the native states. We also predict the mechanisms of force-induced unfolding of the coiled-coil spectrin (a three-helix bundle protein) for all 20 structures deposited in the Protein Data Bank. Our approach suggests a natural way to measure the phase diagram in the (f,C) plane, where C is the concentration of denaturants.

Klimov, D. K.; Thirumalai, D.

2000-06-01

282

Xanthurenic Acid Provokes Formation of Unfolded Proteins in Endoplasmic Reticulum of the Lens Epithelial Cells  

Microsoft Academic Search

The role of xanthurenic acid in a cell is unknown, but it is suspected to provoke several diseases. This study shows that accumulation of xanthurenic acid in the lens epithelial cells leads to an overexpression of endoplasmic reticulum (ER) resident stress chaperones proteins, glucose-regulated protein (Grp94), and calreticulin. Both chaperones proteins are overexpressed in the presence of unfolded proteins. A

Halina Z. Malina

1999-01-01

283

Thermally-induced protein unfolding probed by isotope-edited IR spectroscopy  

PubMed Central

Infrared (IR) spectroscopy has been widely utilized for the study of protein folding, unfolding and misfolding processes. We have previously developed a theoretical method for calculating IR spectra of proteins in the amide I region. In this work, we apply this method, in combination with replica-exchange molecular dynamics simulations, to study the equilibrium thermal unfolding transition of the villin headpiece subdomain (HP36). Temperature-dependent IR spectra and spectral densities are calculated. The spectral densities correctly reflect the unfolding conformational changes in the simulation. With the help of isotope labeling, we are able to capture the feature that helix 2 of HP36 loses its secondary structure before global unfolding occurs, in agreement with experiment.

Wang, Lu; Skinner, James L.

2012-01-01

284

Toward a Taxonomy of the Denatured State: Small Angle Scattering Studies of Unfolded Proteins  

SciTech Connect

Despite the critical role the unfolded state plays in defining protein folding kinetics and thermodynamics (Berg et al., 2002; Dunker, 2002; Shortle, 2002; Wright and Dyson, 2002), our understanding of its detailed structure remains rather rudimentary; the heterogeneity of the unfolded ensemble renders difficult or impossible its study by traditional, atomic-level structural methods. Consequently, recent years have seen a significant expansion of small-angle X-ray and neutron scattering (SAXS and SANS, respectively) techniques that provide direct, albeit rotationally and time-averaged, measures of the geometric properties of the unfolded ensemble. These studies have reached a critical mass, allowing us for the first time to define general observations regarding the nature of the geometry - and possibly the chemistry and physics - of unfolded proteins.

Millett, I.S.; Doniach, S.; Plaxco, K.W. (Stanford); (UCSB)

2005-02-15

285

Stretching Single-Domain Proteins: Phase Diagram and Kinetics of Force-Induced Unfolding  

Microsoft Academic Search

Single-molecule force spectroscopy reveals unfolding of domains in titin on stretching. We provide a theoretical framework for these experiments by computing the phase diagrams for force-induced unfolding of single-domain proteins using lattice models. The results show that two-state folders (at zero force) unravel cooperatively, whereas stretching of non-two-state folders occurs through intermediates. The stretching rates of individual molecules show great

D. K. Klimov; D. Thirumalai

1999-01-01

286

Direct Observation of Protein Unfolded State Compaction in the Presence of Macromolecular Crowding  

PubMed Central

Proteins fold and function in cellular environments that are crowded with other macromolecules. As a consequence of excluded volume effects, compact folded states of proteins should be indirectly stabilized due to destabilization of extended unfolded conformations. Here, we assess the role of excluded volume in terms of protein stability, structural dimensions and folding dynamics using a sugar-based crowding agent, dextran 20, and the small ribosomal protein S16 as a model system. To specifically address dimensions, we labeled the protein with BODIPY at two positions and measured Trp-BODIPY distances under different conditions. As expected, we found that dextran 20 (200 mg/ml) stabilized the variants against urea-induced unfolding. At conditions where the protein is unfolded, Förster resonance energy transfer measurements reveal that in the presence of dextran, the unfolded ensemble is more compact and there is residual structure left as probed by far-ultraviolet circular dichroism. In the presence of a crowding agent, folding rates are faster in the two-state regime, and at low denaturant concentrations, a kinetic intermediate is favored. Our study provides direct evidence for protein unfolded-state compaction in the presence of macromolecular crowding along with its energetic and kinetic consequences.

Mikaelsson, Therese; Aden, Jorgen; Johansson, Lennart B.-A.; Wittung-Stafshede, Pernilla

2013-01-01

287

Simulation and Analysis of Differential Scanning Calorimetry Output: Protein Unfolding Studies  

NASA Astrophysics Data System (ADS)

High sensitivity differential scanning calorimetry ( HSDSC ) is a powerful and increasingly used technique for the study of molecular energetics in relation to biopolymers (e.g. proteins and nucleic acids ) or biomacromolecular assemblies ( e.g. liposomes ). HSDSC is, for example, the only method which allows thermodynamic parameters to be obtained directly for protein unfolding. The thermodynamic basis for HSDSC signals obtained for the equilibrium unfolding of proteins as well as the formalism for generating simulations of HSDSC outputs for protein unfolding are discussed. This will allow students ( studying biophysical chemistry, biochemistry, biophysics, molecular biology and allied subjects at undergraduate and postgraduate level ) to obtain an understanding of the type of data analysis required for HSDSC experiments on proteins.

Chowdhry, Babur; Leharne, Stephen

1997-02-01

288

Unfolding proteins with mechanical forces: From toy models to atomistic simulations  

NASA Astrophysics Data System (ADS)

The remarkable combination of strength and toughness, displayed by certain biological materials (e.g. spider silk) and often unmatched by artificial materials, is believed to originate from the mechanical response of individual load-bearing protein domains. Single-molecule pulling experiments carried out during the last decade showed that those proteins, when loaded, respond in a non-equilibrium fashion and can dissipate large amounts of energy though the breaking of sacrificial bonds. In my talk, I will discuss what structural properties correlate with mechanical strength and toughness at the single-molecule level, how thermodynamic stability is related to the mechanical stability, and why both atomistic simulations and simple models seem to fail to reconcile the mechanical responses of the same proteins measured under varied loading regimes. I will further discuss whether it is easier to unfold a protein mechanically by pulling at its ends or by threading it through a narrow pore. The latter process is believed to commonly occur in living organisms as an intermediate step in protein degradation.

Makarov, Dmitrii

2011-03-01

289

A Defective Signal Peptide in a 19-kD ?-Zein Protein Causes the Unfolded Protein Response and an Opaque Endosperm Phenotype in the Maize De*-B30 Mutant1  

PubMed Central

Defective endosperm* (De*)-B30 is a dominant maize (Zea mays) mutation that depresses zein synthesis in the developing endosperm. The mutant kernels have an opaque, starchy phenotype, malformed zein protein bodies, and highly increased levels of binding protein and other chaperone proteins in the endosperm. Immunoblotting revealed a novel ?-zein protein in De*-B30 that migrates between the 22- and 19-kD ?-zein bands. Because the De*-B30 mutation maps in a cluster of 19-kD ?-zein genes, we characterized cDNA clones encoding these proteins from a developing endosperm library. This led to the identification of a 19-kD ?-zein cDNA in which proline replaces serine at the 15th position of the signal peptide. Although the corresponding gene does not appear to be highly expressed in De*-B30, it was found to be tightly linked with the mutant phenotype in a segregating F2 population. Furthermore, when the protein was synthesized in yeast cells, the signal peptide appeared to be less efficiently processed than when serine replaced proline. To test whether this gene is responsible for the De*-B30 mutation, transgenic maize plants expressing this sequence were created. T1 seeds originating from the transformants manifested an opaque kernel phenotype with enhanced levels of binding protein in the endosperm, similar to De*-B30. These results are consistent with the hypothesis that the De*-B30 mutation causes a defective signal peptide in a 19-kD ?-zein protein.

Kim, Cheol Soo; Hunter, Brenda G.; Kraft, Jeffery; Boston, Rebecca S.; Yans, Sarah; Jung, Rudolf; Larkins, Brian A.

2004-01-01

290

THE SURFACE-MEDIATED UNFOLDING KINETICS OF GLOBULAR PROTEINS IS DEPENDENT ON MOLECULAR WEIGHT AND TEMPERATURE  

SciTech Connect

The adsorption and unfolding pathways of proteins on rigid surfaces are essential in numerous complex processes associated with biomedical engineering, nanotechnology, and chromatography. It is now well accepted that the kinetics of unfolding are characterized by chemical and physical interactions dependent on protein deformability and structure, as well as environmental pH, temperature, and surface chemistry. Although this fundamental process has broad implications in medicine and industry, little is known about the mechanism because of the atomic lengths and rapid time scales involved. Therefore, the unfolding kinetics of myoglobin, ?-glucosidase, and ovalbumin were investigated by adsorbing the globular proteins to non-porous cationic polymer beads. The protein fractions were adsorbed at different residence times (0, 9, 10, 20, and 30 min) at near-physiological conditions using a gradient elution system similar to that in high-performance liquid chromatography. The elution profi les and retention times were obtained by ultraviolet/visible spectrophotometry. A decrease in recovery was observed with time for almost all proteins and was attributed to irreversible protein unfolding on the non-porous surfaces. These data, and those of previous studies, fi t a positively increasing linear trend between percent unfolding after a fi xed (9 min) residence time (71.8%, 31.1%, and 32.1% of myoglobin, ?-glucosidase, and ovalbumin, respectively) and molecular weight. Of all the proteins examined so far, only myoglobin deviated from this trend with higher than predicted unfolding rates. Myoglobin also exhibited an increase in retention time over a wide temperature range (0°C and 55°C, 4.39 min and 5.74 min, respectively) whereas ovalbumin and ?-glucosidase did not. Further studies using a larger set of proteins are required to better understand the physiological and physiochemical implications of protein unfolding kinetics. This study confi rms that surface-mediated unfolding can be described by experimental techniques, thereby allowing for the better elucidation of the relationships between the structure and function of soluble proteins as well as other macromolecules.

Patananan, A.N.; Goheen, S.C.

2008-01-01

291

The Surface-Mediated Unfolding Kinetics of Globular Proteins is Dependent on Molecular Weight and Temperature  

SciTech Connect

The adsorption and unfolding of proteins on rigid surfaces is characterized by numerous chemical and physical interactions such as hydrogen bonds, disulfide bridges, hydrophobic effects, and London forces. The kinetics of unfolding is dependent on pH, temperature, surface chemistry, as well as protein deformability and structure. In practical applications, this fundamental process has broad implications in biomedical engineering (i.e. artificial implants, drug delivery, and surgical equipment), nanotechnology, maritime construction, and chromatography. However, little is known about the mechanisms behind unfolding because of the atomic lengths and rapid time scales associated with the surface-mediated pathway. Therefore, the unfolding kinetics of myoglobin, ?-glucosidase, and ovalbumin were investigated by adsorbing the proteins to non-porous cationic polymer beads. The protein fractions were adsorbed at different residence times (0, 9, 10, 20, and 30 min) at near-physiological conditions using a gradient elution system similar to that in high-performance liquid chromatography (HPLC). The elution profiles and retention times were obtained by UV/Vis spectrophotometry. A decrease in recovery was observed with time for almost all proteins and was attributed to protein unfolding on the non-porous surfaces. This data, and those of previous studies, fit a linear trend between percent unfolding after a fixed (9 min) residence time (71.8%, 31.1%, and 32.1% of myoglobin, ?-glucosidase, and ovalbumin, respectively) and molecular weight. Of all the proteins examined so far, only myoglobin deviated from this trend. Myoglobin also exhibited an increase in retention time over a wide temperature range (0?C and 55?C, 4.39 min and 5.74 min, respectively) whereas ovalbumin and ?-glucosidase did not. Further studies using a larger set of proteins are required to better understand the physiological and physiochemical implications of protein unfolding kinetics. This study confirms that unfolding can be described by experimental techniques, thereby allowing for the better elucidation of the relationships between the structure and function of soluble proteins as well as other macromolecules.

Patananan, Alexander; Goheen, Steven C.

2008-12-01

292

Direct quantification of the attempt frequency determining the mechanical unfolding of ubiquitin protein.  

PubMed

Understanding protein dynamics requires a comprehensive knowledge of the underlying potential energy surface that governs the motion of each individual protein molecule. Single molecule mechanical studies have provided the unprecedented opportunity to study the individual unfolding pathways along a well defined coordinate, the end-to-end length of the protein. In these experiments, unfolding requires surmounting an energy barrier that separates the native from the extended state. The calculation of the absolute value of the barrier height has traditionally relied on the assumption of an attempt frequency, ?(‡). Here we used single molecule force-clamp spectroscopy to directly determine the value of ?(‡) for mechanical unfolding by measuring the unfolding rate of the small protein ubiquitin at varying temperatures. Our experiments demonstrate a significant effect of the temperature on the mechanical rate of unfolding. By extrapolating the unfolding rate in the absence of force for different temperatures, varying within the range spanning from 5 to 45 °C, we measured a value for the activation barrier of ?G(‡) = 71 ± 5 kJ/mol and an exponential prefactor ?(‡) ?4 × 10(9) s(-1). Although the measured prefactor value is 3 orders of magnitude smaller than the value predicted by the transition state theory (?6 × 10(12) s(-1)), it is 400-fold higher than that encountered in analogous experiments studying the effect of temperature on the reactivity of a protein-embedded disulfide bond (?10(7) M(-1) s(-1)). This approach will allow quantitative characterization of the complete energy landscape of a folding polypeptide from highly extended states, of capital importance for proteins with elastic function. PMID:21768096

Popa, Ionel; Fernández, Julio M; Garcia-Manyes, Sergi

2011-09-01

293

Forced unfolding of proteins within cells -- a proteomic method  

NASA Astrophysics Data System (ADS)

Many cellular activities are mediated by conformational changes in proteins or else involve rearrangement of protein assemblies. These motions are now commonly investigated in vitro as well as at the single-molecule level. But we sought to develop an in-cell method to study these motions and to do so on a proteomic scale. We have been especially interested in studying molecular responses in cells under stress, and we initially developed a labeling technique in the simplest human cell, the red blood cell. The premise is to label cysteines with cell-viable, thiol-reactive fluorophores in both stressed and unstressed cells. Then, differential labeling of proteins would indicate that under stress, previously buried cysteine residues become exposed and thus accessible to the fluorescent probe. Fluorescence imaging and saparations provide initial clues to structures and proteins, but Mass Spectrometry precisely maps the sites that are exposed. Subsequent work on recombinants and in modeling is then used to explain the cell-derived findings, and the method has now been applied to several nucleated cell types.

Chase, Brian; Discher, Dennis

2009-03-01

294

Pressure Equilibrium and Jump Study on Unfolding of 23-kDa Protein from Spinach Photosystem II  

PubMed Central

Pressure-induced unfolding of 23-kDa protein from spinach photosystem II has been systematically investigated at various experimental conditions. Thermodynamic equilibrium studies indicate that the protein is very sensitive to pressure. At 20°C and pH 5.5, 23-kDa protein shows a reversible two-state unfolding transition under pressure with a midpoint near 160 MPa, which is much lower than most natural proteins studied to date. The free energy (?Gu) and volume change (?Vu) for the unfolding are 5.9 kcal/mol and ?160 ml/mol, respectively. It was found that NaCl and sucrose significantly stabilize the protein from unfolding and the stabilization is associated not only with an increase in ?Gu but also with a decrease in ?Vu. The pressure-jump studies of 23-kDa protein reveal a negative activation volume for unfolding (?66.2 ml/mol) and a positive activation volume for refolding (84.1 ml/mol), indicating that, in terms of system volume, the protein transition state lies between the folded and unfolded states. Examination of the temperature effect on the unfolding kinetics indicates that the thermal expansibility of the transition state and the unfolded state of 23-kDa protein are closer to each other and they are larger than that of the native state. The diverse pressure-refolding pathways of 23-kDa protein in some conditions were revealed in pressure-jump kinetics.

Tan, Cui-Yan; Xu, Chun-He; Wong, Jun; Shen, Jian-Ren; Sakuma, Shinsuke; Yamamoto, Yasusi; Lange, Reinhard; Balny, Claude; Ruan, Kang-Cheng

2005-01-01

295

Protein Unfolding Coupled to Ligand Binding: Differential Scanning Calorimetry Simulation Approach  

NASA Astrophysics Data System (ADS)

The aim of this work is to present the physicochemical basis underlying the changes in protein thermostability upon ligand binding. The article is addressed to advanced undergraduate and postgraduate chemistry students with an interest in protein biophysics. In addition, this article provides a useful tool for both learning and teaching biophysics because it links fundamental concepts: thermodynamics, chemical equilibrium, and protein stability. The influence of protein ligand interactions on thermally-induced protein denaturation was monitored by differential scanning calorimetry (DSC). The changes in DSC output (thermogram) emerge by linking binding equilibrium with reversible protein unfolding thermodynamics. We derive the formalism for the description of protein unfolding in the presence of ligand that can bind to a single site on either native, unfolded, or both protein states. In addition to a rigorous mathematical description of the involved equilibria, the model provides the general formulation for simulating thermograms and calculating the changes in protein species during heating. First, we describe ligand interaction and emphasize the relationship between protein stability parameters and redistribution of species in equilibrium. After that, we describe the origin of bimodal thermograms, and finally, the effect on thermogram shape of protein concentration at constant ligand/protein mole ratio.

Soledad Celej, María; Fidelio, Gerardo Daniel; Dassie, Sergio Alberto

2005-01-01

296

A Hyperbolic Cosine Latent Trait Model For Unfolding Dichotomous Single-Stimulus Responses  

Microsoft Academic Search

Social-psychological variables are typically measured using either cumulative or unfolding response processes. In the former, the greater the location of a person relative to the location of a stimulus on the continuum, the greater the proba bility of a positive response; in the latter, the closer the location of the person to the location of the statement, irrespective of direction,

David Andrich; Guanzhong Luo

1993-01-01

297

Protein denaturation in vacuo: intrinsic unfolding pathways associated with the native tertiary structure of lysozyme  

NASA Astrophysics Data System (ADS)

Using computer-simulated molecular dynamics, we study the effect of sequence mutation on the unfolding mechanism of a native fold. The system considered is the native fold of hen egg-white lysozyme, exposed to centrifugal unfolding in vacuo. This unfolding bias elicits configurational transitions that imitate the behaviour of anhydrous proteins diffusing after electrospraying from neutral-pH solutions. By changing the sequences threaded onto the native fold of lysozyme, we probe the role of disulfide bridges and the effect of a global mutation. We find that the initial denaturing steps share common characteristics for the tested sequences. Recurrent features are: (i) the presence of dumbbell conformers with significant residual secondary structure, (ii) the ubiquitous formation of hairpins and two-stranded ?-sheets regardless of disulfide bridges, and (iii) an unfolding pattern where the reduction in folding complexity is highly correlated with the decrease in chain compactness. These findings appear to be intrinsic to the shape of the native fold, suggesting that similar unfolding pathways may be accessible to many protein sequences.

Arteca, Gustavo A.; Tapia, O.

298

[Pharmacological studies on neurodegenerative diseases focusing on refolding and degradation of unfolded proteins in the endoplasmic reticulum].  

PubMed

The endoplasmic reticulum (ER) has physiological roles in the quality control of proteins. Various stresses (e.g., oxidation, aging) to the ER cause accumulation of unfolded/misfolded proteins in the ER lumen, followed by unfolded protein responses (UPR) such as refolding of unfolded protein by chaperons, ER-associated degradation (ERAD), and termination of protein synthesis. In this study, we identified protein-disulfide isomerase (PDI) upregulation by hypoxic stress in the ER of rat brains/astroglial cells. PDI overexpression attenuates hypoxia-induced neuronal apoptosis. In the brain autopsy of patients with sporadic Alzheimer's and Parkinson diseases, PDI was found to be S-nitrosylated, which reduced chaperone activity of PDI, suggesting the involvement of PDI in these diseases. In addition, we identified the novel E3 ubiquitin ligase HRD1 and observed that HRD1 activates degradation of Parkin-associated endothelin receptor-like receptor (Pael-R). HRD1 suppresses ER stress and Pael-R-induced apoptosis. Furthermore, HRD1 ubiquitinates amyloid precursor protein (APP), resulting in the decrease in amyloid ? (A?) generation. Suppression of HRD1 expression causes APP accumulation and A? generation. HRD1 protein significantly decreased in the cerebral cortex of patients with Alzheimer's disease. HRD1 decrease in the brain of patients with Alzheimer's disease could be due to the insolubilization of HRD1 by oxidative stress. Subsequently, we observed that 4-phenylbutyric acid (4-PBA) possesses chaperone activity, which prevents protein aggregation and that 4-(4-methoxyphenyl)butanoic acid, a 4-PBA derivative, increases protective ability against ER stress-induced neuronal death. We believe that 4-PBA and its derivatives are potential candidates for pharmacological intervention for ER stress-induced neurodegenerative diseases. PMID:24694815

Nomura, Yasuyuki

2014-01-01

299

Precursory signatures of protein folding/unfolding: From time series correlation analysis to atomistic mechanisms  

NASA Astrophysics Data System (ADS)

Folded conformations of proteins in thermodynamically stable states have long lifetimes. Before it folds into a stable conformation, or after unfolding from a stable conformation, the protein will generally stray from one random conformation to another leading thus to rapid fluctuations. Brief structural changes therefore occur before folding and unfolding events. These short-lived movements are easily overlooked in studies of folding/unfolding for they represent momentary excursions of the protein to explore conformations in the neighborhood of the stable conformation. The present study looks for precursory signatures of protein folding/unfolding within these rapid fluctuations through a combination of three techniques: (1) ultrafast shape recognition, (2) time series segmentation, and (3) time series correlation analysis. The first procedure measures the differences between statistical distance distributions of atoms in different conformations by calculating shape similarity indices from molecular dynamics simulation trajectories. The second procedure is used to discover the times at which the protein makes transitions from one conformation to another. Finally, we employ the third technique to exploit spatial fingerprints of the stable conformations; this procedure is to map out the sequences of changes preceding the actual folding and unfolding events, since strongly correlated atoms in different conformations are different due to bond and steric constraints. The aforementioned high-frequency fluctuations are therefore characterized by distinct correlational and structural changes that are associated with rate-limiting precursors that translate into brief segments. Guided by these technical procedures, we choose a model system, a fragment of the protein transthyretin, for identifying in this system not only the precursory signatures of transitions associated with ? helix and ? hairpin, but also the important role played by weaker correlations in such protein folding dynamics.

Hsu, P. J.; Cheong, S. A.; Lai, S. K.

2014-05-01

300

Reversible unfolding of a thermophilic membrane protein in phospholipid/detergent mixed micelles.  

PubMed

Folding mechanisms and stability of membrane proteins are poorly understood because of the known difficulties in finding experimental conditions under which reversible denaturation could be possible. In this work, we describe the equilibrium unfolding of Archaeoglobus fulgidus CopA, an 804-residue alpha-helical membrane protein that is involved in transporting Cu(+) throughout biological membranes. The incubation of CopA reconstituted in phospholipid/detergent mixed micelles with high concentrations of guanidinium hydrochloride induced a reversible decrease in fluorescence quantum yield, far-UV ellipticity, and loss of ATPase and phosphatase activities. Refolding of CopA from this unfolded state led to recovery of full biological activity and all the structural features of the native enzyme. CopA unfolding showed typical characteristics of a two-state process, with DeltaG(w) degrees =12.9 kJ mol(-)(1), m=4.1 kJ mol(-1) M(-1), C(m)=3 M, and DeltaCp(w) degrees =0.93 kJ mol(-1) K(-1). These results point out to a fine-tuning mechanism for improving protein stability. Circular dichroism spectroscopic analysis of the unfolded state shows that most of the secondary and tertiary structures were disrupted. The fraction of Trp fluorescence accessible to soluble quenchers shifted from 0.52 in the native state to 0.96 in the unfolded state, with a significant spectral redshift. Also, hydrophobic patches in CopA, mainly located in the transmembrane region, were disrupted as indicated by 1-anilino-naphtalene-8-sulfonate fluorescence. Nevertheless, the unfolded state had a small but detectable amount of residual structure, which might play a key role in both CopA folding and adaptation for working at high temperatures. PMID:20114054

Roman, Ernesto A; Argüello, José M; González Flecha, F Luis

2010-03-26

301

Protein folding, unfolding and aggregation. Pressure induced intermediate states on the refolding pathway of horseradish peroxidase  

NASA Astrophysics Data System (ADS)

We studied the refolding and aggregation of pressure unfolded proteins. Horseradish peroxidase was found to be very stable and no partially folded intermediates were populated during the refolding. However, the removal of the haem group or the Ca2+ ions or reduction of the disulfide bridge destabilized the protein, resulting in a significant amount of aggregation prone intermediate conformation. Substitution of the haem for fluorescent porphyrin however did not influence the refolding of the protein.

Smeller, László; Fidy, Judit; Heremans, Karel

2004-04-01

302

Chemistry on a Single Protein, Vascular Cell Adhesion Molecule1, during Forced Unfolding  

Microsoft Academic Search

Proteins of many types experience tensile forces in their normal function, and vascular cell adhesion mole- cule-1 (VCAM-1) is typical in this. VCAM has seven Ig domains, and each has a disulfide bond (-S-S-) buried in its core that covalently stabilizes about half of each domain against unfolding. VCAM is extended here by single molecule atomic force microscopy in the

Nishant Bhasin; Philippe Carl; Sandy Harper; Gang Feng; Hui Lu; David W. Speicher

2004-01-01

303

Single molecule unfolding and stretching of protein domains inside a solid-state nanopore by electric field  

PubMed Central

Single molecule methods have provided a significantly new look at the behavior of biomolecules in both equilibrium and non-equilibrium conditions. Most notable are the stretching experiments performed by atomic force microscopes and laser tweezers. Here we present an alternative single molecule method that can unfold a protein domain, observed at electric fields greater than 106?V/m, and is fully controllable by the application of increasing voltages across the membrane of the pore. Furthermore this unfolding mechanism is characterized by measuring both the residence time of the protein within the nanopore and the current blockade. The unfolding data supports a gradual unfolding mechanism rather than the cooperative transition observed by classical urea denaturation experiments. Lastly it is shown that the voltage-mediated unfolding is a function of the stability of the protein by comparing two mutationally destabilized variants of the protein.

Freedman, Kevin J.; Haq, S. Raza; Edel, Joshua B.; Jemth, Per; Kim, Min Jun

2013-01-01

304

Single-molecule protein unfolding and translocation by an ATP-fueled proteolytic machine  

PubMed Central

All cells employ ATP-powered proteases for protein-quality control and regulation. In the ClpXP protease, ClpX is a AAA+ machine that recognizes specific protein substrates, unfolds these molecules, and then translocates the denatured polypeptide through a central pore and into ClpP for degradation. Here, we use optical-trapping nanometry to probe the mechanics of enzymatic unfolding and translocation of single molecules of a multidomain substrate. Our experiments demonstrate the capacity of ClpXP and ClpX to perform mechanical work under load, reveal very fast and highly cooperative unfolding of individual substrate domains, suggest a translocation step size of 5–8 amino acids, and support a power-stroke model of denaturation in which successful enzyme-mediated unfolding of stable domains requires coincidence between mechanical pulling by the enzyme and a transient stochastic reduction in protein stability. We anticipate that single-molecule studies of the mechanical properties of other AAA+ proteolytic machines will reveal many shared features with ClpXP.

Aubin-Tam, Marie-Eve; Olivares, Adrian O.; Sauer, Robert T.; Baker, Tania A.; Lang, Matthew J.

2011-01-01

305

Amyloid Fibril Formation Can Proceed from Different Conformations of a Partially Unfolded Protein  

PubMed Central

Protein misfolding and aggregation are interconnected processes involved in a wide variety of nonneuropathic, systemic, and neurodegenerative diseases. More generally, if mutations in sequence or changes in environmental conditions lead to partial unfolding of the native state of a protein, it will often aggregate, sometimes into well-defined fibrillar structures. A great deal of interest has been directed at discovering the characteristic features of metastable partially unfolded states that precede the aggregated states of proteins. In this work, human muscle acylphosphatase (AcP) has been first destabilized, by addition of urea or by means of elevated temperatures, and then incubated in the presence of different concentrations of 2,2,2, trifluoroethanol ranging from 5% to 25% (v/v). The results show that AcP is able to form both fibrillar and nonfibrillar aggregates with a high ?-sheet content from partially unfolded states with very different structural features. Moreover, the presence of ?-helical structure in such a state does not appear to be a fundamental determinant of the ability to aggregate. The lack of ready aggregation under some of the conditions examined here is attributable primarily to the intrinsic properties of the solutions rather than to specific structural features of the partially unfolded states that precede aggregation. Aggregation appears to be favored when the solution conditions promote stable intermolecular interactions, particularly hydrogen bonds. In addition, the structures of the resulting aggregates are largely independent of the conformational properties of their soluble precursors.

Calamai, Martino; Chiti, Fabrizio; Dobson, Christopher M.

2005-01-01

306

Bile salts act as effective protein-unfolding agents and instigators of disulfide stress in vivo.  

PubMed

Commensal and pathogenic bacteria must deal with many different stress conditions to survive in and colonize the human gastrointestinal tract. One major challenge that bacteria encounter in the gut is the high concentration of bile salts, which not only aid in food absorption but also act as effective physiological antimicrobials. The mechanism by which bile salts limit bacterial growth is still largely unknown. Here, we show that bile salts cause widespread protein unfolding and aggregation, affecting many essential proteins. Simultaneously, the bacterial cytosol becomes highly oxidizing, indicative of disulfide stress. Strains defective in reducing oxidative thiol modifications, restoring redox homeostasis, or preventing irreversible protein aggregation under disulfide stress conditions are sensitive to bile salt treatment. Surprisingly, cholate and deoxycholate, two of the most abundant and very closely related physiological bile salts, vary substantially in their destabilizing effects on proteins in vitro and cause protein unfolding of different subsets of proteins in vivo. Our results provide a potential mechanistic explanation for the antimicrobial effects of bile salts, help explain the beneficial effects of bile salt mixtures, and suggest that we have identified a physiological source of protein-unfolding disulfide stress conditions in bacteria. PMID:24706920

Cremers, Claudia M; Knoefler, Daniela; Vitvitsky, Victor; Banerjee, Ruma; Jakob, Ursula

2014-04-22

307

Unfolding of a model protein on ion exchange and mixed mode chromatography surfaces.  

PubMed

Recent studies with proteins indicate that conformational changes and aggregation can occur during ion exchange chromatography (IEC). Such behavior is not usually expected, but could lead to decreased yield and product degradation from both IEC and multi mode chromatography (MMC) that has ligands of both hydrophobic and charged functionalities. In this study, we used hydrogen exchange mass spectrometry to investigate unfolding of the model protein BSA on IEC and MMC surfaces under different solution conditions at 25°C. Increased solvent exposure, indicating greater unfolding relative to that in solution, was found for protein adsorbed on cationic IEC and MMC surfaces in the pH range of 3.0 to 4.5, where BSA has decreased stability in solution. There was no effect of anionic surfaces at pH values in the range from 6.0 to 9.0. Differences of solvent exposure of whole molecules when adsorbed and in solution suggest that adsorbed BSA unfolds at lower pH values and may show aggregation, depending upon pH and the surface type. Measurements on digested peptides showed that classifications of stability can be made for various regions; these are generally retained as pH is changed. When salt was added to MMC systems, where electrostatic interactions would be minimized, less solvent exposure was seen, implying that it is the cationic moieties, rather than the hydrophobic ligands, which cause greater surface unfolding at low salt concentrations. These results suggest that proteins of lower stability may exhibit unfolding and aggregation during IEC and MMC separations, as they can with hydrophobic interaction chromatography. PMID:24997510

Gospodarek, Adrian M; Hiser, Diana E; O'Connell, John P; Fernandez, Erik J

2014-08-15

308

Microsecond dynamics of an unfolded protein by a line confocal tracking of single molecule fluorescence  

PubMed Central

We present a new method for high speed tracking of fluorescence time series from single proteins. The method uses a fast sample flow and a modified confocal microscopy, line confocal microscopy, and achieves the time resolution of less than 20??s. The obtained time series from the B domain of protein A labeled with donor and acceptor fluorophores suggest conformational heterogeneity and dynamic fluctuations in the unfolded state.

Oikawa, Hiroyuki; Suzuki, Yuta; Saito, Masataka; Kamagata, Kiyoto; Arai, Munehito; Takahashi, Satoshi

2013-01-01

309

Microsecond simulations of the folding/unfolding thermodynamics of the Trp-cage mini protein  

PubMed Central

We study the unbiased folding/unfolding thermodynamics of the Trp-cage miniprotein using detailed molecular dynamics simulations of an all-atom model of the protein in explicit solvent, using the Amberff99SB force field. Replica-exchange molecular dynamics (REMD) simulations are used to sample the protein ensembles over a broad range of temperatures covering the folded and unfolded states, and at two densities. The obtained ensembles are shown to reach equilibrium in the 1 ?s per replica timescale. The total simulation time employed in the calculations exceeds 100 ?s. Ensemble averages of the fraction folded, pressure, and energy differences between the folded and unfolded states as a function of temperature are used to model the free energy of the folding transition, ?G(P,T), over the whole region of temperature and pressures sampled in the simulations. The ?G(P,T) diagram describes an ellipse over the range of temperatures and pressures sampled, predicting that the system can undergo pressure induced unfolding and cold denaturation at low temperatures and high pressures, and unfolding at low pressures and high temperatures. The calculated free energy function exhibits remarkably good agreement with the experimental folding transition temperature (Tf = 321 K), free energy and specific heat changes. However, changes in enthalpy and entropy are significantly different than the experimental values. We speculate that these differences may be due to the simplicity of the semi-empirical force field used in the simulations and that more elaborate force fields may be required to describe appropriately the thermodynamics of proteins.

Day, Ryan; Paschek, Dietmar; Garcia, Angel E.

2012-01-01

310

Exploring the role of internal friction in the dynamics of unfolded proteins using simple polymer models  

NASA Astrophysics Data System (ADS)

Recent experiments showed that the reconfiguration dynamics of unfolded proteins are often adequately described by simple polymer models. In particular, the Rouse model with internal friction (RIF) captures internal friction effects as observed in single-molecule fluorescence correlation spectroscopy (FCS) studies of a number of proteins. Here we use RIF, and its non-free draining analog, Zimm model with internal friction, to explore the effect of internal friction on the rate with which intramolecular contacts can be formed within the unfolded chain. Unlike the reconfiguration times inferred from FCS experiments, which depend linearly on the solvent viscosity, the first passage times to form intramolecular contacts are shown to display a more complex viscosity dependence. We further describe scaling relationships obeyed by contact formation times in the limits of high and low internal friction. Our findings provide experimentally testable predictions that can serve as a framework for the analysis of future studies of contact formation in proteins.

Cheng, Ryan R.; Hawk, Alexander T.; Makarov, Dmitrii E.

2013-02-01

311

Apolar and polar solvation thermodynamics related to the protein unfolding process.  

PubMed Central

Thermodynamics related to hydrated water upon protein unfolding is studied over a broad temperature range (5-125 degrees C). The hydration effect arising from the apolar interior is modeled as an increased number of hydrogen bonds between water molecules compared with bulk water. The corresponding contribution from the polar interior is modeled as a two-step process. First, the polar interior breaks hydrogen bonds in bulk water upon unfolding. Second, due to strong bonds between the polar surface and the nearest water molecules, we assume quantization using a simplified two-state picture. The heat capacity change upon hydration is compared with model compound data evaluated previously for 20 different proteins. We obtain good correspondence with the data for both the apolar and the polar interior. We note that the effective coupling constants for both models have small variations among the proteins we have investigated.

Bakk, Audun; H?ye, Johan S; Hansen, Alex

2002-01-01

312

Unfolding and Refolding of Juvenile Hormone Binding Protein  

Microsoft Academic Search

Juvenile hormone (JH) regulates insect development. JH present in the hemolymph is bound to a specific glycoprotein, juvenile hormone binding protein (JHBP), which serves as a carrier to deploy the hormone to target tissues. In this report structural changes of JHBP from Galleria mellonella induced by guanidine hydrochloride have been investigated by a combination of size-exclusion chromatography, protein activity measurements,

Piotr Dobryszycki; Robert Ko?odziejczyk; Daniel Krowarsch; Jacek Gapi?ski; Andrzej O?yhar; Marian Kochman

2004-01-01

313

High-Energy Intermediates in Protein Unfolding Characterized by Thiol Labeling under Nativelike Conditions.  

PubMed

A protein unfolding reaction usually appears to be so dominated by a large free energy barrier that identifying and characterizing high-energy intermediates and, hence, dissecting the unfolding reaction into multiple structural transitions have proven to be a challenge. In particular, it has been difficult to identify any detected high-energy intermediate with the dry (DMG) and wet (WMG) molten globules that have been implicated in the unfolding reactions of at least some proteins. In this study, a native-state thiol labeling methodology was used to identify high-energy intermediates, as well as to delineate the barriers to the disruption of side chain packing interactions and to site-specific solvent exposure in different regions of the small protein, single-chain monellin (MNEI). Labeling studies of four single-cysteine-containing variants of MNEI have identified three high-energy intermediates, populated to very low extents under nativelike conditions. A significant dispersion in the opening rates of the cysteine side chains has allowed multiple steps, leading to the loss of side chain packing, to be resolved temporally. A detailed structural analysis of the positions of the four cysteine residue positions, which are buried to different depths within the protein, has suggested a direct correlation with the structure of a DMG, detected in previous studies. It is observed that side chain packing within the core of the protein is maintained, while that at the surface is disrupted, in the DMG. The core of the protein becomes solvent-exposed only in a WMG populated after the rate-limiting step of unfolding at high denaturant concentrations. PMID:24832528

Malhotra, Pooja; Udgaonkar, Jayant B

2014-06-10

314

Antigen retrieval causes protein unfolding: evidence for a linear epitope model of recovered immunoreactivity.  

PubMed

Antigen retrieval (AR), in which formalin-fixed paraffin-embedded tissue sections are briefly heated in buffers at high temperature, often greatly improves immunohistochemical staining. An important unresolved question regarding AR is how formalin treatment affects the conformation of protein epitopes and how heating unmasks these epitopes for subsequent antibody binding. The objective of the current study was to use model proteins to determine the effect of formalin treatment on protein conformation and thermal stability in relation to the mechanism of AR. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to identify the presence of protein formaldehyde cross-links, and circular dichroism spectropolarimetry was used to determine the effect of formalin treatment and high-temperature incubation on the secondary and tertiary structure of the model proteins. Results revealed that for some proteins, formalin treatment left the native protein conformation unaltered, whereas for others, formalin denatured tertiary structure, yielding a molten globule protein. In either case, heating to temperatures used in AR methods led to irreversible protein unfolding, which supports a linear epitope model of recovered protein immunoreactivity. Consequently, the core mechanism of AR likely centers on the restoration of normal protein chemical composition coupled with improved accessibility to linear epitopes through protein unfolding. PMID:21411808

Fowler, Carol B; Evers, David L; O'Leary, Timothy J; Mason, Jeffrey T

2011-04-01

315

Real-time investigation of protein unfolding at an air-water interface at the 1 s time scale  

PubMed Central

Protein unfolding at an air–water interface has been demonstrated such that the X-ray reflectivity can be measured with an acquisition time of 1?s using a recently developed simultaneous multiple-angle–wavelength-dispersive X-ray reflectometer. This has enabled the electron density profile of the adsorbed protein molecules to be obtained in real time. A globular protein, lysozyme, adsorbed at the air–water interface is found to unfold into a flat shape within 1?s.

Yano, Yohko F.; Arakawa, Etsuo; Voegeli, Wolfgang; Matsushita, Tadashi

2013-01-01

316

Opioid peptides derived from food proteins suppress aggregation and promote reactivation of partly unfolded stressed proteins.  

PubMed

A new view of the opioid peptides is presented. The potential of small peptides derived from precursor food proteins, to bind to partly unfolded stressed proteins to prevent their irreversible aggregation and inactivation has been demonstrated in various in vitro test systems: dithiothreitol-induced aggregation of alpha-lactalbumin (LA), heat-induced aggregation of alcohol dehydrogenase (ADH), and aggregation and inactivation of bovine erythrocyte carbonic anhydrase (CA) in the process of its refolding after removal of stress conditions. Using dynamic light scattering (DLS), turbidimetry, fluorescence, and circular dichroism measurements protective effects of the synthetic opioid peptides: exorphin C from wheat gluten (Tyr-Pro-Ile-Ser-Leu), rubiscolin-5 from spinach ribulose-bisphosphate-carboxylase/oxygenase (Rubisco) (Tyr-Pro-Leu-Asp-Leu), and hemorphin-6 from bovine hemoglobin (Tyr-Pro-Trp-Thr-Gln-Arg) have been revealed. We have demonstrated the concentration-dependent suppression of light scattering intensity of aggregates of LA and ADH in the presence of the peptides, the population of nanoparticles with higher hydrodynamic radii being shifted to the lower ones, accompanied by an increase in the lag period of aggregation. The presence of the peptides in the refolding solution was shown to assist reactivation of CA and enhance the yield of the CA soluble protein. The results suggest that bioactive food protein fragments may be regarded as exogenous supplements to the endogenous defense mechanisms of the human organism under stress conditions. PMID:19954758

Artemova, N V; Bumagina, Z M; Kasakov, A S; Shubin, V V; Gurvits, B Ya

2010-02-01

317

Enhancing cellular uptake of GFP via unfolded supercharged protein tags.  

PubMed

One of the barriers to the development of protein therapeutics is effective delivery to mammalian cells. The proteins must maintain a careful balance of polar moieties to enable administration and distribution and hydrophobic character to minimize cell toxicity. Numerous strategies have been applied to this end, from appending additional cationic peptides to supercharging the protein itself, sometimes with limited success. Here we present a strategy that combines these methods, by equipping a protein with supercharged elastin-like polypeptide (ELP) tags. We monitored cellular uptake and cell viability for GFP reporter proteins outfitted with a range of ELP tags and demonstrated enhanced uptake that correlates with the number of positive charges, while maintaining remarkably low cytotoxicity and resistance to degradation in the cell. GFP uptake proceeded mainly through caveolae-mediated endocytosis and we observed GFP emission inside the cells over extended time (up to 48 h). Low toxicity combined with high molecular weights of the tag opens the way to simultaneously optimize cell uptake and pharmacokinetic parameters. Thus, cationic supercharged ELP tags show great potential to improve the therapeutic profile of protein drugs leading to more efficient and safer biotherapeutics. PMID:23478039

Pesce, Diego; Wu, Yuzhou; Kolbe, Anke; Weil, Tanja; Herrmann, Andreas

2013-06-01

318

Concepts in Spectral Unfolding.  

National Technical Information Service (NTIS)

The report discusses the problem of unfolding spectral information from a set of measured data, using response functions of the experimental apparatus. General concepts, applicable to a number of unfolding problems, are developed in terms of a basic Fredh...

G. I. Kerley

1969-01-01

319

Folding and unfolding of a non-fluorescent mutant of green fluorescent protein  

PubMed Central

Green fluorescent protein (GFP), from the Pacific jellyfish A. victoria, has numerous uses in biotechnology and cell and molecular biology as a protein marker because of its specific chromophore, which is spontaneously created after proper protein folding. After formation, the chromophore is very stable and remains intact during protein unfolding, meaning that the GFP unfolding process is not the reverse of the original folding reaction; i.e., the principles of microscopic reversibility do not apply. We have generated the mutant S65T/G67A-GFP, which is unable to form the cyclic chromophore, with the goal of investigating the folding, unfolding and competing aggregation of GFP under fully reversible conditions. Our studies have been performed in the presence of GdnHCl. The GFP conformation was monitored using intrinsic tryptophan fluorescence, and fluorescence of bis-ANS. Light scattering was used to follow GFP aggregation. We conclude from these fluorescence measurements, that S65T/G67A-GFP folding is largely reversible. During equilibrium folding, the first step is formation of molten globule, prone to aggregation.

Kutrowska, Beata Wielgus; Narczyk, Marta; Buszko, Anna; Bzowska, Agnieszka; Clark, Patricia L.

2009-01-01

320

Universality in the Timescales of Internal Loop Formation in Unfolded Proteins and Single-Stranded Oligonucleotides  

PubMed Central

Understanding the rate at which various parts of a molecular chain come together to facilitate the folding of a biopolymer (e.g., a protein or RNA) into its functional form remains an elusive goal. Here we use experiments, simulations, and theory to study the kinetics of internal loop closure in disordered biopolymers such as single-stranded oligonucleotides and unfolded proteins. We present theoretical arguments and computer simulation data to show that the relationship between the timescale of internal loop formation and the positions of the monomers enclosing the loop can be recast in a form of a universal master dependence. We also perform experimental measurements of the loop closure times of single-stranded oligonucleotides and show that both these and previously reported internal loop closure kinetics of unfolded proteins are well described by this theoretically predicted dependence. Finally, we propose that experimental deviations from the master dependence can then be used as a sensitive probe of dynamical and structural order in unfolded proteins and other biopolymers.

Cheng, Ryan R.; Uzawa, Takanori; Plaxco, Kevin W.; Makarov, Dmitrii E.

2010-01-01

321

Structural characteristic of folding/unfolding intermediate of pokeweed anti-viral protein revealed by time-resolved fluorescence.  

PubMed

The structural feature of unfolding intermediate of pokeweed anti-viral protein (PAP) was characterized using time-resolved fluorescence spectroscopic methods to elucidate protein folding/unfolding process. CD and fluorescence spectra consistently demonstrated that the unfolding of PAP completed at 4 M of guanidine hydrochloride (GuHCl). The fluorescence resonance energy transfer (FRET) and time-resolve fluorescence depolarization analysis of Trp208 and Trp237 located in the C-terminal domain of PAP suggested that peculiar unfolding intermediate populated before reaching to the unfolding state. The FRET distance of Trp237 to Tyr182 was extended to more than 28 Å with keeping the compact conformation in the unfolding intermediate state populated in the presence of 2 M GuHCl. On the other hand, Trp208 maintained the energy transfer pair with Tyr72 near the active site, although the rotational freedom was increased a little. There results suggest that the most distinguished structural feature of the unfolding intermediate of PAP is the separation of C-terminal domain from N-terminal domain. FRET and fluorescence depolarization studies also showed that C-terminal domain would be more separated to liberate the segmental motions of Trp208 and Trp237 distinctly at the unfolding state. PMID:23319009

Matsumoto, Shuzo; Taniguchi, Yuka; Fukunaga, Yukihiro; Nakashima, Hiromichi; Watanabe, Keiichi; Yamashita, Shoji; Nishimoto, Etsuko

2013-05-01

322

Measurement of energy landscape roughness of folded and unfolded proteins  

PubMed Central

The dynamics of protein conformational changes, from protein folding to smaller changes, such as those involved in ligand binding, are governed by the properties of the conformational energy landscape. Different techniques have been used to follow the motion of a protein over this landscape and thus quantify its properties. However, these techniques often are limited to short timescales and low-energy conformations. Here, we describe a general approach that overcomes these limitations. Starting from a nonnative conformation held by an aromatic disulfide bond, we use time-resolved spectroscopy to observe nonequilibrium backbone dynamics over nine orders of magnitude in time, from picoseconds to milliseconds, after photolysis of the disulfide bond. We find that the reencounter probability of residues that initially are in close contact decreases with time following an unusual power law that persists over the full time range and is independent of the primary sequence. Model simulations show that this power law arises from subdiffusional motion, indicating a wide distribution of trapping times in local minima of the energy landscape, and enable us to quantify the roughness of the energy landscape (4–5 kBT). Surprisingly, even under denaturing conditions, the energy landscape remains highly rugged with deep traps (>20 kBT) that result from multiple nonnative interactions and are sufficient for trapping on the millisecond timescale. Finally, we suggest that the subdiffusional motion of the protein backbone found here may promote rapid folding of proteins with low contact order by enhancing contact formation between nearby residues.

Milanesi, Lilia; Waltho, Jonathan P.; Hunter, Christopher A.; Shaw, Daniel J.; Beddard, Godfrey S.; Reid, Gavin D.; Dev, Sagarika; Volk, Martin

2012-01-01

323

Telomerase recruitment by the telomere end binding protein-? facilitates G-quadruplex DNA unfolding in ciliates  

Microsoft Academic Search

The telomeric G-overhangs of the ciliate Stylonychia lemnae fold into a G-quadruplex DNA structure in vivo. Telomeric G-quadruplex formation requires the presence of two telomere end binding proteins, TEBP? and TEBP?, and is regulated in a cell-cycle dependent manner. Unfolding of this structure in S phase is dependent on the phosphorylation of TEBP?. Here we show that TEBP? phosphorylation is

Katrin Paeschke; Stefan Juranek; Tomas Simonsson; Anne Hempel; Daniela Rhodes; Hans Joachim Lipps

2008-01-01

324

ClpX shifts into high gear to unfold stable proteins.  

PubMed

Protein degradation by the ClpXP protease requires collaboration among the six AAA+ domains of ClpX. Using single-molecule optical tweezers, Sen et al. show that ClpX uses a coordinated succession of power strokes to translocate polypeptides in ATP-tunable bursts before reloading with nucleotide. This strategy allows ClpX to kinetically capture transiently unfolded intermediates. PMID:24243009

Maurizi, Michael R; Stan, George

2013-10-24

325

Unfolding of DNA quadruplexes induced by HIV1 nucleocapsid protein  

Microsoft Academic Search

The human immunodeficiency virus type 1 nucleo- capsid protein (NC) is a nucleic acid chaperone that catalyzes the rearrangement of nucleic acids into their thermodynamically most stable structures. In the present study, a combination of optical and ther- modynamic techniques were used to characterize the influence of NC on the secondary structure, thermal stability and energetics of monomolecular DNA quad-

Besik I. Kankia; George Barany; Karin Musier-Forsyth

2005-01-01

326

Unfolding Thermodynamics of Cysteine-Rich Proteins and Molecular Thermal-Adaptation of Marine Ciliates  

PubMed Central

Euplotes nobilii and Euplotes raikovi are phylogenetically closely allied species of marine ciliates, living in polar and temperate waters, respectively. Their evolutional relation and the sharply different temperatures of their natural environments make them ideal organisms to investigate thermal-adaptation. We perform a comparative study of the thermal unfolding of disulfide-rich protein pheromones produced by these ciliates. Recent circular dichroism (CD) measurements have shown that the two psychrophilic (E. nobilii) and mesophilic (E. raikovi) protein families are characterized by very different melting temperatures, despite their close structural homology. The enhanced thermal stability of the E. raikovi pheromones is realized notwithstanding the fact that these proteins form, as a rule, a smaller number of disulfide bonds. We perform Monte Carlo (MC) simulations in a structure-based coarse-grained (CG) model to show that the higher stability of the E. raikovi pheromones is due to the lower locality of the disulfide bonds, which yields a lower entropy increase in the unfolding process. Our study suggests that the higher stability of the mesophilic E. raikovi phermones is not mainly due to the presence of a strongly hydrophobic core, as it was proposed in the literature. In addition, we argue that the molecular adaptation of these ciliates may have occurred from cold to warm, and not from warm to cold. To provide a testable prediction, we identify a point-mutation of an E. nobilii pheromone that should lead to an unfolding temperature typical of that of E. raikovi pheromones.

Cazzolli, Giorgia; Skrbic, Tatjana; Guella, Graziano; Faccioli, Pietro

2013-01-01

327

FoldIndex: a simple tool to predict whether a given protein sequence is intrinsically unfolded.  

PubMed

An easy-to-use, versatile and freely available graphic web server, FoldIndex is described: it predicts if a given protein sequence is intrinsically unfolded implementing the algorithm of Uversky and co-workers, which is based on the average residue hydrophobicity and net charge of the sequence. FoldIndex has an error rate comparable to that of more sophisticated fold prediction methods. Sliding windows permit identification of large regions within a protein that possess folding propensities different from those of the whole protein. PMID:15955783

Prilusky, Jaime; Felder, Clifford E; Zeev-Ben-Mordehai, Tzviya; Rydberg, Edwin H; Man, Orna; Beckmann, Jacques S; Silman, Israel; Sussman, Joel L

2005-08-15

328

Unfolding of proteins monitored by electrospray ionization mass spectrometry: a comparison of positive and negative ion modes  

Microsoft Academic Search

Electrospray ionization (ESI) mass spectrometry (MS) in both the positive and negative ion mode has been used to study protein\\u000a unfolding transitions of lysozyme, cytochrome c (cyt c), and ubiquitin in solution. As expected, ESI of unfolded lysozyme leads to the formation of substantially higher charge\\u000a states than the tightly folded protein in both modes of operation. Surprisingly, the acid-induced

Lars Konermann; D. J. Douglas

1998-01-01

329

Protein stabilization by osmolytes from hyperthermophiles: effect of mannosylglycerate on the thermal unfolding of recombinant nuclease a from Staphylococcus aureus studied by picosecond time-resolved fluorescence and calorimetry.  

PubMed

2-O-alpha-Mannosylglycerate, a negatively charged osmolyte widely distributed among (hyper)thermophilic microorganisms, is known to provide notable protection to proteins against thermal denaturation. To study the mechanism responsible for protein stabilization, pico-second time-resolved fluorescence spectroscopy was used to characterize the thermal unfolding of a model protein, Staphylococcus aureus recombinant nuclease A (SNase), in the presence or absence of mannosylglycerate. The fluorescence decay times are signatures of the protein state, and the pre-exponential coefficients are used to evaluate the molar fractions of the folded and unfolded states. Hence, direct determination of equilibrium constants of unfolding from molar fractions was carried out. Van't Hoff plots of the equilibrium constants provided reliable thermodynamic data for SNase unfolding. Differential scanning calorimetry was used to validate this thermodynamic analysis. The presence of 0.5 m potassium mannosylglycerate caused an increase of 7 degrees C in the SNase melting temperature and a 2-fold increase in the unfolding heat capacity. Despite the considerable degree of stabilization rendered by this solute, the nature and population of protein states along unfolding were not altered in the presence of mannosylglycerate, denoting that the unfolding pathway of SNase was unaffected. The stabilization of SNase by mannosylglycerate arises from decreased unfolding entropy up to 65 degrees C and from an enthalpy increase above this temperature. In molecular terms, stabilization is interpreted as resulting from destabilization of the denatured state caused by preferential exclusion of the solute from the protein hydration shell upon unfolding, and stabilization of the native state by specific interactions. The physiological significance of charged solutes in hyperthermophiles is discussed. PMID:15347691

Faria, Tiago Q; Lima, João C; Bastos, Margarida; Maçanita, António L; Santos, Helena

2004-11-19

330

Limited proteolysis of natively unfolded protein 4E-BP1 in the presence of trifluoroethanol.  

PubMed

Natively unfolded (intrinsically disordered (ID) proteins) have been attracting an increasing attention due to their involvement in many regulatory processes. Natively unfolded proteins can fold upon binding to their metabolic partners. Coupled folding and binding events usually involve only relatively short motifs (binding motifs). These binding motifs which are able to fold should have an increased propensity to form a secondary structure. The aim of the present work was to probe the conformation of the intrinsically disordered protein 4E-BP1 in the native and partly folded states by limited proteolysis and to reveal regions with a high propensity to form an ordered structure. Trifuoroethanol (TFE) in low concentrations (up to 15 vol%) was applied to increase the helical population of protein regions with a high intrinsic propensity to fold. When forming helical structures, these regions lose mobility and become more protected from proteases than random/unfolded protein regions. Limited proteolysis followed by mass spectrometry analysis allows identification of the regions with decreased mobility in TFE solutions. Trypsin and V8 proteases were used to perform limited proteolysis of the 4E-BP1 protein in buffer and in solutions with low TFE concentrations at 37°C and at elevated temperatures (42 and 50°C). Comparison of the results obtained with the previously established 4E-BP1 structure and the binding motif illustrates the ability of limited proteolysis in the presence of a folding assistant (TFE) to map the regions with high and low propensities to form a secondary structure revealing potential binding motifs inside the intrinsically disordered protein. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 591-602, 2014. PMID:24122746

Hackl, Ellen V

2014-06-01

331

Stochastic but Highly Coordinated Protein Unfolding and Translocation by the ClpXP Proteolytic Machine.  

PubMed

ClpXP and other AAA+ proteases recognize, mechanically unfold, and translocate target proteins into a chamber for proteolysis. It is not known whether these remarkable molecular machines operate by a stochastic or sequential mechanism or how power strokes relate to the ATP-hydrolysis cycle. Single-molecule optical trapping allows ClpXP unfolding to be directly visualized and reveals translocation steps of ?1-4 nm in length, but how these activities relate to solution degradation and the physical properties of substrate proteins remains unclear. By studying single-molecule degradation using different multidomain substrates and ClpXP variants, we answer many of these questions and provide evidence for stochastic unfolding and translocation. We also present a mechanochemical model that accounts for single-molecule, biochemical, and structural results for our observation of enzymatic memory in translocation stepping, for the kinetics of translocation steps of different sizes, and for probabilistic but highly coordinated subunit activity within the ClpX ring. PMID:25083874

Cordova, Juan Carlos; Olivares, Adrian O; Shin, Yongdae; Stinson, Benjamin M; Calmat, Stephane; Schmitz, Karl R; Aubin-Tam, Marie-Eve; Baker, Tania A; Lang, Matthew J; Sauer, Robert T

2014-07-31

332

Unfolding the response of a zero-degree magnetic spectrometer from measurements of the ? resonance  

NASA Astrophysics Data System (ADS)

The magnetic spectrometer FRagment Separator at GSI has been used to investigate the in-medium ?-resonance excitation in peripheral heavy-ion reactions. The resolving power of this spectrometer makes it possible to disentangle the longitudinal-momentum loss induced by the excitation of the ? resonance in the projectile residues produced in isobaric charge-exchange collisions. However, beam emittance, electromagnetic interactions of projectile and residual nuclei in the target, and the accuracy of the tracking detectors limit the final resolution. The characterization of the ? resonance requires then to unfold the measured longitudinal-momentum distribution from the response of the spectrometer. In this work, we use an unfolding procedure based on the Richardson-Lucy method with a regularization technique to optimize the stability of the solution against statistical fluctuations. The method is validated using measurements of isobaric charge-changing collisions with a 136Xe beam at 500 A MeV.

Vargas, J.; Benlliure, J.; Caamaño, M.

2013-04-01

333

Unfolding and translocation pathway of substrate protein controlled by structure in repetitive allosteric cycles of the ClpY ATPase  

PubMed Central

Clp ATPases are ring-shaped AAA+ motors in the degradation pathway that perform critical actions of unfolding and translocating substrate proteins (SPs) through narrow pores to deliver them to peptidase components. These actions are effected by conserved diaphragm-forming loops found in the central channel of the Clp ATPase hexamer. Conformational changes, that take place in the course of repetitive ATP-driven cycles, result in mechanical forces applied by the central channel loops onto the SP. We use coarse-grained simulations to elucidate allostery-driven mechanisms of unfolding and translocation of a tagged four-helix bundle protein by the ClpY ATPase. Unfolding is initiated at the tagged C-terminal region via an obligatory intermediate. The resulting nonnative conformation is competent for translocation, which proceeds on a different time scale than unfolding and involves sharp stepped transitions. Completion of the translocation process requires assistance from the ClpQ peptidase. These mechanisms contrast nonallosteric mechanical unfolding of the SP. In atomic force microscopy experiments, multiple unfolding pathways are available and large mechanical forces are required to unravel the SP relative to those exerted by the central channel loops of ClpY. SP threading through a nonallosteric ClpY nanopore involves simultaneous unfolding and translocation effected by strong pulling forces.

Kravats, Andrea; Jayasinghe, Manori; Stan, George

2011-01-01

334

Concerted dihedral rotations give rise to internal friction in unfolded proteins.  

PubMed

Protein chains undergo conformational diffusion during folding and dynamics, experiencing both thermal kicks and viscous drag. Recent experiments have shown that the corresponding friction can be separated into wet friction, which is determined by the solvent viscosity, and dry friction, where frictional effects arise due to the interactions within the protein chain. Despite important advances, the molecular origins underlying dry friction in proteins have remained unclear. To address this problem, we studied the dynamics of the unfolded cold-shock protein at different solvent viscosities and denaturant concentrations. Using extensive all-atom molecular dynamics simulations we estimated the internal friction time scales and found them to agree well with the corresponding experimental measurements (Soranno et al. Proc. Natl. Acad. Sci. U.S.A. 2012, 109, 17800-17806). Analysis of the reconfiguration dynamics of the unfolded chain further revealed that hops in the dihedral space provide the dominant mechanism of internal friction. Furthermore, the increased number of concerted dihedral moves at physiological conditions suggest that, in such conditions, the concerted motions result in higher frictional forces. These findings have important implications for understanding the folding kinetics of proteins as well as the dynamics of intrinsically disordered proteins. PMID:24844314

Echeverria, Ignacia; Makarov, Dmitrii E; Papoian, Garegin A

2014-06-18

335

Protein folding and unfolding by force microscopy: a novel inverse methodology  

NASA Astrophysics Data System (ADS)

It has been shown recently that it is possible to reverse-engineer the folding of complex proteins by an AFM-based force versus distance methodology. In essence, the protein is attached to a functionalised surface, then linked to a functionalized tip of a force-sensing/imposing lever, and finally stretched by withdrawal of the probe. The experiments are technically demanding, and subject to a number of artefacts. Moreover, the unfolding process cannot readily be reversed. A 'reverse" methodology is possible in principle and is likely to provide greater insight into the folding/unfolding sequence. The protein in question will now be attached to a functionalized superparamagnetic bead in solution. A functionalized probe is then introduced into solution whereupon the protein (and its bead) attaches itself to the tip of the probe, while being tracked optically. The system is then submerged in an inhomogeneous magnetic dipole field, giving rise to a magnetic dipole-dipole interaction with the bead. The resultant force will stretch the protein, while being monitored at a force resolution of ca. 1 pN by the deflection of the lever. The significant aspect of the scheme is that the probe acts as a position-sensitive element as well as being a force-sensing device. The field gradient and its rate of change within the interaction volume can readily be controlled to 1 part in 106, and can be reversed. Thus the unfolding/folding sequence can be retraced for the same molecule. Moreover, information about kinetics will be accessible from varying the rate of change in field strength, and the effects of ambient fluid conditions can be investigated in real time with a flow-through arrangement. The proposed methodology is likely to be more userfriendly as a result of dispensing with the functionalized surface, and by being able to determine optically that a single functionalized bead has arrived at the probe tip. Elements of the experimental arrangement are currently at the design stage.

Watson, Gregory S.; Watson, Jolanta A.; Brown, Christopher L.; Myhra, Sverre

2005-02-01

336

Unfolding of quadruplex structure in the G-rich strand of the minisatellite repeat by the binding protein UP1  

PubMed Central

The mouse hypervariable minisatellite (MN) Pc-1 consists of tandem repeats of d(GGCAG) and flanked sequences. We have previously demonstrated that single-stranded d(GGCAG)n folds into the intramolecular folded-back quadruplex structure under physiological conditions. Because DNA polymerase progression in vitro is blocked at the repeat, the characteristic intramolecular quadruplex structure of the repeat, at least in part, could be responsible for the hypermutable feature of Pc-1 and other MNs with similar repetitive units. On the other hand, we have isolated six MN Pc-1 binding proteins (MNBPs) from nuclear extracts of NIH 3T3 cells. Here, we describe one of those MNBPs, MNBP-B, that binds to the single-stranded d(GGCAG)n. Amino acid sequences of seven proteolytic peptide fragments of MNBP-B were determined, and the cDNA clones were isolated. MNBP-B was proven identical to the single-stranded DNA-binding protein, UP1. Recombinant UP1 bound to single-stranded d(GGCAG)n and other G-rich repetitive sequences, such as d(GTCAGG)n and d(GTTAGG)n. In addition, UP1 was demonstrated by CD spectrum analysis to unfold the intramolecular quadruplex structure of d(GGCAG)5 and d(TTAGGG)4 and to abrogate the arrest of DNA synthesis at the d(GGG)n site. This ability of UP1 suggests that unfolding of quadruplex DNA is required for DNA synthesis processes.

Fukuda, Hirokazu; Katahira, Masato; Tsuchiya, Naoto; Enokizono, Yoshiaki; Sugimura, Takashi; Nagao, Minako; Nakagama, Hitoshi

2002-01-01

337

Out but not in: the large transmembrane ?-barrel protein FhuA unfolds but cannot refold via ?-hairpins.  

PubMed

How transmembrane ?-barrel proteins insert and fold into membranes and by which factors they destabilize, unfold, and misfold represents a field of intense studies. Here, we use single-molecule force spectroscopy to characterize the un- and refolding of the ferric hydroxamate uptake receptor (FhuA), which is one of the largest ?-barrel proteins of the outer membrane of Escherichia coli. Applied to mechanical stress, FhuA undergoes a complex unfolding pathway in which each of the 11 ?-hairpins unfolds one after the other until the entire ?-barrel has unfolded. Once unfolded and relaxed, the FhuA polypeptide cannot fold back into the lipid membrane and adopts various misfolded conformations. Such misfolding is in contrast to the reversible refolding behavior of much smaller ?-barrel outer membrane proteins OmpA and OmpG that occurs at similar experimental conditions. The results suggest that large ?-barrel proteins that show more complex (un-)folding pathways require cofactors for proper insertion and folding into the membrane. PMID:23159125

Thoma, Johannes; Bosshart, Patrick; Pfreundschuh, Moritz; Müller, Daniel J

2012-12-01

338

Prediction of protein relative enthalpic stability from molecular dynamics simulations of the folded and unfolded states.  

PubMed

For proteins of known structure, the relative enthalpic stability with respect to wild-type, ??H(U), can be estimated by direct computation of the folded and unfolded state energies. We propose a model by which the change in stability upon mutation can be predicted from all-atom molecular dynamics simulations for the folded state and a peptide-based model for the unfolded state. The unfolding enthalpies are expressed in terms of environmental and hydration-solvent reorganization contributions that readily allow a residue-specific analysis of ??H(U). The method is applied to estimate the relative enthalpic stability of variants with buried charged groups in T4 lysozyme. The predicted relative stabilities are in good agreement with experimental data. Environmental factors are observed to contribute more than hydration to the overall ??H(U). The residue-specific analysis finds that the effects of burying charge are both localized and long-range. The enthalpy for hydration-solvent reorganization varies considerably among different amino-acid types, but because the variant folded state structures are similar to those of the wild-type, the hydration-solvent reorganization contribution to ??H(U) is localized at the mutation site, in contrast to environmental contributions. Overall, mutation of apolar and polar amino acids to charged amino acids are destabilizing, but the reasons are complex and differ from site to site. PMID:23083720

Dadarlat, Voichita M; Gorenstein, Lev A; Post, Carol Beth

2012-10-17

339

Prediction of Protein Relative Enthalpic Stability from Molecular Dynamics Simulations of the Folded and Unfolded States  

PubMed Central

For proteins of known structure, the relative enthalpic stability with respect to wild-type, ??HU, can be estimated by direct computation of the folded and unfolded state energies. We propose a model by which the change in stability upon mutation can be predicted from all-atom molecular dynamics simulations for the folded state and a peptide-based model for the unfolded state. The unfolding enthalpies are expressed in terms of environmental and hydration-solvent reorganization contributions that readily allow a residue-specific analysis of ??HU. The method is applied to estimate the relative enthalpic stability of variants with buried charged groups in T4 lysozyme. The predicted relative stabilities are in good agreement with experimental data. Environmental factors are observed to contribute more than hydration to the overall ??HU. The residue-specific analysis finds that the effects of burying charge are both localized and long-range. The enthalpy for hydration-solvent reorganization varies considerably among different amino-acid types, but because the variant folded state structures are similar to those of the wild-type, the hydration-solvent reorganization contribution to ??HU is localized at the mutation site, in contrast to environmental contributions. Overall, mutation of apolar and polar amino acids to charged amino acids are destabilizing, but the reasons are complex and differ from site to site.

Dadarlat, Voichita M.; Gorenstein, Lev A.; Post, Carol Beth

2012-01-01

340

XBP1, Unfolded Protein Response, and Endocrine Responsiveness.  

National Technical Information Service (NTIS)

Almost 50% of all ER+ breast tumors will not respond to endocrine therapy. Resistance to endocrine therapy remains a significant clinical problem and advanced ER+ breast cancer is largely an incurable disease. Endocrine manipulation in sensitive cells can...

R. Clark

2011-01-01

341

XBP1, Unfolded Protein Response, and Endocrine Responsiveness.  

National Technical Information Service (NTIS)

Almost 50% of all ER+ breast tumors will not respond to endocrine therapy. Resistance to endocrine therapy remains a significant clinical problem and advanced ER+ breast cancer is largely an incurable disease. Endocrine manipulation in sensitive cells can...

R. Clarke

2012-01-01

342

XBP1, Unfolded Protein Response, and Endocrine Responsiveness.  

National Technical Information Service (NTIS)

Almost 50% of all ER+ breast tumors will not respond to endocrine therapy. Resistance to endocrine therapy remains a significant clinical problem and advanced ER+ breast cancer is largely an incurable disease. Endocrine manipulation in sensitive cells can...

R. Clarke

2009-01-01

343

Local unfolding is required for the site-specific protein modification by transglutaminase.  

PubMed

The transglutaminase (TGase) from Streptomyces mobaraensis catalyzes transamidation reactions in a protein substrate leading to the modification of the side chains of Gln and Lys residues according to the A-CONH(2) + H(2)N-B ? A-CONH-B + NH(3) reaction, where both A and B can be a protein or a ligand. A noteworthy property of TGase is its susbstrate specificity, so that often only a few specific Gln or Lys residues can be modified in a globular protein. The molecular features of a globular protein dictating the site-specific reactions mediated by TGase are yet poorly understood. Here, we have analyzed the reactivity toward TGase of apomyoglobin (apoMb), ?-lactalbumin (?-LA), and fragment 205-316 of thermolysin. These proteins are models of protein structure and folding that have been studied previously using the limited proteolysis technique to unravel regions of local unfolding in their amino acid sequences. The three proteins were modified by TGase at the level of Gln or Lys residues with dansylcadaverine or carbobenzoxy-l-glutaminylglycine, respectively. Despite these model proteins containing several Gln and Lys residues, the sites of TGase derivatization occur over restricted chain regions of the protein substrates. In particular, the TGase-mediated modifications occur in the "helix F" region in apoMb, in the ?-domain in apo-?-LA in its molten globule state, and in the N-terminal region in fragment 205-316 of thermolysin. Interestingly, the sites of limited proteolysis are located in the same chain regions of these proteins, thus providing a clear-cut demonstration that chain flexibility or local unfolding overwhelmingly dictates the site-specific modification by both TGase and a protease. PMID:23083324

Spolaore, Barbara; Raboni, Samanta; Ramos Molina, Amparo; Satwekar, Abhijeet; Damiano, Nunzio; Fontana, Angelo

2012-10-30

344

Genetic regulation of spy gene expression in Escherichia coli in the presence of protein unfolding agent ethanol.  

PubMed

In a living cell, folding of proteins is assisted by molecular chaperones and other folding helpers. In Escherichia coli (E. coli), recently an ATP independent chaperon 'Spy' was discovered which is highly up-regulated in the presence of protein unfolding agents like ethanol, butanol and tannic acid. Two response regulators; BaeR and CpxR have been recognized as transcriptional regulators of spy gene. However, the mechanism of genetic regulation of spy under protein denaturants like ethanol has not been studied in detail so far. Based on a combination of genetic, molecular biology and biochemical experimental data, we propose that BaeR protein is the primary regulator of spy gene in response to ethanol stress in E. coli. In addition, we expanded the experimental spectrum and validated that regulation of spy gene in the presence of zinc and copper metal stress is primarily via BaeR and CpxR regulators respectively. We also performed in-silico analysis to identify the homologs of Spy protein and their cognate regulatory elements in bacterial species belonging to enterobacteriaceae family. Based on the unique ATP-independent chaperone nature and genetic regulation of spy we also propose its importance in biosensor development and facilitated production of properly folded recombinant proteins. PMID:24999585

Srivastava, Santosh Kumar; Lambadi, Paramesh Ramulu; Ghosh, Tamoghna; Pathania, Ranjana; Navani, Naveen Kumar

2014-09-10

345

A camel passes through the eye of a needle: protein unfolding activity of Clp ATPases  

PubMed Central

Summary Clp ATPases are protein machines involved in protein degradation and disaggregation. The common structural feature of Clp ATPases is the formation of ring-shaped oligomers. Recent work has shown that the function of all Clp ATPases is based on an energy-dependent threading of substrates through the narrow pore at the center of the ring. This review gives an outline of known mechanistic principles of threading machines that unfold protein substrates either before their degradation (ClpA, ClpX, HslU) or during their reactivation from aggregates (ClpB). The place of Clp ATPases within a broad AAA+ superfamily of ATPases associated with various cellular activities suggests that similar mechanisms can be used by other protein machines to induce conformational rearrangements in a wide variety of substrates.

Zolkiewski, Michal

2007-01-01

346

Recognition and binding of human telomeric g-quadruplex DNA by unfolding protein 1.  

PubMed

The specific recognition by proteins of G-quadruplex structures provides evidence of a functional role for in vivo G-quadruplex structures. As previously reported, the ribonucleoprotein, hnRNP Al, and it is proteolytic derivative, unwinding protein 1 (UP1), bind to and destabilize G-quadruplex structures formed by the human telomeric repeat d(TTAGGG)n. UP1 has been proposed to be involved in the recruitment of telomerase to telomeres for chain extension. In this study, a detailed thermodynamic characterization of the binding of UP1 to a human telomeric repeat sequence, the d[AGGG(TTAGGG)3] G-quadruplex, is presented and reveals key insights into the UP1-induced unfolding of the G-quadruplex structure. The UP1-G-quadruplex interactions are shown to be enthalpically driven, exhibiting large negative enthalpy changes for the formation of both the Na(+) and K(+) G-quadruplex-UP1 complexes (?H values of -43 and -19 kcal/mol, respectively). These data reveal three distinct enthalpic contributions from the interactions of UP1 with the Na(+) form of G-quadruplex DNA. The initial interaction is characterized by a binding affinity of 8.5 × 10(8) M(-1) (strand), 200 times stronger than the binding of UP1 to a single-stranded DNA with a comparable but non-quadruplex-forming sequence [4.1 × 10(6) M(-1) (strand)]. Circular dichroism spectroscopy reveals the Na(+) form of the G-quadruplex to be completely unfolded by UP1 at a binding ratio of 2:1 (UP1:G-quadruplex DNA). The data presented here demonstrate that the favorable energetics of the initial binding event are closely coupled with and drive the unfolding of the G-quadruplex structure. PMID:24831962

Hudson, Jason S; Ding, Lei; Le, Vu; Lewis, Edwin; Graves, David

2014-05-27

347

Cooperative Unfolding of Compact Conformations of the Intrinsically Disordered Protein Osteopontin  

PubMed Central

Intrinsically disordered proteins (IDPs) constitute a class of biologically active proteins that lack defined tertiary and often secondary structure. The IDP Osteopontin (OPN), a cytokine involved in metastasis of several types of cancer, is shown to simultaneously sample extended, random coil-like conformations and stable, cooperatively folded conformations. By a combination of two magnetic resonance methods, electron paramagnetic resonance and nuclear magnetic resonance spectroscopy, we demonstrate that the OPN ensemble exhibits not only characteristics of an extended and flexible polypeptide, as expected for an IDP, but also simultaneously those of globular proteins, in particular sigmoidal structural denaturation profiles. Both types of states, extended and cooperatively folded, are populated simultaneously by OPN in its apo state. The heterogeneity of the structural properties of IDPs is thus shown to even involve cooperative folding and unfolding events.

2013-01-01

348

OneG: A Computational Tool for Predicting Cryptic Intermediates in the Unfolding Kinetics of Proteins under Native Conditions  

PubMed Central

Understanding the relationships between conformations of proteins and their stabilities is one key to address the protein folding paradigm. The free energy change (?G) of unfolding reactions of proteins is measured by traditional denaturation methods and native hydrogen-deuterium (H/D) exchange methods. However, the free energy of unfolding (?GU) and the free energy of exchange (?GHX) of proteins are not in good agreement, though the experimental conditions of both methods are well matching to each other. The anomaly is due to any one or combinations of the following reasons: (i) effects of cis-trans proline isomerisation under equilibrium unfolding reactions of proteins (ii) inappropriateness in accounting the baselines of melting curves (iii) presence of cryptic intermediates, which may elude the melting curve analysis and (iv) existence of higher energy metastable states in the H/D exchange reactions of proteins. Herein, we have developed a novel computational tool, OneG, which accounts the discrepancy between ?GU and ?GHX of proteins by systematically accounting all the four factors mentioned above. The program is fully automated and requires four inputs: three-dimensional structures of proteins, ?GU, ?GU* and residue-specific ?GHX determined under EX2-exchange conditions in the absence of denaturants. The robustness of the program has been validated using experimental data available for proteins such as cytochrome c and apocytochrome b562 and the data analyses revealed that cryptic intermediates of the proteins detected by the experimental methods and the cryptic intermediates predicted by the OneG for those proteins were in good agreement. Furthermore, using OneG, we have shown possible existence of cryptic intermediates and metastable states in the unfolding pathways of cardiotoxin III and cobrotoxin, respectively, which are homologous proteins. The unique application of the program to map the unfolding pathways of proteins under native conditions have been brought into fore and the program is publicly available at http://sblab.sastra.edu/oneg.html

Richa, Tambi; Sivaraman, Thirunavukkarasu

2012-01-01

349

Evidence for an unfolding/threading mechanism for protein disaggregation by Saccharomyces cerevisiae Hsp104.  

PubMed

Saccharomyces cerevisiae Hsp104, a hexameric member of the Hsp100/Clp subfamily of AAA+ ATPases with two nucleotide binding domains (NBD1 and 2), refolds aggregated proteins in conjunction with Hsp70 molecular chaperones. Hsp104 may act as a "molecular crowbar" to pry aggregates apart and/or may extract proteins from aggregates by unfolding and threading them through the axial channel of the Hsp104 hexamer. Targeting Tyr-662, located in a Gly-Tyr-Val-Gly motif that forms part of the axial channel loop in NBD2, we created conservative (Phe and Trp) and non-conservative (Ala and Lys) amino acid substitutions. Each of these Hsp104 derivatives was comparable to the wild type protein in their ability to hydrolyze ATP, assemble into hexamers, and associate with heat-shock-induced aggregates in living cells. However, only those with conservative substitutions complemented the thermotolerance defect of a Deltahsp104 yeast strain and promoted refolding of aggregated protein in vitro. Monitoring fluorescence from Trp-662 showed that titration of fully assembled molecules with either ATP or ADP progressively quenches fluorescence, suggesting that nucleotide binding determines the position of the loop within the axial channel. A Glu to Lys substitution at residue 645 in the NBD2 axial channel strongly alters the nucleotide-induced change in fluorescence of Trp-662 and specifically impairs in protein refolding. These data establish that the structural integrity of the axial channel through NBD2 is required for Hsp104 function and support the proposal that Hsp104 and ClpB use analogous unfolding/threading mechanisms to promote disaggregation and refolding that other Hsp100s use to promote protein degradation. PMID:15128736

Lum, Ronnie; Tkach, Johnny M; Vierling, Elizabeth; Glover, John R

2004-07-01

350

The Janus face of the archaeal Cdc48/p97 homologue VAT: protein folding versus unfolding.  

PubMed

Members of the AAA family of ATPases have been implicated in chaperone-like activities. We used the archaeal Cdc48/p97 homologue VAT as a model system to investigate the effect of an AAA protein on the folding and unfolding of two well-studied, heterologous substrates, cyclophilin and penicillinase. We found that, depending on the Mg2+ concentration, VAT assumes two states with maximum rates of ATP hydrolysis that differ by an order of magnitude. In the low-activity state, VAT accelerated the refolding of penicillinase, whereas in the high-activity state, it accelerated its unfolding. Both reactions were ATP-dependent. In its interaction with cyclophilin, VAT was ATP-independent and only promoted refolding. The N-terminal domain of VAT, which lacks ATPase activity, also accelerated the refolding of cyclophilin but showed no effect on penicillinase. VAT appears to be structurally equivalent over its entire length to Sec18/NSF, suggesting that these results apply more broadly to group II AAA proteins. PMID:10543442

Golbik, R; Lupas, A N; Koretke, K K; Baumeister, W; Peters, J

1999-09-01

351

Paramagnetic relaxation enhancements in unfolded proteins: Theory and application to drkN SH3 domain  

PubMed Central

Site-directed spin labeling in combination with paramagnetic relaxation enhancement (PRE) measurements is one of the most promising techniques for studying unfolded proteins. Since the pioneering work of Gillespie and Shortle (J Mol Biol 1997;268:158), PRE data from unfolded proteins have been interpreted using the theory that was originally developed for rotational spin relaxation. At the same time, it can be readily recognized that the relative motion of the paramagnetic tag attached to the peptide chain and the reporter spin such as 1HN is best described as a translation. With this notion in mind, we developed a number of models for the PRE effect in unfolded proteins: (i) mutual diffusion of the two tethered spheres, (ii) mutual diffusion of the two tethered spheres subject to a harmonic potential, (iii) mutual diffusion of the two tethered spheres subject to a simulated mean-force potential (Smoluchowski equation); (iv) explicit-atom molecular dynamics simulation. The new models were used to predict the dependences of the PRE rates on the 1HN residue number and static magnetic field strength; the results are appreciably different from the Gillespie–Shortle model. At the same time, the Gillespie–Shortle approach is expected to be generally adequate if the goal is to reconstruct the distance distributions between 1HN spins and the paramagnetic center (provided that the characteristic correlation time is known with a reasonable accuracy). The theory has been tested by measuring the PRE rates in three spin-labeled mutants of the drkN SH3 domain in 2M guanidinium chloride. Two modifications introduced into the measurement scheme—using a reference compound to calibrate the signals from the two samples (oxidized and reduced) and using peak volumes instead of intensities to determine the PRE rates—lead to a substantial improvement in the quality of data. The PRE data from the denatured drkN SH3 are mostly consistent with the model of moderately expanded random-coil protein, although part of the data point toward a more compact structure (local hydrophobic cluster). At the same time, the radius of gyration reported by Choy et al. (J Mol Biol 2002;316:101) suggests that the protein is highly expanded. This seemingly contradictory evidence can be reconciled if one assumes that denatured drkN SH3 forms a conformational ensemble that is dominated by extended conformations, yet also contains compact (collapsed) species. Such behavior is apparently more complex than predicted by the model of a random-coil protein in good solvent/poor solvent.

Xue, Yi; Podkorytov, Ivan S; Rao, D Krishna; Benjamin, Nathan; Sun, Honglei; Skrynnikov, Nikolai R

2009-01-01

352

Paramagnetic relaxation enhancements in unfolded proteins: theory and application to drkN SH3 domain.  

PubMed

Site-directed spin labeling in combination with paramagnetic relaxation enhancement (PRE) measurements is one of the most promising techniques for studying unfolded proteins. Since the pioneering work of Gillespie and Shortle (J Mol Biol 1997;268:158), PRE data from unfolded proteins have been interpreted using the theory that was originally developed for rotational spin relaxation. At the same time, it can be readily recognized that the relative motion of the paramagnetic tag attached to the peptide chain and the reporter spin such as (1)H(N) is best described as a translation. With this notion in mind, we developed a number of models for the PRE effect in unfolded proteins: (i) mutual diffusion of the two tethered spheres, (ii) mutual diffusion of the two tethered spheres subject to a harmonic potential, (iii) mutual diffusion of the two tethered spheres subject to a simulated mean-force potential (Smoluchowski equation); (iv) explicit-atom molecular dynamics simulation. The new models were used to predict the dependences of the PRE rates on the (1)H(N) residue number and static magnetic field strength; the results are appreciably different from the Gillespie-Shortle model. At the same time, the Gillespie-Shortle approach is expected to be generally adequate if the goal is to reconstruct the distance distributions between (1)H(N) spins and the paramagnetic center (provided that the characteristic correlation time is known with a reasonable accuracy). The theory has been tested by measuring the PRE rates in three spin-labeled mutants of the drkN SH3 domain in 2M guanidinium chloride. Two modifications introduced into the measurement scheme-using a reference compound to calibrate the signals from the two samples (oxidized and reduced) and using peak volumes instead of intensities to determine the PRE rates-lead to a substantial improvement in the quality of data. The PRE data from the denatured drkN SH3 are mostly consistent with the model of moderately expanded random-coil protein, although part of the data point toward a more compact structure (local hydrophobic cluster). At the same time, the radius of gyration reported by Choy et al. (J Mol Biol 2002;316:101) suggests that the protein is highly expanded. This seemingly contradictory evidence can be reconciled if one assumes that denatured drkN SH3 forms a conformational ensemble that is dominated by extended conformations, yet also contains compact (collapsed) species. Such behavior is apparently more complex than predicted by the model of a random-coil protein in good solvent/poor solvent. PMID:19544584

Xue, Yi; Podkorytov, Ivan S; Rao, D Krishna; Benjamin, Nathan; Sun, Honglei; Skrynnikov, Nikolai R

2009-07-01

353

Escherichia coli ribosomal protein S1 unfolds structured mRNAs onto the ribosome for active translation initiation.  

PubMed

Regulation of translation initiation is well appropriate to adapt cell growth in response to stress and environmental changes. Many bacterial mRNAs adopt structures in their 5' untranslated regions that modulate the accessibility of the 30S ribosomal subunit. Structured mRNAs interact with the 30S in a two-step process where the docking of a folded mRNA precedes an accommodation step. Here, we used a combination of experimental approaches in vitro (kinetic of mRNA unfolding and binding experiments to analyze mRNA-protein or mRNA-ribosome complexes, toeprinting assays to follow the formation of ribosomal initiation complexes) and in vivo (genetic) to monitor the action of ribosomal protein S1 on the initiation of structured and regulated mRNAs. We demonstrate that r-protein S1 endows the 30S with an RNA chaperone activity that is essential for the docking and the unfolding of structured mRNAs, and for the correct positioning of the initiation codon inside the decoding channel. The first three OB-fold domains of S1 retain all its activities (mRNA and 30S binding, RNA melting activity) on the 30S subunit. S1 is not required for all mRNAs and acts differently on mRNAs according to the signals present at their 5' ends. This work shows that S1 confers to the ribosome dynamic properties to initiate translation of a large set of mRNAs with diverse structural features. PMID:24339747

Duval, Mélodie; Korepanov, Alexey; Fuchsbauer, Olivier; Fechter, Pierre; Haller, Andrea; Fabbretti, Attilio; Choulier, Laurence; Micura, Ronald; Klaholz, Bruno P; Romby, Pascale; Springer, Mathias; Marzi, Stefano

2013-12-01

354

Hydration-responsive folding and unfolding in graphene oxide liquid crystal phases.  

PubMed

Graphene oxide is promising as a plate-like giant molecular building block for the assembly of new carbon materials. Its water dispersibility, liquid crystallinity, and ease of reduction offer advantages over other carbon precursors if its fundamental assembly rules can be identified. This article shows that graphene oxide sheets of known lateral dimension form nematic liquid crystal phases with transition points in agreement with the Onsager hard-plate theory. The liquid crystal phases can be systematically ordered into defined supramolecular patterns using surface anchoring, complex fluid flow, and microconfinement. Graphene oxide is seen to exhibit homeotropic surface anchoring at interfaces driven by excluded volume entropy and by adsorption enthalpy associated with its partially hydrophobic basal planes. Surprisingly, some of the surface-ordered graphene oxide phases dry into graphene oxide solids that undergo a dramatic anisotropic swelling upon rehydration to recover their initial size and shape. This behavior is shown to be a unique hydration-responsive folding and unfolding transition. During drying, surface tension forces acting parallel to the layer planes cause a buckling instability that stores elastic energy in accordion-folded structures in the dry solid. Subsequent water infiltration reduces interlayer frictional forces and triggers release of the stored elastic energy in the form of dramatic unidirectional expansion. We explain the folding/unfolding phenomena by quantitative nanomechanics and introduce the potential of liquid crystal-derived graphene oxide phases as new stimuli-response materials. PMID:21877716

Guo, Fei; Kim, Franklin; Han, Tae Hee; Shenoy, Vivek B; Huang, Jiaxing; Hurt, Robert H

2011-10-25

355

In vitro unfolding, refolding, and polymerization of human ?D crystallin, a protein involved in cataract formation  

PubMed Central

Human ?D crystallin (H?D-Crys), a major protein of the human eye lens, is a primary component of cataracts. This 174-residue primarily ?-sheet protein is made up of four Greek keys separated into two domains. Mutations in the human gene sequence encoding H?D-Crys are implicated in early-onset cataracts in children, and the mutant protein expressed in Escherichia coli exhibits properties that reflect the in vivo pathology. We have characterized the unfolding, refolding, and competing aggregation of human wild-type H?D-Crys as a function of guanidinium hydrochloride (GuHCl) concentration at neutral pH and 37°C, using intrinsic tryptophan fluorescence to monitor in vitro folding. Wild-type H?D-Crys exhibited reversible refolding above 1.0 M GuHCl. The GuHCl unfolded protein was more fluorescent than its native counterpart despite the absence of metal or ion-tryptophan interactions. Aggregation of refolding intermediates of H?D-Crys was observed in both equilibrium and kinetic refolding processes. The aggregation pathway competed with productive refolding at denaturant concentrations below 1.0 M GuHCl, beyond the major conformational transition region. Atomic force microscopy of samples under aggregating conditions revealed the sequential appearance of small nuclei, thin protofibrils, and fiber bundles. The H?D-Crys fibrous aggregate species bound bisANS appreciably, indicating the presence of exposed hydrophobic pockets. The mechanism of H?D-Crys aggregation may provide clues to understanding age-onset cataract formation in vivo.

Kosinski-Collins, Melissa S.; King, Jonathan

2003-01-01

356

Role of Protein Stabilizers on the Conformation of the Unfolded State of Cytochrome c and Its Early Folding Kinetics  

PubMed Central

An insight into the conformation and dynamics of unfolded and early intermediate states of a protein is essential to understand the mechanism of its aggregation and to design potent inhibitor molecules. Fluorescence correlation spectroscopy has been used to study the effects of several model protein stabilizers on the conformation of the unfolded state and early folding dynamics of tetramethyl rhodamine-labeled cytochrome c from Saccharomyces cerevisiae at single molecular resolution. Special attention has been given to arginine, which is a widely used stabilizer for improving refolding yield of different proteins. The value of the hydrodynamic radius (rH) obtained by analyzing the intensity fluctuations of the diffusing molecules has been found to increase in a two-state manner as the protein is unfolded by urea. The results further show that the presence of arginine and other protein stabilizers favors a relatively structured conformation of the unfolded states (rH of 29 ?) over an extended one (rH of 40 ?), which forms in their absence. Also, the time constant of a kinetic component (?R) of about 30 ?s has been observed by analyzing the correlation functions, which represents forma