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1

The unfolded protein response  

Microsoft Academic Search

The unfolded protein response (UPR) is a signal transduction network activated by inhibition of protein folding in the endoplasmic\\u000a reticulum (ER). The UPR coordinates adaptive responses to this stress situation, including induction of ER resident molecular\\u000a chaperone and protein foldase expression to increase the protein folding capacity of the ER, induction of phospholipid synthesis,\\u000a attenuation of general translation, and upregulation

Martin Schröder

2006-01-01

2

Translation Attenuation Mechanism in Unfolded Protein Response  

NASA Astrophysics Data System (ADS)

Endoplasmic Reticulum is a cellular organelle where membrane and extracellular proteins are folded with the help of chaperons. Insulin is one example of such extracellular proteins. Unfolded Protein Response (UPR) is a cell response to an increased level of unfolded proteins in ER. In pancreatic ?-cells failure in UPR leads to accumulation of unfolded insulin in Endoplasmic reticulum and eventual cell death. This is thought to be one of the causes of type two diabetes.

Trusina, Ala; Papa, Feroz; Tang, Chao

3

Targeting the unfolded protein response in disease.  

PubMed

Stress induced by the accumulation of unfolded proteins in the endoplasmic reticulum (ER) is a feature of specialized secretory cells and is also observed in many diseases, including cancer, diabetes, autoimmune conditions, liver disorders, obesity and neurodegenerative disorders. Cellular adaptation to ER stress is achieved by the activation of the unfolded protein response, which is an integrated signal transduction pathway that modulates many aspects of ER physiology. When these mechanisms of adaptation are insufficient to handle the unfolded protein load, cells undergo apoptosis. Here, we discuss recent advances in the design of novel compounds and therapeutic strategies to manipulate levels of ER stress in disease. PMID:23989796

Hetz, Claudio; Chevet, Eric; Harding, Heather P

2013-08-30

4

Analysis of unfolded protein response in Arabidopsis.  

PubMed

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. PMID:23913037

Chen, Yani; Brandizzi, Federica

2013-01-01

5

Evolution of the unfolded protein response.  

PubMed

The unfolded protein response (UPR) is a network of signaling pathways that responds to stress in the endoplasmic reticulum (ER). The general output of the UPR is to upregulate genes involved in ER function, thus restoring and/or increasing the capacity of the ER to fold and process proteins. In parallel, many organisms have mechanisms for limiting the load on the ER by attenuating translation or degrading ER-targeted mRNAs. Despite broad conservation of these signaling pathways across eukaryotes, interesting variations demonstrate a variety of mechanisms for managing ER stress. How do early-diverging protozoa respond to stress when they lack traditional transcriptional regulation? What is the role of the ER stress sensor Ire1 in fungal species that are missing its main target? Here I describe how diverse species have optimized the UPR to fit their needs. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum. PMID:23369734

Hollien, Julie

2013-01-28

6

Signal integration in the endoplasmic reticulum unfolded protein response  

Microsoft Academic Search

The endoplasmic reticulum (ER) responds to the accumulation of unfolded proteins in its lumen (ER stress) by activating intracellular signal transduction pathways — cumulatively called the unfolded protein response (UPR). Together, at least three mechanistically distinct arms of the UPR regulate the expression of numerous genes that function within the secretory pathway but also affect broad aspects of cell fate

David Ron; Peter Walter

2007-01-01

7

The role of the unfolded protein response in the heart  

Microsoft Academic Search

The misfolding of nascent proteins, or the unfolding of proteins after synthesis is complete, can occur in response to numerous environmental stresses, or as a result of mutations that de-stabilize protein structure. Cells have developed elaborate protein quality control systems that recognize improperly folded proteins and either refold them or facilitate their degradation. One such quality control system is the

Christopher C. Glembotski

2008-01-01

8

The unfolded protein response in nutrient sensing and differentiation  

Microsoft Academic Search

Eukaryotic cells coordinate protein-folding reactions in the endoplasmic reticulum with gene expression in the nucleus and messenger RNA translation in the cytoplasm. As the rate of protein synthesis increases, protein folding can be compromised, so cells have evolved signal-transduction pathways that control transcription and translation — the 'unfolded protein response'. Recent studies indicate that these pathways also coordinate rates of

Donalyn Scheuner; Martin Schröder; Xiaohua Shen; Kyungho Lee; Chuan Yin Liu; Stacey M. Arnold; Randal J. Kaufman

2002-01-01

9

Endoplasmic reticulum stress sensing in the unfolded protein response.  

PubMed

Secretory and transmembrane proteins enter the endoplasmic reticulum (ER) as unfolded proteins and exit as either folded proteins in transit to their target organelles or as misfolded proteins targeted for degradation. The unfolded protein response (UPR) maintains the protein-folding homeostasis within the ER, ensuring that the protein-folding capacity of the ER meets the load of client proteins. Activation of the UPR depends on three ER stress sensor proteins, Ire1, PERK, and ATF6. Although the consequences of activation are well understood, how these sensors detect ER stress remains unclear. Recent evidence suggests that yeast Ire1 directly binds to unfolded proteins, which induces its oligomerization and activation. BiP dissociation from Ire1 regulates this oligomeric equilibrium, ultimately modulating Ire1's sensitivity and duration of activation. The mechanistic principles of ER stress sensing are the focus of this review. PMID:23388626

Gardner, Brooke M; Pincus, David; Gotthardt, Katja; Gallagher, Ciara M; Walter, Peter

2013-03-01

10

Endothelin-1, the Unfolded Protein Response, and Persistent Inflammation  

PubMed Central

Endothelin-1 is a potent vasoactive peptide that occurs in chronically high levels in humans with pulmonary hypertension and in animal models of the disease. Recently, the unfolded protein response was implicated in a variety of diseases, including pulmonary hypertension. In addition, evidence is increasing for pathological, persistent inflammation in the pathobiology of this disease. We investigated whether endothelin-1 might engage the unfolded protein response and thus link inflammation and the production of hyaluronic acid by pulmonary artery smooth muscle cells. Using immunoblot, real-time PCR, immunofluorescence, and luciferase assays, we found that endothelin-1 induces both a transcriptional and posttranslational activation of the three major arms of the unfolded protein response. The pharmacologic blockade of endothelin A receptors, but not endothelin B receptors, attenuated the observed release, as did a pharmacologic blockade of extracellular signal–regulated kinases 1 and 2 (ERK-1/2) signaling. Using short hairpin RNA and ELISA, we observed that the release by pulmonary artery smooth muscle cells of inflammatory modulators, including hyaluronic acid, is associated with endothelin-1–induced ERK-1/2 phosphorylation and the unfolded protein response. Furthermore, the synthesis of hyaluronic acid induced by endothelin-1 is permissive for persistent THP-1 monocyte binding. These results suggest that endothelin-1, in part because it induces the unfolded protein response in pulmonary artery smooth muscle cells, triggers proinflammatory processes that likely contribute to vascular remodeling in pulmonary hypertension.

Belchenko, Dmitry D.; Nguyen, Cecilia M.; Colvin, Kelley L.; Ivy, D. Dunbar; Stenmark, K. R.

2012-01-01

11

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

12

Xenobiotic perturbation of ER stress and the unfolded protein response.  

PubMed

The proper folding, assembly, and maintenance of cellular proteins is a highly regulated process and is critical for cellular homeostasis. Multiple cellular compartments have adapted their own systems to ensure proper protein folding, and quality control mechanisms are in place to manage stress due to the accumulation of unfolded proteins. When the accumulation of unfolded proteins exceeds the capacity to restore homeostasis, these systems can result in a cell death response. Unfolded protein accumulation in the endoplasmic reticulum (ER) leads to ER stress with activation of the unfolded protein response (UPR) governed by the activating transcription factor 6 (ATF6), inositol requiring enzyme-1 (IRE1), and PKR-like endoplasmic reticulum kinase (PERK) signaling pathways. Many xenobiotics have been shown to influence ER stress and UPR signaling with either pro-survival or pro-death features. The ultimate outcome is dependent on many factors including the mechanism of action of the xenobiotic, concentration of xenobiotic, duration of exposure (acute vs. chronic), cell type affected, nutrient levels, oxidative stress, state of differentiation, and others. Assessing perturbations in activation or inhibition of ER stress and UPR signaling pathways are likely to be informative parameters to measure when analyzing mechanisms of action of xenobiotic-induced toxicity. PMID:23334697

Lafleur, Marc A; Stevens, James L; Lawrence, Jeffrey W

2013-01-18

13

Cytomegalovirus Downregulates IRE1 to Repress the Unfolded Protein Response  

PubMed Central

During viral infection, a massive demand for viral glycoproteins can overwhelm the capacity of the protein folding and quality control machinery, leading to an accumulation of unfolded proteins in the endoplasmic reticulum (ER). To restore ER homeostasis, cells initiate the unfolded protein response (UPR) by activating three ER-to-nucleus signaling pathways, of which the inositol-requiring enzyme 1 (IRE1)-dependent pathway is the most conserved. To reduce ER stress, the UPR decreases protein synthesis, increases degradation of unfolded proteins, and upregulates chaperone expression to enhance protein folding. Cytomegaloviruses, as other viral pathogens, modulate the UPR to their own advantage. However, the molecular mechanisms and the viral proteins responsible for UPR modulation remained to be identified. In this study, we investigated the modulation of IRE1 signaling by murine cytomegalovirus (MCMV) and found that IRE1-mediated mRNA splicing and expression of the X-box binding protein 1 (XBP1) is repressed in infected cells. By affinity purification, we identified the viral M50 protein as an IRE1-interacting protein. M50 expression in transfected or MCMV-infected cells induced a substantial downregulation of IRE1 protein levels. The N-terminal conserved region of M50 was found to be required for interaction with and downregulation of IRE1. Moreover, UL50, the human cytomegalovirus (HCMV) homolog of M50, affected IRE1 in the same way. Thus we concluded that IRE1 downregulation represents a previously undescribed viral strategy to curb the UPR.

Stahl, Sebastian; Burkhart, Julia M.; Hinte, Florian; Tirosh, Boaz; Mohr, Hermine; Zahedi, Rene P.; Sickmann, Albert; Ruzsics, Zsolt; Budt, Matthias; Brune, Wolfram

2013-01-01

14

Cytomegalovirus Downregulates IRE1 to Repress the Unfolded Protein Response.  

PubMed

During viral infection, a massive demand for viral glycoproteins can overwhelm the capacity of the protein folding and quality control machinery, leading to an accumulation of unfolded proteins in the endoplasmic reticulum (ER). To restore ER homeostasis, cells initiate the unfolded protein response (UPR) by activating three ER-to-nucleus signaling pathways, of which the inositol-requiring enzyme 1 (IRE1)-dependent pathway is the most conserved. To reduce ER stress, the UPR decreases protein synthesis, increases degradation of unfolded proteins, and upregulates chaperone expression to enhance protein folding. Cytomegaloviruses, as other viral pathogens, modulate the UPR to their own advantage. However, the molecular mechanisms and the viral proteins responsible for UPR modulation remained to be identified. In this study, we investigated the modulation of IRE1 signaling by murine cytomegalovirus (MCMV) and found that IRE1-mediated mRNA splicing and expression of the X-box binding protein 1 (XBP1) is repressed in infected cells. By affinity purification, we identified the viral M50 protein as an IRE1-interacting protein. M50 expression in transfected or MCMV-infected cells induced a substantial downregulation of IRE1 protein levels. The N-terminal conserved region of M50 was found to be required for interaction with and downregulation of IRE1. Moreover, UL50, the human cytomegalovirus (HCMV) homolog of M50, affected IRE1 in the same way. Thus we concluded that IRE1 downregulation represents a previously undescribed viral strategy to curb the UPR. PMID:23950715

Stahl, Sebastian; Burkhart, Julia M; Hinte, Florian; Tirosh, Boaz; Mohr, Hermine; Zahedi, René P; Sickmann, Albert; Ruzsics, Zsolt; Budt, Matthias; Brune, Wolfram

2013-08-08

15

Plant transducers of the endoplasmic reticulum unfolded protein response.  

PubMed

The unfolded protein response (UPR) activates a set of genes to overcome accumulation of unfolded proteins in the endoplasmic reticulum (ER), a condition termed ER stress, and constitutes an essential part of ER protein quality control that ensures efficient maturation of secretory and membrane proteins in eukaryotes. Recent studies on Arabidopsis and rice identified the signaling pathway in which the ER membrane-localized ribonuclease IRE1 (inositol-requiring enzyme 1) catalyzes unconventional cytoplasmic splicing of mRNA, thereby producing the active transcription factor Arabidopsis bZIP60 (basic leucine zipper 60) and its ortholog in rice. Here we review recent findings identifying the molecular components of the plant UPR, including IRE1/bZIP60 and the membrane-bound transcription factors bZIP17 and bZIP28, and implicating its importance in several physiological phenomena such as pathogen response. PMID:22796463

Iwata, Yuji; Koizumi, Nozomu

2012-07-14

16

Unfolded protein response in cancer: the Physician's perspective  

PubMed Central

The unfolded protein response (UPR) is a cascade of intracellular stress signaling events in response to an accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum (ER). Cancer cells are often exposed to hypoxia, nutrient starvation, oxidative stress and other metabolic dysregulation that cause ER stress and activation of the UPR. Depending on the duration and degree of ER stress, the UPR can provide either survival signals by activating adaptive and antiapoptotic pathways, or death signals by inducing cell death programs. Sustained induction or repression of UPR pharmacologically may thus have beneficial and therapeutic effects against cancer. In this review, we discuss the basic mechanisms of UPR and highlight the importance of UPR in cancer biology. We also update the UPR-targeted cancer therapeutics currently in clinical trials.

2011-01-01

17

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

18

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

19

Redox signaling loops in the unfolded protein response.  

PubMed

The endoplasmic reticulum (ER) is the first compartment of secretory pathway. It plays a major role in ER chaperone-assisted folding and quality control, including post-translational modification such as disulfide bond formation of newly synthesized secretory proteins. Protein folding and assembly takes place in the ER, where redox conditions are distinctively different from the other organelles and are favorable for disulfide formation. These reactions generate the production of reactive oxygen species (ROS) as a byproduct of thiol/disulfide exchange reaction among ER oxidoreductin 1 (Ero1), protein disulfide isomerase (PDI) and ER client proteins, during the formation of disulfide bonds in nascent or incorrectly folded proteins. When uncontrolled, this phenomenon perturbs ER homeostasis, thus aggravating the accumulation of improperly folded or unfolded proteins in this compartment (ER stress). This results in the activation of an adaptive mechanism named the unfolded protein response (UPR). In mammalian cells, the UPR is mediated by three ER-resident membrane proteins (PERK, IRE1 and ATF6) and regulates the expression of the UPR target genes, which themselves encode ER chaperones, folding enzymes, pro-apoptotic proteins and antioxidants, with the objective of restoring ER homeostatic balance. In this review, we will describe redox dependent activation (ER) and amplification (cytosol) loops that control the UPR and the consequences these regulatory loops have on cell fate and physiology. PMID:22481091

Higa, Arisa; Chevet, Eric

2012-03-28

20

Hepatitis C virus envelope proteins regulate CHOP via induction of the unfolded protein response  

Microsoft Academic Search

Unfolded protein response (UPR) is a cellular adaptive response that functions to reduce stress caused by malfolded proteins in the endoplasmic reticulum (ER). UPR can be induced under physiological or pathological conditions and is responsible for the pathogenesis of many human diseases. Hepatitis C virus (HCV) is a single-stranded, positive-sense RNA virus causing chronic diseases. Its genome encodes two envelope

Shiu-Wan Chan; Philip Anthony Egan

2005-01-01

21

Review: retinal degeneration: focus on the unfolded protein response.  

PubMed

Recently published literature has provided evidence that the unfolded protein response (UPR) is involved in the development of retinal degeneration. The scope of these studies encompassed diabetic retinopathy, retinopathy of prematurity, glaucoma, retinal detachment, light-induced retinal degeneration, age-related macular degeneration, and inherited retinal degeneration. Subsequent studies investigating the role of individual UPR markers in retinal pathogenesis and examining the therapeutic potential of reprogramming the UPR as a method for modulating the rate of retinal degeneration have been initiated. Manipulation of UPR markers has been made possible by the use of knockout mice, pharmacological agents, and viral vector-mediated augmentation of gene expression. Future research will aim at identifying specific inhibitors and/or inducers of UPR regulatory markers as well as expand the list of UPR-related animal models. Additionally, adeno-associated virus-mediated gene delivery is a safe and effective method for modulating gene expression, and thus is a useful research tool for manipulating individual UPR markers in affected retinas and a promising delivery vector for gene therapy in retinal degenerative disorders. PMID:24068865

Gorbatyuk, Marina; Gorbatyuk, Oleg

2013-09-20

22

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

2013-04-09

23

Review: Retinal degeneration: Focus on the unfolded protein response  

PubMed Central

Recently published literature has provided evidence that the unfolded protein response (UPR) is involved in the development of retinal degeneration. The scope of these studies encompassed diabetic retinopathy, retinopathy of prematurity, glaucoma, retinal detachment, light-induced retinal degeneration, age-related macular degeneration, and inherited retinal degeneration. Subsequent studies investigating the role of individual UPR markers in retinal pathogenesis and examining the therapeutic potential of reprogramming the UPR as a method for modulating the rate of retinal degeneration have been initiated. Manipulation of UPR markers has been made possible by the use of knockout mice, pharmacological agents, and viral vector-mediated augmentation of gene expression. Future research will aim at identifying specific inhibitors and/or inducers of UPR regulatory markers as well as expand the list of UPR-related animal models. Additionally, adeno-associated virus-mediated gene delivery is a safe and effective method for modulating gene expression, and thus is a useful research tool for manipulating individual UPR markers in affected retinas and a promising delivery vector for gene therapy in retinal degenerative disorders.

Gorbatyuk, Oleg

2013-01-01

24

Testicular hyperthermia induces Unfolded Protein Response signaling activation in spermatocyte.  

PubMed

The testes of most mammals are sensitive to temperature. To survive and adapt under conditions that promote endoplasmic reticulum (ER) stress such as heat shock, cells have a self-protective mechanism against ER stress that has been termed the "Unfolded Protein Response" (UPR). However, the cellular and molecular events underlying spermatogenesis with testicular hyperthermia involved in the UPR signaling pathway under ER stress remain poorly understood. In the present study, we verified that UPR signaling via phospho-eIF2?/ATF4/GADD34, p90ATF6, and phospho-IRE1?/XBP-1 is activated with testicular hyperthermia (43 °C, 15 min/day) and induced ER stress-mediated apoptosis associated with CHOP, phospho-JNK, and caspase-3 after repetitive periods of hyperthermia. Levels of phospho-eIF2? protein of mouse spermatocytes in the testis were rapidly increased by one cycle of testicular hyperthermia. ATF4/GADD34 and p90ATF6 expression gradually increased and decreased, respectively, with repetitive cycles of hyperthermia. Spliced XBP1 mRNA as a marker of IRE1 activity was increased after one, three cycles of hyperthermia and decreased by five cycles of hyperthermia. Although the levels of anti-apoptotic phospho-JNK (p54) were gradually decreased after three cycles of hyperthermia, CHOP expression was rapidly increased. After five cycles of testicular hyperthermia, the levels of cleaved caspase-3 and TUNEL-positive apoptotic spermatocytes cells were significantly increased. Our data demonstrated that testicular hyperthermia induces UPR signaling and repetitive cycles of hyperthermia lead to apoptosis of spermatocytes in mouse testis. These results suggest a link between the UPR signaling pathway and testicular hyperthermia. PMID:23611781

Kim, Jung-Hak; Park, Sun-Ji; Kim, Tae-Shin; Park, Hyo-Jin; Park, Junghyung; Kim, Bo Kyung; Kim, Gyeong-Ryul; Kim, Jin-Man; Huang, Song Mei; Chae, Jung-Il; Park, Choon-Keun; Lee, Dong-Seok

2013-04-20

25

Recent advances in understanding the control of secretory proteins by the unfolded protein response in plants.  

PubMed

The membrane transport system is built on the proper functioning of the endoplasmic reticulum (ER). The accumulation of unfolded proteins in the ER lumen (ER stress) disrupts ER homeostasis and disturbs the transport system. In response to ER stress, eukaryotic cells activate intracellular signaling (named the unfolded protein response, UPR), which contributes to the quality control of secretory proteins. On the other hand, the deleterious effects of UPR on plant health and growth characteristics have frequently been overlooked, due to limited information on this mechanism. However, recent studies have shed light on the molecular mechanism of plant UPR, and a number of its unique characteristics have been elucidated. This study briefly reviews the progress of understanding what is happening in plants under ER stress conditions. PMID:23629671

Hayashi, Shimpei; Wakasa, Yuhya; Takaiwa, Fumio

2013-04-29

26

Recent Advances in Understanding the Control of Secretory Proteins by the Unfolded Protein Response in Plants  

PubMed Central

The membrane transport system is built on the proper functioning of the endoplasmic reticulum (ER). The accumulation of unfolded proteins in the ER lumen (ER stress) disrupts ER homeostasis and disturbs the transport system. In response to ER stress, eukaryotic cells activate intracellular signaling (named the unfolded protein response, UPR), which contributes to the quality control of secretory proteins. On the other hand, the deleterious effects of UPR on plant health and growth characteristics have frequently been overlooked, due to limited information on this mechanism. However, recent studies have shed light on the molecular mechanism of plant UPR, and a number of its unique characteristics have been elucidated. This study briefly reviews the progress of understanding what is happening in plants under ER stress conditions.

Hayashi, Shimpei; Wakasa, Yuhya; Takaiwa, Fumio

2013-01-01

27

The unfolded protein response in plants: A fundamental adaptive cellular response to internal and external stresses.  

PubMed

In eukaryotic cells, proteins that enter the secretory pathway are translated on membrane-bound ribosomes and translocated into the endoplasmic reticulum (ER), where they are subjected to chaperone-assisted folding, post-translational modification and assembly. During the evolution of the eukaryotic cell, a homeostatic mechanism was developed to maintain the functions of the ER in the face of various internal and external stresses. The most severe stresses imposed on eukaryotic cells can induce ER stress that can overwhelm the processing capacity of the ER, leading to the accumulation of unfolded proteins in the ER lumen. To cope with this accumulation of unfolded proteins, the unfolded protein response (UPR) is activated to alter transcriptional programs through inositol-requiring enzyme 1 (IRE1) and bZIP17/28 in plants. In addition to transcriptional induction of UPR genes, quality control (QC), translational attenuation, ER-associated degradation (ERAD) and ER stress-induced apoptosis are also conserved as fundamental adaptive cellular responses to ER stress in plants. This article is part of a Special Issue entitled: Translational Plant Proteomics. PMID:23624343

Wahyu Indra Duwi Fanata; Lee, Sang Yeol; Lee, Kyun Oh

2013-04-25

28

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

29

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

30

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

31

Inhibitors of the unfolded protein response and methods for their use  

US Patent & Trademark Office Database

Compounds that are inhibitors of the unfolded protein response and endonuclease IRE1 are provided, together with compositions comprising such compounds, and methods for their use in the treatment of various disorders, such as cancer, autoimmune disorders, and diabetes. Also provided are packaged pharmaceuticals comprising these compositions. The compositions may be administered in combination with another therapeutic agent.

2013-02-12

32

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

33

A Janus-faced role of the unfolded protein response in antitumor immunity  

PubMed Central

The unfolded protein response (UPR) has been established as a cell-intrinsic mechanism of survival for malignant cells facing microenvironmental stressors. Recent evidence indicates that the UPR also modulates antitumor immunity. Here, we discuss the bi-faced role of the UPR as it both promotes and antagonizes antitumor T-cell immunity.

Mahadevan, Navin R.; Rodvold, Jeffrey J.; Zanetti, Maurizio

2013-01-01

34

The Unfolded Protein Response Is Triggered by a Plant Viral Movement Protein1[W][OA  

PubMed Central

Infection with Potato virus X (PVX) in Nicotiana benthamiana plants leads to increased transcript levels of several stress-related host genes, including basic-region leucine zipper 60 (bZIP60), SKP1, ER luminal binding protein (BiP), protein disulfide isomerase (PDI), calreticulin (CRT), and calmodulin (CAM). bZIP60 is a key transcription factor that responds to endoplasmic reticulum (ER) stress and induces the expression of ER-resident chaperones (BiP, PDI, CRT, and CAM). SKP1 is a component of SCF (for SKP1-Cullin-F box protein) ubiquitin ligase complexes that target proteins for proteasomal degradation. Expression of PVX TGBp3 from a heterologous vector induces the same set of genes in N. benthamiana and Arabidopsis (Arabidopsis thaliana) leaves. Virus-induced gene silencing was employed to knock down the expression of bZIP60 and SKP1, and the number of infection foci on inoculated leaves was reduced and systemic PVX accumulation was altered. Silencing bZIP60 led to the suppression of BiP and SKP1 transcript levels, suggesting that bZIP60 might be an upstream signal transducer. Overexpression of TGBp3 led to localized necrosis, but coexpression of TGBp3 with BiP abrogated necrosis, demonstrating that the unfolded protein response alleviates ER stress-related cell death. Steady-state levels of PVX replicase and TGBp2 (which reside in the ER) proteins were unaltered by the presence of TGBp3, suggesting that TGBp3 does not contribute to their turnover. Taken together, PVX TGBp3-induced ER stress leads to up-regulation of bZIP60 and unfolded protein response-related gene expression, which may be important to regulate cellular cytotoxicity that could otherwise lead to cell death if viral proteins reach high levels in the ER.

Ye, Changming; Dickman, Martin B.; Whitham, Steven A.; Payton, Mark; Verchot, Jeanmarie

2011-01-01

35

Protein Misfolding Induces Hypoxic Preconditioning via a Subset of the Unfolded Protein Response Machinery?  

PubMed Central

Prolonged cellular hypoxia results in energy failure and ultimately cell death. However, less-severe hypoxia can induce a cytoprotective response termed hypoxic preconditioning (HP). The unfolded protein response pathway (UPR) has been known for some time to respond to hypoxia and regulate hypoxic sensitivity; however, the role of the UPR, if any, in HP essentially has been unexplored. We have shown previously that a sublethal hypoxic exposure of the nematode Caenorhabditis elegans induces a protein chaperone component of the UPR (L. L. Anderson, X. Mao, B. A. Scott, and C. M. Crowder, Science 323:630-633, 2009). Here, we show that HP induces the UPR and that the pharmacological induction of misfolded proteins is itself sufficient to stimulate a delayed protective response to hypoxic injury that requires the UPR pathway proteins IRE-1, XBP-1, and ATF-6. HP also required IRE-1 but not XBP-1 or ATF-6; instead, GCN-2, which is known to suppress translation and induce an adaptive transcriptional response under conditions of UPR activation or amino acid deprivation, was required for HP. The phosphorylation of the translation factor eIF2?, an established mechanism of GCN-2-mediated translational suppression, was not necessary for HP. These data suggest a model where hypoxia-induced misfolded proteins trigger the activation of IRE-1, which along with GCN-2 controls an adaptive response that is essential to HP.

Mao, Xianrong R.; Crowder, C. Michael

2010-01-01

36

Hepatitis C Virus Envelope Protein E1 Binds PERK and Represses the Unfolded Protein Response  

PubMed Central

Unfolded protein response (UPR) is a cellular adaptive response which functions to reduce stress caused by misfolded proteins in the endoplasmic reticulum (ER). We and others have previously shown that infection with hepatitis C virus (HCV) or expression of the viral proteins can trigger the UPR. HCV is a single-stranded positive-sense RNA virus causing chronic diseases in humans. Its genome encodes two envelope proteins E1 and E2 that mature in the ER to form non-covalently bound native complex and disulphide-bonded aggregates. Apart from the ER targeting proteins, cytosolic forms have been documented. We have previously shown that the ER-targeting E1 and E2 are capable of eliciting the UPR whereas others have shown that the cytosolic-targeting E2 can bind to the ER stress kinase PERK to dampen the UPR. In this report, we further show that the other envelope protein E1, in its cytosolic form, can also bind PERK and dampen the UPR. Using GST-pulldown assay, we show that E1 binds to the cytoplasmic domain of PERK, suggesting interaction of E1 and PERK takes place in the cytoplasm. Using reporter gene assay and Western blotting, we show that cytosolic E1 can repress UPR-induced BiP and CHOP promoter activity and reduce UPR-induced CHOP expression level. Altogether these results suggest opposing functions of ER- and cytosolic forms of HCV envelope proteins depending on their subcellular localization.

Egan, Philip A; Sobkowiak, Michal; Chan, Shiu-Wan

2013-01-01

37

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

38

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

PubMed

Selective small-molecule inhibitors represent powerful tools for the dissection of complex biological processes. ES(I) (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), ES(I) 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. PMID:22145777

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

2012-03-15

39

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

40

Ubiquitin-Like Protein 5 Positively Regulates Chaperone Gene Expression in the Mitochondrial Unfolded Protein Response  

PubMed Central

Perturbation of the protein-folding environment in the mitochondrial matrix selectively upregulates the expression of nuclear genes encoding mitochondrial chaperones. To identify components of the signal transduction pathway(s) mediating this mitochondrial unfolded protein response (UPRmt), we first isolated a temperature-sensitive mutation (zc32) that conditionally activates the UPRmt in C. elegans and subsequently searched for suppressors by systematic inactivation of genes. RNAi of ubl-5, a gene encoding a ubiquitin-like protein, suppresses activation of the UPRmt markers hsp-60?gfp and hsp-6?gfp by the zc32 mutation and by other manipulations that promote mitochondrial protein misfolding. ubl-5 (RNAi) inhibits the induction of endogenous mitochondrial chaperone encoding genes hsp-60 and hsp-6 and compromises the ability of animals to cope with mitochondrial stress. Mitochondrial morphology and assembly of multi-subunit mitochondrial complexes of biotinylated proteins are also perturbed in ubl-5(RNAi) worms, indicating that UBL-5 also counteracts physiological levels of mitochondrial stress. Induction of mitochondrial stress promotes accumulation of GFP-tagged UBL-5 in nuclei of transgenic worms, suggesting that UBL-5 effects a nuclear step required for mounting a response to the threat of mitochondrial protein misfolding.

Benedetti, Cristina; Haynes, Cole M.; Yang, Yun; Harding, Heather P.; Ron, David

2006-01-01

41

Endoplasmic Reticulum Stress and the Unfolded Protein Response in Nonalcoholic Fatty Liver Disease  

PubMed Central

Abstract The underlying causes of nonalcoholic fatty liver disease (NAFLD) are unclear, although recent evidence has implicated the endoplasmic reticulum (ER) in both the development of steatosis and progression to nonalcoholic steatohepatitis. Disruption of ER homeostasis, often termed “ER stress,” has been observed in liver and adipose tissue of humans with NAFLD and/or obesity. Importantly, the signaling pathway activated by disruption of ER homeostasis, the unfolded protein response, has been linked to lipid biosynthesis, insulin action, inflammation, and apoptosis. Therefore, understanding the mechanisms that disrupt ER homeostasis in NAFLD and the role of ER-mediated signaling have become topics of intense investigation. The present review will examine the ER and the unfolded protein response in the context of NAFLD. Antioxid. Redox Signal. 15, 505–521.

Gentile, Christopher L.; Frye, Melinda

2011-01-01

42

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

43

A carrier fusion significantly induces unfolded protein response in heterologous protein production by Aspergillus oryzae.  

PubMed

In heterologous protein production by filamentous fungi, target proteins are expressed as fusions with homologous secretory proteins, called carriers, for higher production yields. Although carrier fusion is thought to overcome the bottleneck in transcriptional and (post)translational processes during heterologous protein production, there is limited knowledge of its physiological effects on the host strain. In this study, we performed DNA microarray analysis by comparing gene expression patterns of two Aspergillus oryzae strains expressing either carrier- or non-carrier-fused bovine chymosin (CHY). When CHY was expressed as a fusion with ?-amylase (AmyB), the production level increased by approximately 2-fold as compared with the non-carrier-fused CHY. DNA microarray analysis revealed that the carrier fusion significantly up-regulated many genes involved in endoplasmic reticulum (ER) protein-folding and secretion. Consistently, hacA transcripts were efficiently spliced in the strain expressing the carrier-fused CHY, indicating an unfolded protein response (UPR). The carrier-fused CHY was detected intracellularly without processing at the Kex2 cleavage site, which is likely recognized in the Golgi, and the carrier fusion delayed extracellular CHY production in the early growth phase as compared with the non-carrier-fused expression. Taken together, our data suggest a proposal that the carrier fusion temporarily accumulates the carrier-fused CHY in the ER and significantly induces UPR. PMID:21822643

Ohno, Ayako; Maruyama, Jun-ichi; Nemoto, Takashi; Arioka, Manabu; Kitamoto, Katsuhiko

2011-08-07

44

The unfolded protein response and its role in intestinal homeostasis and inflammation  

PubMed Central

The unfolded protein response (UPR) is a signaling pathway from the endoplasmic reticulum (ER) to the nucleus that protects cells from the stress caused by misfolded or unfolded proteins [1, 2]. As such, ER stress is an ongoing challenge for all cells given the central biologic importance of secretion as part of normal physiologic functions. This is especially the case for cells that are highly dependent upon secretory function as part of their major duties. Within mucosal tissues, the intestinal epithelium is especially dependent upon an intact UPR for its normal activities [3]. This review will discuss the UPR and the special role that it provides in the functioning of the intestinal epithelium and, when dysfunctional, its implications for understanding mucosal homeostasis and intestinal inflammation, as occurs in inflammatory bowel disease (IBD).

Kaser, Arthur; Flak, Magdalena B.; Tomczak, Michal F.; Blumberg, Richard S.

2011-01-01

45

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

46

Differential requirement of unfolded protein response pathway for calreticulin expression in Caenorhabditis elegans.  

PubMed

Accumulation of unfolded proteins in the endoplasmic reticulum triggers the unfolded protein response (UPR) pathway, which increases the expression of chaperones to maintain the homeostasis. Calreticulin is a calcium-binding chaperone located in the lumen of endoplasmic reticulum (ER). Here we show that in response to a UPR inducing reagent, tunicamycin, the expression of calreticulin (crt-1) is specifically up-regulated in Caenorhabditis elegans. Tunicamycin (TM) induced expression of the crt-1 requires IRE-1 and XBP-1 but is ATF-6 and PEK-1 independent. Analysis of the crt-1 promoter reveals a putative XBP-1 binding site at the -284 to -278 bp region, which was shown to be necessary for TM-mediated induction. Genetic analysis of crt-1 mutants and mutants of UPR pathway genes show various degrees of developmental arrest upon TM treatment. Our results suggest that the TM-induced UPR pathway culminates in the up-regulation of crt-1, which protects the worm from deleterious accumulation of unfolded proteins in the ER. Knockdown of the crt-1, pdi-2, or pdi-3 increased the crt-1 expression, whereas knockdown of the hsp-3 or hsp-4 did not have any effect on crt-1 expression, indicating the existence of complex compensatory networks to cope up with ER stress. PMID:17651753

Lee, Dukgyu; Singaravelu, Gunasekaran; Park, Byung-Jae; Ahnn, Joohong

2007-07-03

47

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

48

The Unfolded Protein Response Regulates Multiple Aspects of Secretory and Membrane Protein Biogenesis and Endoplasmic Reticulum Quality Control  

Microsoft Academic Search

The unfolded protein response (UPR) is an intracellular signaling pathway that relays signals from the lumen of the ER to activate target genes in the nu- cleus. We devised a genetic screen in the yeast Saccha- romyces cerevisiae to isolate mutants that are depen- dent on activation of the pathway for viability. Using this strategy, we isolated mutants affecting various

Davis T. W. Ng; Eric D. Spear; Peter Walter

2000-01-01

49

Protein unfolding: Rigidity lost  

PubMed Central

We relate the unfolding of a protein to its loss of structural stability or rigidity. Rigidity and flexibility are well defined concepts in mathematics and physics, with a body of theorems and algorithms that have been applied successfully to materials, allowing the constraints in a network to be related to its deformability. Here we simulate the weakening or dilution of the noncovalent bonds during protein unfolding, and identify the emergence of flexible regions as unfolding proceeds. The transition state is determined from the inflection point in the change in the number of independent bond-rotational degrees of freedom (floppy modes) of the protein as its mean atomic coordination decreases. The first derivative of the fraction of floppy modes as a function of mean coordination is similar to the fraction-folded curve for a protein as a function of denaturant concentration or temperature. The second derivative, a specific heat-like quantity, shows a peak around a mean coordination of ?r? = 2.41 for the 26 diverse proteins we have studied. As the protein denatures, it loses rigidity at the transition state, proceeds to a state where just the initial folding core remains stable, then becomes entirely denatured or flexible. This universal behavior for proteins of diverse architecture, including monomers and oligomers, is analogous to the rigid to floppy phase transition in network glasses. This approach provides a unifying view of the phase transitions of proteins and glasses, and identifies the mean coordination as the relevant structural variable, or reaction coordinate, along the unfolding pathway.

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

2002-01-01

50

Protein unfolding: Rigidity lost  

NASA Astrophysics Data System (ADS)

We relate the unfolding of a protein to its loss of structural stability or rigidity. Rigidity and flexibility are well defined concepts in mathematics and physics, with a body of theorems and algorithms that have been applied successfully to materials, allowing the constraints in a network to be related to its deformability. Here we simulate the weakening or dilution of the noncovalent bonds during protein unfolding, and identify the emergence of flexible regions as unfolding proceeds. The transition state is determined from the inflection point in the change in the number of independent bond-rotational degrees of freedom (floppy modes) of the protein as its mean atomic coordination decreases. The first derivative of the fraction of floppy modes as a function of mean coordination is similar to the fraction-folded curve for a protein as a function of denaturant concentration or temperature. The second derivative, a specific heat-like quantity, shows a peak around a mean coordination of r = 2.41 for the 26 diverse proteins we have studied. As the protein denatures, it loses rigidity at the transition state, proceeds to a state where just the initial folding core remains stable, then becomes entirely denatured or flexible. This universal behavior for proteins of diverse architecture, including monomers and oligomers, is analogous to the rigid to floppy phase transition in network glasses. This approach provides a unifying view of the phase transitions of proteins and glasses, and identifies the mean coordination as the relevant structural variable, or reaction coordinate, along the unfolding pathway.

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

2002-03-01

51

Transient Aggregation of Ubiquitinated Proteins Is a Cytosolic Unfolded Protein Response to Inflammation and Endoplasmic Reticulum Stress*  

PubMed Central

Failure to maintain protein homeostasis (proteostasis) leads to accumulation of unfolded proteins and contributes to the pathogenesis of many human diseases. Accumulation of unfolded proteins in the endoplasmic reticulum (ER) elicits unfolded protein response (UPR) that serves to attenuate protein translation, and increase protein refolding or degradation. In contrast to UPR in the ER, the regulatory molecules operative in cytosolic responses and their potential relation to ER stress are not well elucidated. Aggresome-like induced structures (ALIS) have been described as transient aggregation of ubiquitinated proteins in the cytosol. In this study, we show that cells respond to inflammation, infection or ER stress by cytosolic formation of ALIS, indicating that ALIS formation represents an early event in cellular adjustment to altered proteostasis that occurs under these conditions. This response was aided by rapid transcriptional up-regulation of polyubiqutin-binding protein p62. NF-?B and mTOR activation were also required for ALIS formation. Importantly, we show a cross talk between UPR in the ER and cytosolic ALIS. Down-regulation of ER UPR in XBP1 deficient cells increases cyotosolic ALIS formation. Furthermore, lysosomal activity but not macroautophagy is responsible for ALIS clearance. This study reveals the underlying regulatory mechanisms of ALIS formation and clearance, and provides a previously unrecognized common adaptive mechanism for cellular responses against inflammation and ER stress.

Liu, Xian-De; Ko, Soyoung; Xu, Yi; Fattah, Elmoataz Abdel; Xiang, Qian; Jagannath, Chinnaswamy; Ishii, Tetsuro; Komatsu, Masaaki; Eissa, N. Tony

2012-01-01

52

Gene regulatory network of unfolded protein response genes in endoplasmic reticulum stress.  

PubMed

In the endoplasmic reticulum (ER), secretory and membrane proteins are properly folded and modified, and the failure of these processes leads to ER stress. At the same time, unfolded protein response (UPR) genes are activated to maintain homeostasis. Despite the thorough characterization of the individual gene regulation of UPR genes to date, further investigation of the mutual regulation among UPR genes is required to understand the complex mechanism underlying the ER stress response. In this study, we aimed to reveal a gene regulatory network formed by UPR genes, including immunoglobulin heavy chain-binding protein (BiP), X-box binding protein 1 (XBP1), C/EBP [CCAAT/enhancer-binding protein]-homologous protein (CHOP), PKR-like endoplasmic reticulum kinase (PERK), inositol-requiring 1 (IRE1), activating transcription factor 6 (ATF6), and ATF4. For this purpose, we focused on promoter-luciferase reporters for BiP, XBP1, and CHOP genes, which bear an ER stress response element (ERSE), and p5?×?ATF6-GL3, which bears an unfolded protein response element (UPRE). We demonstrated that the luciferase activities of the BiP and CHOP promoters were upregulated by all the UPR genes, whereas those of the XBP1 promoter and p5?×?ATF6-GL3 were upregulated by all the UPR genes except for BiP, CHOP, and ATF4 in HeLa cells. Therefore, an ERSE- and UPRE-centered gene regulatory network of UPR genes could be responsible for the robustness of the ER stress response. Finally, we revealed that BiP protein was degraded when cells were treated with DNA-damaging reagents, such as etoposide and doxorubicin; this finding suggests that the expression level of BiP is tightly regulated at the post-translational level, rather than at the transcriptional level, in the presence of DNA damage. PMID:22802018

Takayanagi, Sayuri; Fukuda, Riga; Takeuchi, Yuuki; Tsukada, Sakiko; Yoshida, Kenichi

2012-07-18

53

Activation of the unfolded protein response during anoxia exposure in the turtle Trachemys scripta elegans.  

PubMed

Red-eared slider turtles, Trachemys scripta elegans, can survive for several weeks without oxygen when submerged in cold water. We hypothesized that anaerobiosis is aided by adaptive up-regulation of the unfolded protein response (UPR), a stress-responsive pathway that is activated by accumulation of unfolded proteins in the endoplasmic reticulum (ER) and functions to restore ER homeostasis. RT-PCR, western immunoblotting and DNA-binding assays were used to quantify the responses and/or activation status of UPR-responsive genes and proteins in turtle tissues after animal exposure to 5 or 20 h of anoxic submergence at 4 °C. The phosphorylation state of protein kinase-like ER kinase (PERK) (a UPR-regulated kinase) and eukaryotic initiation factor 2 (eIF2?) increased by 1.43-2.50 fold in response to anoxia in turtle heart, kidney, and liver. Activation of the PERK-regulated transcription factor, activating transcription factor 4 (ATF4), during anoxia was documented by elevated atf4 transcripts and total ATF4 protein (1.60-2.43 fold), increased nuclear ATF4 content, and increased DNA-binding activity (1.44-2.32 fold). ATF3 and GADD34 (downstream targets of ATF4) also increased by 1.38-3.32 fold in heart and liver under anoxia, and atf3 transcripts were also elevated in heart. Two characteristic chaperones of the UPR, GRP78, and GRP94, also responded positively to anoxia with strong increases in both the transcript and protein levels. The data demonstrate that the UPR is activated in turtle heart, kidney, and liver in response to anoxia, suggesting that this pathway mediates an integrated stress response to protect tissues during oxygen deprivation. PMID:23124854

Krivoruchko, Anastasia; Storey, Kenneth B

2012-11-03

54

Protein C Mutation (A267T) Results in ER Retention and Unfolded Protein Response Activation  

PubMed Central

Background Protein C (PC) deficiency is associated with a high risk of venous thrombosis. Recently, we identified the PC-A267T mutation in a patient with PC deficiency and revealed by in vitro studies decreased intracellular and secreted levels of the mutant. The aim of the present study was to characterize the underlying mechanism(s). Methodology/Principal Findings CHO-K1 cells stably expressing the wild-type (PC-wt) or the PC mutant were generated. In order to examine whether the PC mutant was subjected to increased intracellular degradation, the cells were treated with several inhibitors of various degradation pathways and pulse-chase experiments were performed. Protein-chaperone complexes were analyzed by treating the cells with a cross-linker followed by Western blotting (WB). Expression levels of the immunoglobulin-binding protein (BiP) and the phosphorylated eukaryotic initiation factor 2? (P-eIF2?), both common ER stress markers, were determined by WB to examine if the mutation induced ER stress and unfolded protein response (UPR) activation. We found no major differences in the intracellular degradation between the PC variants. The PC mutant was retained in the endoplasmic reticulum (ER) and had increased association with the Grp-94 and calreticulin chaperones. Retention of the PC-A267T in ER resulted in UPR activation demonstrated by increased expression levels of the ER stress markers BiP and P-eIF2? and caused also increased apoptotic activity in CHO-K1 cells as evidenced by elevated levels of DNA fragmentation. Conclusions/Significance The reduced intracellular level and impaired secretion of the PC mutant were due to retention in ER. In contrast to other PC mutations, retention of the PC-A267T in ER resulted in minor increased proteasomal degradation, rather it induced ER stress, UPR activation and apoptosis.

Tjeldhorn, Lena; Iversen, Nina; Sandvig, Kirsten; Bergan, Jonas; Sandset, Per Morten; Skretting, Grethe

2011-01-01

55

Linking membrane dynamics and trafficking to autophagy and the unfolded protein response.  

PubMed

Cellular stressors typically induce two protective counter-responses-autophagy and the unfolded protein response (UPR). It is conceivable that these two endoplasmic reticulum (ER) membrane-based processes would intersect/interact somehow with the constitutive housekeeping process of exocytic membrane traffic from the ER. How exactly might this occur? Recent evidence indicates that a conserved Rab protein, Rab1/Ypt1p, has functional roles in UPR and autophagy. This molecular switch and its associated effectors may therefore serve to link up a network of cellular responses to stress through changes in membrane dynamics and protein turnover. The notion provides further explanations as to why elevation of Rab1/Ypt1p levels could counter the cytotoxicity of ?-synuclein, and a similar mode of protection may well be at work against other stresses. PMID:23460446

Chua, Christelle En Lin; Tang, Bor Luen

2013-08-01

56

Fine tuning of the Unfolded Protein Response: Assembling the IRE1? interactome  

PubMed Central

Endoplasmic reticulum (ER) stress is a hallmark feature of secretory cells and many diseases including cancer, neurodegeneration, and diabetes. Adaptation to protein folding stress is mediated by the activation of an integrated signal transduction pathway known as the unfolded protein response (UPR). The UPR signals through three distinct stress sensors located at the ER membrane, IRE1?, ATF6 and PERK. Although PERK and IRE1? share functionally similar ER-luminal sensing domains and both are simultaneously activated in cellular paradigms of ER stress in vitro, they are selectively engaged in vivo by the physiological stress of unfolded proteins. The differences in terms of tissue-specific regulation of the UPR may be explained by the formation of distinct regulatory protein complexes. This concept is supported by the recent identification of adaptor and modulator proteins that directly interact with IRE1?. In this review we discuss recent evidence supporting a model where IRE1? signaling emerges as a highly regulated process, controlled by the formation of a dynamic scaffold onto which many regulatory components assemble.

Hetz, Claudio; Glimcher, Laurie H.

2011-01-01

57

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

58

Abnormal Expression of Collagen IV in Lens Activates Unfolded Protein Response Resulting in Cataract*  

PubMed Central

Human diseases caused by mutations in extracellular matrix genes are often associated with an increased risk of cataract and lens capsular rupture. However, the underlying mechanisms of cataract pathogenesis in these conditions are still unknown. Using two different mouse models, we show that the accumulation of collagen chains in the secretory pathway activates the stress signaling pathway termed unfolded protein response (UPR). Transgenic mice expressing ectopic Col4a3 and Col4a4 genes in the lens exhibited activation of IRE1, ATF6, and PERK associated with expansion of the endoplasmic reticulum and attenuation of general protein translation. The expression of the transgenes had adverse effects on lens fiber cell differentiation and eventually induced cell death in a group of transgenic fiber cells. In Col4a1+/?ex40 mutant mice, the accumulation of mutant chains also caused low levels of UPR activation. However, cell death was not induced in mutant lenses, suggesting that low levels of UPR activation are not proapoptotic. Collectively, the results provide in vivo evidence for a role of UPR in cataract formation in response to accumulation of terminally unfolded proteins in the endoplasmic reticulum.

Firtina, Zeynep; Danysh, Brian P.; Bai, Xiaoyang; Gould, Douglas B.; Kobayashi, Takehiro; Duncan, Melinda K.

2009-01-01

59

A molecular mechanism for glaucoma: endoplasmic reticulum stress and the unfolded protein response.  

PubMed

Primary open angle glaucoma (POAG) is a common late-onset neurodegenerative disease. Ocular hypertension represents a major risk factor, but POAG etiology remains poorly understood. Some cases of early-onset congenital glaucoma and adult POAG are linked to mutations in myocilin, a secreted protein of poorly defined function. Transgenic overexpression of myocilin in Drosophila and experiments in mice and human populations implicate the unfolded protein response (UPR) in the pathogenesis of glaucoma. We postulate that compromised ability of the UPR to eliminate misfolded mutant or damaged proteins, including myocilin, causes endoplasmic reticulum stress, resulting in functional impairment of trabecular meshwork cells that regulate intraocular pressure. This mechanism of POAG is reminiscent of other age-dependent neurodegenerative diseases that involve accumulation of protein aggregates. PMID:23876925

Anholt, Robert R H; Carbone, Mary Anna

2013-07-19

60

The mitochondrial unfolded protein response activator ATFS-1 protects cells from inhibition of the mevalonate pathway.  

PubMed

Statins are cholesterol-lowering drugs that inhibit 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase, the rate-limiting enzyme in the synthesis of cholesterol via the mevalonate pathway. This pathway also produces coenzyme Q (a component of the respiratory chain), dolichols (important for protein glycosylation), and isoprenoids (lipid moieties responsible for the membrane association of small GTPases). We previously showed that the nematode Caenorhabditis elegans is useful to study the noncholesterol effects of statins because its mevalonate pathway lacks the sterol synthesis branch but retains all other branches. Here, from a screen of 150,000 mutagenized genomes, we isolated four C. elegans mutants resistant to statins by virtue of gain-of-function mutations within the first six amino acids of the protein ATFS-1, the key regulator of the mitochondrial unfolded protein response that includes activation of the chaperones HSP-6 and HSP-60. The atfs-1 gain-of-function mutants are also resistant to ibandronate, an inhibitor of an enzyme downstream of HMG-CoA reductase, and to gliotoxin, an inhibitor acting on a subbranch of the pathway important for protein prenylation, and showed improved mitochondrial function and protein prenylation in the presence of statins. Additionally, preinduction of the mitochondrial unfolded protein response in wild-type worms using ethidium bromide or paraquat triggered statin resistance, and similar observations were made in Schizosaccharomyces pombe and in a mammalian cell line. We conclude that statin resistance through maintenance of mitochondrial homeostasis is conserved across species, and that the cell-lethal effects of statins are caused primarily through impaired protein prenylation that results in mitochondria dysfunction. PMID:23530189

Rauthan, Manish; Ranji, Parmida; Aguilera Pradenas, Nataly; Pitot, Christophe; Pilon, Marc

2013-03-25

61

Investigating the involvement of the ATF6? pathway of the unfolded protein response in adipogenesis  

PubMed Central

The unfolded protein response (UPR) is activated by endoplasmic reticulum stress resulting from an accumulation of unfolded or mis-folded proteins. The UPR is divided into three arms, involving the activation of ATF-6, PERK and IRE-1, that together act to restrict new protein synthesis and increase the production of chaperones. Recent studies have implicated the PERK and IRE-1 components of the UPR in adipocyte differentiation. In this study, we investigate the importance of ATF6? during adipogenesis using stable knockdown of this protein in the model adipogenic cell line, C3H10T1/2. Reduction of ATF6? expression by >70% resulted in impaired expression of key adipogenic genes and reduced lipid accumulation following the induction of adipogenesis. In contrast, loss of ATF6? did not impair the ability of cells to undergo osteogenic differentiation. Overall, our data indicate that all three arms of the UPR, including ATF6?, must be intact to permit adipogenesis to occur.

Lowe, C E; Dennis, R J; Obi, U; O'Rahilly, S; Rochford, J J

2012-01-01

62

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

63

The ire1 and ptc2 genes involved in the unfolded protein response pathway in the filamentous fungus Trichoderma reesei  

Microsoft Academic Search

A signal transduction pathway called the unfolded protein response is activated when increased levels of misfolded proteins or incorrectly assembled subunits accumulate in the endoplasmic reticulum (ER). The expression of several genes for ER-resident foldases and chaperones, as well as genes encoding proteins that are involved in functions associated with the secretory process, are induced by this pathway. This paper

M. Valkonen; M. Penttilä; M. Saloheimo

2004-01-01

64

Unfolded protein response is involved in the pathology of human congenital hypothyroid goiter and rat non-goitrous congenital hypothyroidism  

Microsoft Academic Search

The unfolded protein response (UPR) is an intracellular signaling pathway that regulates the protein folding and processing capacity of the endoplasmic reticulum (ER). The UPR is induced by the pharmacological agents that perturb ER functions but is also activated upon excessive accumulation of the mutant secretory proteins that are unable to attain correct three-dimensional structure and are thus retained in

M Baryshev; E Sargsyan; G Wallin; A Lejnieks; S Furudate; A Hishinuma; S Mkrtchian

2004-01-01

65

Zhangfei/CREB-ZF - A Potential Regulator of the Unfolded Protein Response  

PubMed Central

Cells respond to perturbations in the microenvironment of the endoplasmic reticulum (ER), and to the overloading of its capacity to process secretory and membrane-associate proteins, by activating the Unfolded Protein Response (UPR). Genes that mediate the UPR are regulated by three basic leucine-zipper (bLZip) motif-containing transcription factors – Xbp1s, ATF4 and ATF6. A failure of the UPR to achieve homeostasis and its continued stimulation leads to apoptosis. Mechanisms must therefore exist to turn off the UPR if it successfully restores normalcy. The bLZip protein Zhangfei/CREBZF/SMILE is known to suppress the ability of several, seemingly structurally unrelated, transcription factors. These targets include Luman/CREB3 and CREBH, ER-resident bLZip proteins known to activate the UPR in some cell types. Here we show that Zhangfei had a suppressive effect on most UPR genes activated by the calcium ionophore thapsigargin. This effect was at least partially due to the interaction of Zhangfei with Xbp1s. The leucine zipper of Zhangfei was required for this interaction, which led to the subsequent proteasomal degradation of Xbp1s. Zhangfei suppressed the ability of Xbp1s to activate transcription from a promoter containing unfolded protein response elements and significantly reduced the ability to Xbp1s to activate the UPR as measured by RNA and protein levels of UPR-related genes. Finally, specific suppression of endogenous Zhangfei in thapsigargin-treated primary rat sensory neurons with siRNA directed to Zhangfei transcripts, led to a significant increase in transcripts and proteins of UPR genes, suggesting a potential role for Zhangfei in modulating the UPR.

Zhang, Rui; Rapin, Noreen; Ying, Zhengxin; Shklanka, Erika; Bodnarchuk, Timothy W.; Verge, Valerie M. K.; Misra, Vikram

2013-01-01

66

Selective inhibition of the unfolded protein response: targeting catalytic sites for Schiff base modification.  

PubMed

Constitutive protein misfolding in the endoplasmic reticulum (ER) can lead to cellular toxicity and disease. Consequently, the protein folding environment within the ER is highly optimised and tightly regulated by the unfolded protein response (UPR). The apparent convergence of myriad diseases upon proteostasis in the ER has triggered a broad effort to identify selective inhibitors of the UPR. In particular, the most ancient component of this cellular stress pathway, the transmembrane protein IRE1, represents an appealing target for pharmacological intervention. Several inhibitors of IRE1 have recently been reported, each containing an aldehyde moiety that forms an unusual, highly selective Schiff base with a single key lysine (K907) within the RNase domain. Here we review the progress made in chemical genetic manipulation of IRE1 and the unfolded protein response and discuss computational strategies to rationalise the selectivity of covalently active small molecules for their targets. As an exemplar, we provide additional evidence that K907 of IRE1 is buried within a particularly unusual environment that facilitates Schiff base formation. New free-energy calculations within a molecular dynamics (MD) simulation framework show that the pKa of K907 is reduced by ?3.6 pKa units, relative to the model pKa of lysine in water. This significant pKa perturbation provides additional insights into the precise requirements for inhibition and for RNase catalysis by IRE1. Our computational method may represent a general approach for identifying potential covalent inhibitory lysine sites within buried protein cavities. PMID:23884086

Tomasio, Susana M; Harding, Heather P; Ron, David; Cross, Benedict C S; Bond, Peter J

2013-08-27

67

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

68

Unfolded protein response in keratinocytes: impact on normal and abnormal keratinization.  

PubMed

The unfolded protein response (UPR) is a signaling pathway from the endoplasmic reticulum (ER) to the nucleus that protects cells from stress caused by misfolded or unfolded proteins. As such, ER stress is an ongoing challenge for all cells, given the central biologic importance of secretion as part of normal physiologic functions. Mild UPR is activated by mild ER stress, which occurs under normal conditions. Abnormal UPR is activated by severe ER stress, which occurs under pathological conditions. Abnormal UPR activation is associated with a number of diseases, including diabetes mellitus and Alzheimer's disease. Within skin tissues, keratinocytes in the epidermis are especially dependent upon a mild UPR for normal differentiation in the course of their differentiation into secretory cells in the uppermost granular layers. Association between abnormal UPR activation and hereditary keratoses, including Darier's disease, keratosis linearis with ichthyosis congenita and keratoderma syndrome, erythrokeratoderma variabilis, and ichthyosis follicularis with atrichia and photophobia syndrome, have been elucidated recently. This review describes the UPR in normal and abnormal keratinization and discusses the regulation of abnormal UPR activation by chemical chaperones as a potential treatment for one of the hereditary keratoses. PMID:23352280

Sugiura, Kazumitsu

2013-01-04

69

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

70

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

71

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

72

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

73

Targeting the unfolded protein response in neurodegeneration: A new approach to therapy.  

PubMed

Neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and the rarer prion diseases, share a number of key similarities, including aggregation of disease-specific proteins in the brain and neuronal loss. The focus of research in these disorders has centred on pathogenesis caused by individual proteins and their build up in their specific diseases, but there are also likely to be more generic pathways that are active in neurodegeneration across the spectrum of these disorders. The unfolded protein response (UPR) has recently emerged as one such pathway. The UPR is normally a protective cellular response that protects against endoplasmic reticulum (ER) stress, which occurs with the build up of misfolded proteins. Recent evidence indicates that in neurodegenerative disease this pathway becomes constitutively activated, preventing protein translation. UPR activation is found in post mortem brains in a variety of diseases, including AD, PD and prion diseases, and has also been found to be activated in mouse models of neurodegeneration and in various in vitro models. We propose that modulation of the UPR in neurodegeneration is therefore a promising target for future therapeutic treatments. This article is part of the Special Issue entitled 'The Synaptic Basis of Neurodegenerative Disorders'. PMID:24035917

Halliday, Mark; Mallucci, Giovanna R

2013-09-10

74

Exposure to tributyltin induces endoplasmic reticulum stress and the unfolded protein response in zebrafish.  

PubMed

Tributyltin (TBT) is a major marine contaminant and causes endocrine disruption, hepatotoxicity, immunotoxicity, and neurotoxicity. However, the molecular mechanisms underlying the toxicity of TBT have not been fully elucidated. We examined whether exposure to TBT induces the endoplasmic reticulum (ER) stress response in zebrafish, a model organism. Zebrafish-derived BRF41 fibroblast cells were exposed to 0.5 or 1?M TBT for 0.5-16h and subsequently lysed and immunoblotted to detect ER stress-related proteins. Zebrafish embryos, grown until 32h post fertilization (hpf), were exposed to 1?M TBT for 16h and used in whole mount in situ hybridization and immunohistochemistry to visualize the expression of ER chaperones and an ER stress-related apoptosis factor. Exposure of the BRF41 cells to TBT caused phosphorylation of the zebrafish homolog of protein kinase RNA-activated-like ER kinase (PERK), eukaryotic translation initiation factor 2 alpha (eIF2?), and inositol-requiring enzyme 1 (IRE1), characteristic splicing of X-box binding protein 1 (XBP1) mRNA, and enhanced expression of activating transcription factor 4 (ATF4) protein. In TBT-exposed zebrafish embryos, ectopic expression of the gene encoding zebrafish homolog of the 78kDa glucose-regulating protein (GRP78) and gene encoding CCAAT/enhancer-binding protein homologous protein (CHOP) was detected in the precursors of the neuromast, which is a sensory organ for detecting water flow and vibration. Our in vitro and in vivo studies revealed that exposure of zebrafish to TBT induces the ER stress response via activation of both the PERK-eIF2? and IRE1-XBP1 pathways of the unfolded protein response (UPR) in an organ-specific manner. PMID:24055755

Komoike, Yuta; Matsuoka, Masato

2013-09-04

75

Vanadyl bisacetylacetonate protects ? cells from palmitate-induced cell death through the unfolded protein response pathway.  

PubMed

Endoplasmic reticulum (ER) stress induced by free fatty acids (FFA) is important to ?-cell loss during the development of type 2 diabetes. To test whether vanadium compounds could influence ER stress and the responses in their mechanism of antidiabetic effects, we investigated the effects and the mechanism of vanadyl bisacetylacetonate [VO(acac)(2)] on ? cells upon treatment with palmitate, a typical saturated FFA. The experimental results showed that VO(acac)(2) could enhance FFA-induced signaling pathways of unfolded protein responses by upregulating the prosurvival chaperone immunoglobulin heavy-chain binding protein/78-kDa glucose-regulated protein and downregulating the expression of apoptotic C/EBP homologous protein, and consequently the reduction of insulin synthesis. VO(acac)(2) also ameliorated FFA-disturbed Ca(2+) homeostasis in ? cells. Overall, VO(acac)(2) enhanced stress adaption, thus protecting ? cells from palmitate-induced apoptosis. This study provides some new insights into the mechanisms of antidiabetic vanadium compounds. PMID:21512771

Gao, Zhonglan; Zhang, Chengyue; Yu, Siwang; Yang, Xiaoda; Wang, Kui

2011-04-22

76

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

2013-08-14

77

Overexpression of human virus surface glycoprotein precursors induces cytosolic unfolded protein response in Saccharomyces cerevisiae  

PubMed Central

Background The expression of human virus surface proteins, as well as other mammalian glycoproteins, is much more efficient in cells of higher eukaryotes rather than yeasts. The limitations to high-level expression of active viral surface glycoproteins in yeast are not well understood. To identify possible bottlenecks we performed a detailed study on overexpression of recombinant mumps hemagglutinin-neuraminidase (MuHN) and measles hemagglutinin (MeH) in yeast Saccharomyces cerevisiae, combining the analysis of recombinant proteins with a proteomic approach. Results Overexpressed recombinant MuHN and MeH proteins were present in large aggregates, were inactive and totally insoluble under native conditions. Moreover, the majority of recombinant protein was found in immature form of non-glycosylated precursors. Fractionation of yeast lysates revealed that the core of viral surface protein aggregates consists of MuHN or MeH disulfide-linked multimers involving eukaryotic translation elongation factor 1A (eEF1A) and is closely associated with small heat shock proteins (sHsps) that can be removed only under denaturing conditions. Complexes of large Hsps seem to be bound to aggregate core peripherally as they can be easily removed at high salt concentrations. Proteomic analysis revealed that the accumulation of unglycosylated viral protein precursors results in specific cytosolic unfolded protein response (UPR-Cyto) in yeast cells, characterized by different action and regulation of small Hsps versus large chaperones of Hsp70, Hsp90 and Hsp110 families. In contrast to most environmental stresses, in the response to synthesis of recombinant MuHN and MeH, only the large Hsps were upregulated whereas sHsps were not. Interestingly, the amount of eEF1A was also increased during this stress response. Conclusions Inefficient translocation of MuHN and MeH precursors through ER membrane is a bottleneck for high-level expression in yeast. Overexpression of these recombinant proteins induces the UPR's cytosolic counterpart, the UPR-Cyto, which represent a subset of proteins involved in the heat-shock response. The involvement of eEF1A may explain the mechanism by which only large chaperones, but not small Hsps are upregulated during this stress response. Our study highlights important differences between viral surface protein expression in yeast and mammalian cells at the first stage of secretory pathway.

2011-01-01

78

Integration of the Unfolded Protein and Oxidative Stress Responses through SKN-1/Nrf  

PubMed Central

The Unfolded Protein Response (UPR) maintains homeostasis in the endoplasmic reticulum (ER) and defends against ER stress, an underlying factor in various human diseases. During the UPR, numerous genes are activated that sustain and protect the ER. These responses are known to involve the canonical UPR transcription factors XBP1, ATF4, and ATF6. Here, we show in C. elegans that the conserved stress defense factor SKN-1/Nrf plays a central and essential role in the transcriptional UPR. While SKN-1/Nrf has a well-established function in protection against oxidative and xenobiotic stress, we find that it also mobilizes an overlapping but distinct response to ER stress. SKN-1/Nrf is regulated by the UPR, directly controls UPR signaling and transcription factor genes, binds to common downstream targets with XBP-1 and ATF-6, and is present at the ER. SKN-1/Nrf is also essential for resistance to ER stress, including reductive stress. Remarkably, SKN-1/Nrf-mediated responses to oxidative stress depend upon signaling from the ER. We conclude that SKN-1/Nrf plays a critical role in the UPR, but orchestrates a distinct oxidative stress response that is licensed by ER signaling. Regulatory integration through SKN-1/Nrf may coordinate ER and cytoplasmic homeostasis.

Glover-Cutter, Kira M.; Lin, Stephanie; Blackwell, T. Keith

2013-01-01

79

Transmembrane bZIP transcription factors in ER stress signaling and the unfolded protein response.  

PubMed

Regulated intramembrane proteolysis (RIP) of the transmembrane transcription factor ATF6 represents a key step in effecting adaptive response to the presence of unfolded or malfolded protein in the endoplasmic reticulum. Recent studies have highlighted new ATF6-related transmembrane transcription factors. It is likely that current models for ER stress signaling are incomplete and that the expansion of the bZIP transmembrane family reflects selectivity in many aspects of these responses, including the type and duration of any particular stress, the cell type in which it occurs, and the integration with other aspects of cell-type-specific organization or additional intrinsic pathways, and the integration and communication between these pathways, not only in a cell-type-specific manner, but also between different tissues and organs. This review summarizes current information on the bZIP-transmembrane proteins and discusses outstanding questions on the elucidation of the stress signals, the repertoire of components involved in regulating different aspects of the forward transport, cleavage, nuclear import, transcriptional activity, and turnover of each of these factors, and dissection of the integration of the various outputs into broad coordinated responses. PMID:17887918

Bailey, Daniel; O'Hare, Peter

2007-12-01

80

Recombinant Antibody Production in Arabidopsis Seeds Triggers an Unfolded Protein Response1[W][OA  

PubMed Central

Among the many plant-based production systems that are being tested for molecular farming, seeds are very attractive, as they provide a stable environment in which the accumulating recombinant proteins can be stored. However, it is not known exactly how high production levels of recombinant antibodies influence the endogenous transcriptome and proteome of the developing seed. To address this question, we studied the transcriptomic status in developing Arabidopsis (Arabidopsis thaliana) seeds 13 d post anthesis of three transgenic lines, producing varying levels of recombinant VHH or single-chain Fv antibody fragments fused to the human immunoglobulin G1-derived Fc fragment under the control of the ?-PHASEOLIN seed-specific promoter. Using genome-wide Tiling arrays, we demonstrated that only a small proportion of the transcriptome was significantly changed in each of the lines compared with the wild type. Strikingly, in all three lines, we found a large overlap of up-regulated genes corresponding to protein folding, glycosylation/modification, translocation, vesicle transport, and protein degradation, suggestive of a state of cellular stress called the unfolded protein response. Moreover, the gene up-regulation amplitude was similar in all three lines. We hypothesize that the production of recombinant antibodies in the endoplasmic reticulum triggers endoplasmic reticulum stress, causing a disturbance of the normal cellular homeostasis.

De Wilde, Kirsten; De Buck, Sylvie; Vanneste, Kevin; Depicker, Ann

2013-01-01

81

Stress management: How the unfolded protein response impacts fatty liver disease.  

PubMed

Induction of the unfolded protein response (UPR) is recognized as central to fatty liver disease (FLD) pathophysiology. This pathway may be a potential therapeutic target for FLD, as well as other diseases. However, fundamental questions as to how UPR contributes to FLD remain unanswered. Conflicting data suggest that this pathway can both protect against and augment this disease. Here, we review the relationship between protein secretion, endoplasmic reticulum function (ER), and UPR activation. The UPR serves to maintain secretory pathway homeostasis by enhancing the protein folding environment in the ER, and we review data investigating the role for individual UPR players in fatty liver (steatosis). We explore a novel concept in the field that all cases of UPR activation do not equal "ER stress". Rather, different types of UPRs that can either protect against or cause FLD are discussed. Refining our current understanding of this complex pathway is particularly important, as drugs that affect the protein folding environment in the ER and affect UPR activation are being successful in clinical trials for FLD. PMID:22732510

Imrie, Dru; Sadler, Kirsten C

2012-06-22

82

Recombinant antibody production in Arabidopsis seeds triggers an unfolded protein response.  

PubMed

Among the many plant-based production systems that are being tested for molecular farming, seeds are very attractive, as they provide a stable environment in which the accumulating recombinant proteins can be stored. However, it is not known exactly how high production levels of recombinant antibodies influence the endogenous transcriptome and proteome of the developing seed. To address this question, we studied the transcriptomic status in developing Arabidopsis (Arabidopsis thaliana) seeds 13 d post anthesis of three transgenic lines, producing varying levels of recombinant VHH or single-chain Fv antibody fragments fused to the human immunoglobulin G1-derived Fc fragment under the control of the ?-PHASEOLIN seed-specific promoter. Using genome-wide Tiling arrays, we demonstrated that only a small proportion of the transcriptome was significantly changed in each of the lines compared with the wild type. Strikingly, in all three lines, we found a large overlap of up-regulated genes corresponding to protein folding, glycosylation/modification, translocation, vesicle transport, and protein degradation, suggestive of a state of cellular stress called the unfolded protein response. Moreover, the gene up-regulation amplitude was similar in all three lines. We hypothesize that the production of recombinant antibodies in the endoplasmic reticulum triggers endoplasmic reticulum stress, causing a disturbance of the normal cellular homeostasis. PMID:23188806

De Wilde, Kirsten; De Buck, Sylvie; Vanneste, Kevin; Depicker, Ann

2012-11-27

83

PKR Protects Colonic Epithelium Against Colitis Through the Unfolded Protein Response and Prosurvival Signaling  

PubMed Central

Background The dsRNA-activated protein kinase (PKR) phosphorylates the ? subunit of eukaryotic translation initiation factor 2 (eIF2?), a global regulator of protein synthesis in mammals. In addition, PKR activates several signal transduction pathways including STAT3 and AKT. PKR is activated by a number of inflammatory stimuli that are induced in the inflamed intestine. In this study we intended to determine the role of PKR in colonic epithelial cells during experimental colitis in mice. Methods Age- and sex-matched PKR+/+,+/? and PKR?/? littermate mice were reconstituted with wildtype bone marrow cells and subjected to dextran sodium sulfate (DSS)-induced colitis. Results PKR?/? mice displayed more severe clinical and histological manifestations upon DSS colitis compared with their PKR+/+,+/? litter-mates. In response to DSS colitis, the colonic epithelial cells of PKR?/? mice exhibited impaired activation of the unfolded protein response (UPR) signaling, including eIF2? phosphorylation, endoplasmic reticulum (ER) chaperone response, and ER-associated degradation (ERAD) components, as well as antioxidative stress response. In addition, the phosphorylation of STAT3 and AKT, which are protective against epithelial cell death and colonic inflammation, was also impaired in the colonic epithelial cells of PKR?/? mice upon DSS colitis. Conclusions These data demonstrate that PKR is a physiologically relevant transducer of inflammatory response signaling in colonic epithelial cells. PKR may promote the homeostasis and survival of intestinal epithelial cells (IECs) through eIF2?-mediated UPR activation, as well as the activation of STAT3 and AKT pathways. In the absence of PKR, the survival and proliferation of IECs was impaired, thus exacerbating intestinal inflammation.

Cao, Stewart Siyan; Song, Benbo; Kaufman, Randal J.

2013-01-01

84

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

PubMed Central

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 that SCI triggers a fast ER stress reaction (1–3?h) involving the upregulation of key components of the unfolded protein response (UPR), a process that propagates through the spinal cord. Ablation of X-box-binding protein 1 (XBP1) or activating transcription factor 4 (ATF4) expression, two major UPR transcription factors, leads to a reduced locomotor recovery after experimental SCI. The effects of UPR inactivation were associated with a significant increase in the number of damaged axons and reduced amount of oligodendrocytes surrounding the injury zone. In addition, altered microglial activation and pro-inflammatory cytokine expression were observed in ATF4 deficient mice after SCI. Local expression of active XBP1 into the spinal cord using adeno-associated viruses enhanced locomotor recovery after SCI, and was associated with an increased number of oligodendrocytes. Altogether, our results demonstrate a functional role of the UPR in SCI, offering novel therapeutic targets to treat this invalidating condition.

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

2012-01-01

85

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

PubMed

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 that SCI triggers a fast ER stress reaction (1-3 h) involving the upregulation of key components of the unfolded protein response (UPR), a process that propagates through the spinal cord. Ablation of X-box-binding protein 1 (XBP1) or activating transcription factor 4 (ATF4) expression, two major UPR transcription factors, leads to a reduced locomotor recovery after experimental SCI. The effects of UPR inactivation were associated with a significant increase in the number of damaged axons and reduced amount of oligodendrocytes surrounding the injury zone. In addition, altered microglial activation and pro-inflammatory cytokine expression were observed in ATF4 deficient mice after SCI. Local expression of active XBP1 into the spinal cord using adeno-associated viruses enhanced locomotor recovery after SCI, and was associated with an increased number of oligodendrocytes. Altogether, our results demonstrate a functional role of the UPR in SCI, offering novel therapeutic targets to treat this invalidating condition. PMID:22337234

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

2012-02-16

86

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

2012-08-06

87

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

88

Nitrosative stress-induced s-glutathionylation of protein disulfide isomerase leads to activation of the unfolded protein response.  

PubMed

The rapid proliferation of cancer cells mandates a high protein turnover. The endoplasmic reticulum (ER) is intimately involved in protein processing. An accumulation of unfolded or misfolded proteins in the ER leads to a cascade of transcriptional and translational events collectively called the unfolded protein response (UPR). Protein disulfide isomerase (PDI) is one of the most abundant ER proteins and maintains a sentinel function in organizing accurate protein folding. Treatment of cells with O(2)-[2,4-dinitro-5-(N-methyl-N-4-carboxyphenylamino)phenyl]1-(N,N-dimethylamino)diazen-1-ium-1,2-diolate (PABA/NO) resulted in a dose-dependent increase in intracellular nitric oxide that caused S-glutathionylation of various proteins. Within 4 h, PABA/NO activated the UPR and led to translational attenuation as measured by the phosphorylation and activation of the ER transmembrane kinase, pancreatic ER kinase, and its downstream effector eukaryotic initiation factor 2 in human leukemia (HL60) and ovarian cancer cells (SKOV3). Cleavage of the transcription factor X-box protein 1 and transcriptional activation of the ER resident proteins BiP, PDI, GRP94, and ERO1 (5- to 10-fold induction) also occurred. Immunoprecipitation of PDI showed that whereas nitrosylation was undetectable, PABA/NO treatment caused S-glutathionylation of PDI. Mass spectroscopy analysis showed that single cysteine residues within each of the catalytic sites of PDI had a mass increase [+305.3 Da] consistent with S-glutathionylation. Circular dichroism confirmed that S-glutathionylation of PDI results in alterations in the alpha-helix content of PDI and is concurrent with inhibition of its isomerase activity. Thus, it appears that S-glutathionylation of PDI is an upstream signaling event in the UPR and may be linked with the cytotoxic potential of PABA/NO. PMID:19773442

Townsend, Danyelle M; Manevich, Yefim; He, Lin; Xiong, Ying; Bowers, Robert R; Hutchens, Steven; Tew, Kenneth D

2009-09-22

89

Chlorine induces the unfolded protein response in murine lungs and skin.  

PubMed

Chlorine (Cl2) is an important industrial chemical. Accidental full body exposure to Cl2 poses an environmental, occupational, and public health hazard characterized mainly by injury to the lung, skin, and ocular epithelia. The cellular mechanisms underlying its acute toxicity are incompletely understood. This study examined whether whole body exposure of BALB/c mice to Cl2 in environmental chambers leads to the up-regulation of the unfolded protein response (UPR) in their lungs and skin. Shaved BALB/c mice were exposed to a sublethal concentration of Cl2 (400 ppm for 30 min) and returned to room air for 1 or 6 hours and killed. IL-6 and TNF-? were increased significantly at 1 and 6 hours after Cl2 exposure in the lungs and at 6 hours in the skin. These changes were accompanied by increased UPR signaling (i.e., activation of protein kinase RNA-like endoplasmic reticulum kinase, inositol-requiring enzyme 1 ?, and activating transcription factor 6?) at these time points. The expression of hepcidin, which regulates tissue accumulation and mobilization of iron, was increased in the skin and lungs of Cl2-exposed mice. The data shown herein indicate for the first time the up-regulation of UPR signaling and hepcidin in the skin and lungs of Cl2-exposed mice, which persisted when the mice were returned to room air for 6 hours. PMID:23668485

Li, Changzhao; Weng, Zhiping; Doran, Stephen F; Srivastava, Ritesh K; Afaq, Farrukh; Matalon, Sadis; Athar, Mohammad

2013-08-01

90

Using temporal genetic switches to synchronize the unfolded protein response in cell populations in vivo  

PubMed Central

In recent years, recognition of the importance of protein aggregation in human diseases has increasingly come to the fore and it is clear that many degenerative disorders involve activation of a metabolic signaling cascade known as the unfolded protein response (UPR). The UPR encompasses conserved mechanisms in cells to monitor and react to changes in metabolic flux through the secretory pathway. Such changes reflect an imbalance in cell homeostasis and the UPR integrates several signaling cascades to restore homeostasis. As such, the UPR is simply interpreted as a protection mechanism for cells as they perform their normal functions. A number of groups have suggested that the UPR also can eliminate cells in which homeostasis is lost, for example during disease. This notion has kindled the rather paradoxical concept that inhibiting the UPR will ameliorate degenerative disease. However, several in vivo studies in the nervous system indicate that curtailing UPR function either exacerbates disease or may reduce severity through unexpected or unidentified pathways. Perhaps the notion that the UPR protects cells or eliminates them stems from widespread use of suboptimal paradigms to characterize the UPR; thus, too little is currently known about this homeostatic pathway. Herein, I describe the development of genetic switch technology (GST) to generate a novel model for studying UPR diseases. The model is geared toward obtaining high resolution in vivo detail for oligodendrocytes of the central nervous system, but it can be adapted to study other cell types and other UPR diseases.

Gow, Alexander

2011-01-01

91

Signalling danger: endoplasmic reticulum stress and the unfolded protein response in pancreatic islet inflammation.  

PubMed

Protein synthesis is increased by several-fold in stimulated pancreatic beta cells. Synthesis and folding of (pro)insulin takes place in the endoplasmic reticulum (ER), and beta cells trigger the unfolded protein response (UPR) to upgrade the functional capacity of the ER. Prolonged or excessive UPR activation contributes to beta cell dysfunction and death in type 2 diabetes, but there is another side of the UPR that may be of particular relevance for autoimmune type 1 diabetes, namely, the cross-talk between the UPR and innate immunity/inflammation. Recent evidence, discussed in this review, indicates that both saturated fats and inflammatory mediators such as cytokines trigger the UPR in pancreatic beta cells. The UPR potentiates activation of nuclear factor ?B, a key regulator of inflammation. Two branches of the UPR, namely IRE1/XBP1s and PERK/ATF4/CHOP, mediate the UPR-induced sensitisation of pancreatic beta cells to the proinflammatory effects of cytokines. This can contribute to the upregulation of local inflammatory mechanisms and the aggravation of insulitis. The dialogue between the UPR and inflammation may provide an explanation for the parallel increase in the prevalence of childhood obesity and type 1 diabetes. PMID:23132339

Eizirik, D L; Miani, M; Cardozo, A K

2012-11-07

92

The Unfolded Protein State Revisited  

Microsoft Academic Search

Most studies on proteins have centered on the conformation and stability of the folded state. The unfolded state has essentially\\u000a been neglected because of its reputation of being devoid of biological function, and not well-defined. Recently the importance\\u000a of unfolded segments, as part of the secondary structure of globular proteins and their role in the performance of biological\\u000a functions, has

Patricio A. Carvajal; Tyre C. Lanier

93

Unfolded Protein Response Signaling and MAP Kinase Pathways Underlie Pathogenesis of Arsenic-induced Cutaneous Inflammation  

PubMed Central

Arsenic exposure through drinking water is a major global public health problem and is associated with an enhanced risk of various cancers including skin cancer. In human skin, arsenic induces precancerous melanosis and keratosis, which may progress to basal cell and squamous cell carcinoma. However, the mechanism by which these pathophysiological alterations occur remains elusive. In this study, we showed that sub-chronic arsenic exposure to SKH-1 mice induced unfolded protein response (UPR) signaling regulated by proteins, inositol-requiring enzyme-1 (IRE1), PKR-like endoplasmic reticulum kinase (PERK) and activating transcription factor 6 (ATF6). Arsenic activated all three UPR regulatory proteins in the skin. Arsenic induced IRE1 phosphorylation which resulted in augmented splicing of X-box binding protein 1 (XBP-1) leading to its migration to the nucleus, and also enhanced transcriptional activation of downstream target proteins. Hyperphosphorylation of PERK which induces eukaryotic translation initial factor 2 ? (eIF2?) in a phosphorylation-dependent manner enhanced translation of ATF4, in addition to augmenting proteolytic activation of ATF6 in arsenic-treated skin. A similar increase in the expression of CHOP was observed. Enhanced XBP-1s, ATF4 and ATF6 regulated downstream chaperones GRP94 and GRP78. Additionally, arsenic induced inflammation-related p38/MAPKAPK-2 MAPK signaling and alterations in Th-1/Th-2/Th-17 cytokines/chemokines and their receptors. Antioxidant N-acetyl cysteine blocked arsenic-induced reactive oxygen species, with a concomitant attenuation of UPR and MAPK signaling and pro-inflammatory cytokine/chemokine signatures. Our results identify novel pathways involved in the pathogenesis of arsenic-mediated cutaneous inflammation which may also be related to enhanced cancer risk in arsenic exposed cohorts.

Li, Changzhao; Xu, Jianmin; Li, Fugui; Chaudhary, Sandeep C.; Weng, Zhiping; Wen, Jianming; Elmets, Craig A.; Ahsan, Habibul; Athar, Mohammad

2011-01-01

94

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

95

Antiviral Activity of a Small Molecule Deubiquitinase Inhibitor Occurs via Induction of the Unfolded Protein Response  

PubMed Central

Ubiquitin (Ub) is a vital regulatory component in various cellular processes, including cellular responses to viral infection. As obligate intracellular pathogens, viruses have the capacity to manipulate the ubiquitin (Ub) cycle to their advantage by encoding Ub-modifying proteins including deubiquitinases (DUBs). However, how cellular DUBs modulate specific viral infections, such as norovirus, is poorly understood. To examine the role of DUBs during norovirus infection, we used WP1130, a small molecule inhibitor of a subset of cellular DUBs. Replication of murine norovirus in murine macrophages and the human norovirus Norwalk virus in a replicon system were significantly inhibited by WP1130. Chemical proteomics identified the cellular DUB USP14 as a target of WP1130 in murine macrophages, and pharmacologic inhibition or siRNA-mediated knockdown of USP14 inhibited murine norovirus infection. USP14 is a proteasome-associated DUB that also binds to inositol-requiring enzyme 1 (IRE1), a critical mediator of the unfolded protein response (UPR). WP1130 treatment of murine macrophages did not alter proteasome activity but activated the X-box binding protein-1 (XBP-1) through an IRE1-dependent mechanism. In addition, WP1130 treatment or induction of the UPR also reduced infection of other RNA viruses including encephalomyocarditis virus, Sindbis virus, and La Crosse virus but not vesicular stomatitis virus. Pharmacologic inhibition of the IRE1 endonuclease activity partially rescued the antiviral effect of WP1130. Taken together, our studies support a model whereby induction of the UPR through cellular DUB inhibition blocks specific viral infections, and suggest that cellular DUBs and the UPR represent novel targets for future development of broad spectrum antiviral therapies.

Perry, Jeffrey W.; Ahmed, Mohammad; Chang, Kyeong-Ok; Donato, Nicholas J.; Showalter, Hollis D.; Wobus, Christiane E.

2012-01-01

96

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

97

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

98

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

99

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

100

Examination of a second node of translational control in the unfolded protein response.  

PubMed

The unfolded protein response (UPR) is a largely cytoprotective signaling cascade that acts to re-establish homeostasis of the endoplasmic reticulum (ER) under conditions of stress by inducing an early and transient block in general protein synthesis and by increasing the folding and degradative capacity of the cell through an extensive transcriptional program. It is well established that the mechanism for the early translational attenuation during ER stress occurs through phosphorylation of eukaryotic initiation factor 2 ? (eIF2?) by activated PERK. Our data demonstrate that when eIF2? is dephosphorylated translation is not fully restored to pre-stressed levels. We found that this correlates with reduced mTOR activity and as a result decreases phosphorylation of 4E-BP1, which negatively regulates assembly of the eIF4F complex and cap-dependent translation. The decrease in mTOR activity and 4E-BP1 phosphorylation is associated with activation of AMP kinase, a negative regulator of mTOR, and in the case of some stress conditions, downregulation of signaling through key components of the PI3K pathway. Furthermore, we show that there is a subset of mRNAs that does not recover from UPR-induced translational repression, including those whose translation is particularly sensitive to loss of eIF4F, such as cyclin D1, Bcl-2 and MMP-9. Together these data implicate reduced mTOR activity and 4E-BP1 hypophosphorylation as a second, more restricted mechanism of translational control occurring somewhat later in the UPR. PMID:23843622

Preston, Amanda M; Hendershot, Linda M

2013-07-10

101

Unfolded protein response regulates cardiac sodium current in systolic human heart failure.  

PubMed

Background- Human heart failure (HF) increases alternative mRNA splicing of the type V, voltage-gated cardiac Na(+) channel ?-subunit (SCN5A), generating variants encoding truncated, nonfunctional channels that are trapped in the endoplasmic reticulum. In this work, we tested whether truncated Na(+) channels activate the unfolded protein response (UPR), contributing to SCN5A electric remodeling in HF. Methods and Results- UPR and SCN5A were analyzed in human ventricular systolic HF tissue samples and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Cells were exposed to angiotensin II (AngII) and hypoxia, known activators of abnormal SCN5A mRNA splicing, or were induced to overexpress SCN5A variants. UPR effectors, protein kinase R-like ER kinase (PERK), calreticulin, and CHOP, were increased in human HF tissues. Induction of SCN5A variants with AngII or hypoxia or the expression of exogenous variants induced the UPR with concomitant downregulation of Na(+) current. PERK activation destabilized SCN5A and, surprisingly, Kv4.3 channel mRNAs but not transient receptor potential cation channel M7 (TRPM7) channel mRNA. PERK inhibition prevented the loss of full-length SCN5A and Kv4.3 mRNA levels resulting from expressing Na(+) channel mRNA splice variants. Conclusions- UPR can be initiated by Na(+) channel mRNA splice variants and is involved in the reduction of cardiac Na(+) current during human HF. Because the effect is not entirely specific to the SCN5A transcript, the UPR may play an important role in downregulation of multiple cardiac genes in HF. PMID:24036084

Gao, Ge; Xie, An; Zhang, Jianhua; Herman, Amanda M; Jeong, Euy-Myoung; Gu, Lianzhi; Liu, Man; Yang, Kai-Chien; Kamp, Timothy J; Dudley, Samuel C

2013-09-13

102

Compromising the Unfolded Protein Response Induces Autophagy-Mediated Cell Death in Multiple Myeloma Cells  

PubMed Central

Objective To determine whether the Unfolded Protein Response (UPR) sensors (PERK, ATF6 and IRE-1) can be targeted to promote death of Multiple Myeloma (MM) cells. Methods We have knocked-down separately each UPR stress sensor in human MM cell lines using RNA interference and followed MM cell death by monitoring the membrane, mitochondrial and nuclear alterations. Involvement of caspases in MM cell death consecutive to UPR sensor knock-down was analyzed by western blotting, measurement of their enzymatic activity using fluorigenic substrates and susceptibility to a pan-caspase inhibitor. Activation of the autophagic process was measured directly by detection of autophagosomes (electronic microscopy), monodansylcadaverine staining, production of the cleaved form of the microtubule-associated protein 1A/1B light chain 3 (LC3) and indirectly by analyzing the impact of pharmacological inhibitors of autophagy such as 3MA and bafilomycin A1. Results We show that extinction of a single UPR stress sensor (PERK) induces a non-apoptotic form of cell death in MM cells that requires autophagy for its execution. We also show that this cytotoxic autophagic process represses the apoptosis program by reducing the cytosolic release of the apoptogenic factors Smac/DIABLO and cytochrome c. Interpretation Altogether our findings suggest that autophagy can contribute to execution of death in mammalian cells that are exposed to mild ER stress. They also suggest that the autophagic process can regulate the intrinsic apoptotic pathway by inhibiting production of death effectors by the mitochondria, thus preventing formation of a functional apoptosome. Altogether these findings give credit to the idea that UPR sensors can be envisaged as therapeutic targets for the treatment of MM.

Michallet, Anne-Sophie; Mondiere, Paul; Taillardet, Morgan; Leverrier, Yann; Genestier, Laurent; Defrance, Thierry

2011-01-01

103

The transcription factor HACA mediates the unfolded protein response in Aspergillus niger , and up-regulates its own transcription  

Microsoft Academic Search

The unfolded protein response (UPR) involves a complex signalling pathway in which the transcription factor HACA plays a central role. Here we report the cloning and characterisation of the hacA gene and its product from Aspergillus niger. ER (endoplasmic reticulum) stress results in the splicing of an unconventional 20-nt intron from the A. niger hacA mRNA, and is associated with

H. J. Mulder; M. Saloheimo; M. Penttilä; S. M. Madrid

2004-01-01

104

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

105

Regulation of immunoglobulin synthesis, modification, and trafficking by the unfolded protein response a quantitative approach.  

PubMed

Plasma cells are professional secretory cells, which function as cellular factories for immunoglobulin synthesis and secretion. Being the sole cell type responsible for antibody secretion they play an essential role in the immune response against a broad spectrum of pathogens. Since plasma cells have a long life span and are able to secrete copious amounts of antibody, their number and repertoire should be tightly regulated. Disruption of their homeostasis may lead to severe diseases, such as immunodeficiency or multiple myeloma. Much of the complications of multiple myeloma are attributed to the antibodies themselves, which accumulate in the bloodstream and lead to kidney and pulmonary insufficiencies. Similar pathologies are common to other plasma cell-related diseases, such as AL amyloidosis and autoimmune diseases, in which Ig molecules accumulate to toxic levels without good means to curtail their production. The process of plasma cell differentiation and maintenance is poorly understood. The discovery that the IRE1/XBP-1 arm of the unfolded protein response (UPR) is necessary to yield full-fledged plasma cells in vivo was a breakthrough in the field. Over the years valuable biochemical information on plasma cell differentiation was obtained by exploring the downstream activities of XBP-1. The most pronounced phenotype of XBP-1 deficiency in plasma cells in vitro is the steep reduction in ? chain synthesis albeit similar levels of its mRNA. Remarkably, the defect is specific to Ig heavy chains as synthesis of other glycoproteins remains normal. Furthermore, when XBP-1 is absent or its mRNA splicing is inhibited the efficiency of protein translocation into the ER is severely impaired. Still, fundamental questions remain unanswered, such as what exactly generates the conditions of endoplasmic reticulum (ER) stress that activates the UPR in the developing plasma cells. Another enigma is how lipid biosynthesis and protein synthesis, both dramatically modulated during differentiation, are coordinated. In this chapter, we will provide detailed methodologies for measurements of Ig synthesis and misinsertion into the ER as readout of ER physiology in the course of plasma cell differentiation. PMID:21329807

Drori, Adi; Tirosh, Boaz

2011-01-01

106

ARMET is a soluble ER protein induced by the unfolded protein response via ERSE-II element.  

PubMed

Arginine rich, mutated in early stage of tumors (ARMET) was first identified as a human gene highly mutated in a variety of cancers. However, little is known about the characteristics of the ARMET protein and its expression. We identified ARMET as a gene upregulated by endoplasmic reticulum (ER) stress. Here, we show that the mouse homologue of ARMET is an 18-kDa soluble ER protein that is mature after cleavage of a signal sequence and has four intramolecular disulfide bonds, including two in CXXC sequences. ER stress stimulated ARMET expression, and the expression patterns of ARMET mRNA and protein in mouse tissues were similar to those of Grp78, an Hsp70-family protein required for quality control of proteins in the ER. A reporter gene assay using a mouse ARMET promoter revealed that the unfolded protein response of the ARMET gene is regulated by an ERSE-II element whose sequence is identical to that of the HERP gene. ARMET is the second fully characterized ERSE-II-dependent gene and likely contributes to quality control of proteins in the ER. PMID:17507765

Mizobuchi, Naomi; Hoseki, Jun; Kubota, Hiroshi; Toyokuni, Shinya; Nozaki, Jun-ichi; Naitoh, Motoko; Koizumi, Akio; Nagata, Kazuhiro

2007-05-14

107

Influence of chronic hyperglycemia on the loss of the unfolded protein response in transplanted islets.  

PubMed

Chronic hyperglycemia contributes to ?-cell dysfunction in diabetes and with islet transplantation, but the mechanisms remain unclear. Recent studies demonstrate that the unfolded protein response (UPR) is critical for ?-cell function. Here, we assessed the influence of hyperglycemia on UPR gene expression in transplanted islets. Streptozotocin-induced diabetic or control nondiabetic mice were transplanted under the kidney capsule with syngeneic islets either sufficient or not to normalize hyperglycemia. Twenty-one days after transplantation, islet grafts were excised and RT-PCR was used to assess gene expression. In islet grafts from diabetic mice, expression levels of many UPR genes of the IRE1/ATF6 pathways, which are important for adaptation to endoplasmic reticulum stress, were markedly reduced compared with that in islet grafts from control mice. UPR genes of the PERK pathway were also downregulated. The normalization of glycemia restored the changes in mRNA expression, suggesting that chronic hyperglycemia contributes to the downregulation of multiple arms of UPR gene expression. Similar correlations were observed between blood glucose and mRNA levels of transcription factors involved in the maintenance of ?-cell phenotype and genes implicated in ?-cell function, suggesting convergent regulation of UPR gene expression and ?-cell differentiation by hyperglycemia. However, the normalization of glycemia was not accompanied by restoration of antioxidant or pro-inflammatory cytokine mRNA levels, which were increased in islet grafts from diabetic mice. These studies demonstrate that chronic hyperglycemia contributes to the downregulation of multiple arms of UPR gene expression in transplanted mouse islets. Failure of the adaptive UPR may contribute to ?-cell dedifferentiation and dysfunction in diabetes. PMID:23833251

Walters, Stacey N; Luzuriaga, Jude; Chan, Jeng Yie; Grey, Shane T; Laybutt, D Ross

2013-09-06

108

Statins inhibit protein lipidation and induce the unfolded protein response in the non-sterol producing nematode Caenorhabditis elegans  

PubMed Central

Statins are compounds prescribed to lower blood cholesterol in millions of patients worldwide. They act by inhibiting HMG-CoA reductase, the rate-limiting enzyme in the mevalonate pathway that leads to the synthesis of farnesyl pyrophosphate, a precursor for cholesterol synthesis and the source of lipid moieties for protein prenylation. The nematode Caenorhabditis elegans possesses a mevalonate pathway that lacks the branch leading to cholesterol synthesis, and thus represents an ideal organism to specifically study the noncholesterol roles of the pathway. Inhibiting HMG-CoA reductase in C. elegans using statins or RNAi leads to developmental arrest and loss of membrane association of a GFP-based prenylation reporter. The unfolded protein response (UPR) is also strongly activated, suggesting that impaired prenylation of small GTPases leads to the accumulation of unfolded proteins and ER stress. UPR induction was also observed upon pharmacological inhibition of farnesyl transferases or RNAi inhibition of a specific isoprenoid transferase (M57.2) and found to be dependent on both ire-1 and xbp-1 but not on pek-1 or atf-6, which are all known regulators of the UPR. The lipid stores and fatty acid composition were unaffected in statin-treated worms, even though they showed reduced staining with Nile red. We conclude that inhibitors of HMG-CoA reductase or of farnesyl transferases induce the UPR by inhibiting the prenylation of M57.2 substrates, resulting in developmental arrest in C. elegans. These results provide a mechanism for the pleiotropic effects of statins and suggest that statins could be used clinically where UPR activation may be of therapeutic benefit.

Morck, Catarina; Olsen, Louise; Kurth, Caroline; Persson, Annelie; Storm, Nadia Jin; Svensson, Emma; Jansson, John-Olov; Hellqvist, Marika; Enejder, Annika; Faergeman, Nils J.; Pilon, Marc

2009-01-01

109

Activation of the unfolded protein response in vascular endothelial cells of nondiabetic obese adults.  

PubMed

Context: Activation of the unfolded protein response (UPR) is emerging as an important molecular signature of cardiometabolic diseases associated with obesity. However, despite the well-established role of the vascular endothelium in obesity-related cardiometabolic dysfunction, it is unclear whether the UPR is activated in endothelial cells of obese adults. Objective: The objective of the study was to determine whether markers of UPR activation are increased in endothelial cells (ECs) of nondiabetic obese adults with impaired endothelial function. Design, Setting, and Participants: Endothelial cells were obtained from antecubital veins of the nondiabetic obese adults [body mass index (BMI) ? 30 kg/m(2), n = 12] with impaired endothelial function and from their nonobese peers (BMI < 30 kg/m(2), n = 14). Main Outcome Variables: UPR activation via expression (quantitative immunofluorescence) of the proximal UPR sensors, inositol-requiring endoplasmic reticulum (ER)-to-nucleus signaling protein 1 (IRE1), RNA-dependent protein kinase-like ER eukaryotic initiation factor-2? kinase (PERK), and activating transcription factor 6 (ATF6), were the main outcome variables. Results: IRE1 expression was greater in obese vs nonobese individuals (0.84 ± 0.09 vs 0.47 ± 0.02 IRE1 intensity/human umbilical vein EC (HUVEC) intensity (n = 10/8, P < .01). Obese individuals also had greater EC activation of UPR stress sensors PERK and ATF6, indicated by increased expression of phosphorylated PERK [p-PERK; 0.49 ± 0.05 vs 0.36 ± 0.03, p-PERK (threonine 981) intensity/HUVEC intensity, n = 10 men, 13 women, P < .05] and nuclear localization of ATF6 (0.38 ± 0.05 vs 0.23 ± 0.02, nuclear ATF6 intensity/HUVEC intensity, n = 5 men, 9 women, P < .01), respectively. Stepwise linear regression analysis revealed that indices of body fat (BMI and waist circumference) were the strongest independent predictors of all 3 UPR mediators, explaining between 18% and 59% of the variance in endothelial cell expression of IRE1, p-PERK, and nuclear ATF6 localization. Conclusion: These results provide novel evidence for UPR activation in the endothelial cells of nondiabetic obese adults with vascular endothelial dysfunction. PMID:23913943

Kaplon, Rachelle E; Chung, Eric; Reese, Lauren; Cox-York, Kimberly; Seals, Douglas R; Gentile, Christopher L

2013-08-02

110

Oxidative protein folding and unfolded protein response elicit differing redox regulation in endoplasmic reticulum and cytosol of yeast.  

PubMed

Oxidative protein folding can exceed the cellular secretion machinery, inducing the unfolded protein response (UPR). Sustained endoplasmic reticulum (ER) stress leads to cell stress and disease, as described for Alzheimer, Parkinson, and diabetes mellitus, among others. It is currently assumed that the redox state of the ER is optimally balanced for formation of disulfide bonds using glutathione as the main redox buffer and that UPR causes a reduction of this organelle. The direct effect of oxidative protein folding in the ER, however, has not yet been dissected from UPR regulation. To measure in vivo redox conditions in the ER and cytosol of the yeast model organism Pichia pastoris we targeted redox-sensitive roGFP variants to the respective organelles. Thereby, we clearly demonstrate that induction of the UPR causes reduction of the cytosol in addition to ER reduction. Similarly, a more reduced redox state of the cytosol, but not of the ER, is observed during oxidative protein folding in the ER without UPR induction, as demonstrated by overexpressing genes of disulfide bond-rich secretory proteins such as porcine trypsinogen or protein disulfide isomerase (PDI1) and ER oxidase (ERO1). Cytosolic reduction seems not to be caused by the action of glutathione reductase (GLR1) and could not be compensated for by overexpression of cytosolic glutathione peroxidase (GPX1). Overexpression of GPX1 and PDI1 oxidizes the ER and increases the secretion of correctly folded proteins, demonstrating that oxidative protein folding per se is enhanced by a more oxidized ER and is counterbalanced by a more reduced cytosol. As the total glutathione concentration of these strains does not change significantly, but the ratio of GSH to GSSG is altered, either transport or redox signaling between the glutathione pools of ER and cytosol is assumed. These data clearly demonstrate that protein folding and ER stress have a severe impact on the cytosolic redox balance, which may be a major factor during development of folding-related diseases. PMID:22406321

Delic, Marizela; Rebnegger, Corinna; Wanka, Franziska; Puxbaum, Verena; Haberhauer-Troyer, Christina; Hann, Stephan; Köllensperger, Gunda; Mattanovich, Diethard; Gasser, Brigitte

2012-03-08

111

Intrinsic Capacities of Molecular Sensors of the Unfolded Protein Response to Sense Alternate Forms of Endoplasmic Reticulum Stress  

PubMed Central

The unfolded protein response (UPR) regulates the protein-folding capacity of the endoplasmic reticulum (ER) according to cellular demand. In mammalian cells, three ER transmembrane components, IRE1, PERK, and ATF6, initiate distinct UPR signaling branches. We show that these UPR components display distinct sensitivities toward different forms of ER stress. ER stress induced by ER Ca2+ release in particular revealed fundamental differences in the properties of UPR signaling branches. Compared with the rapid response of both IRE1 and PERK to ER stress induced by thapsigargin, an ER Ca2+ ATPase inhibitor, the response of ATF6 was markedly delayed. These studies are the first side-by-side comparisons of UPR signaling branch activation and reveal intrinsic features of UPR stress sensor activation in response to alternate forms of ER stress. As such, they provide initial groundwork toward understanding how ER stress sensors can confer different responses and how optimal UPR responses are achieved in physiological settings.

DuRose, Jenny B.; Tam, Arvin B.

2006-01-01

112

The X-box binding protein-1 transcription factor is required for plasma cell differentiation and the unfolded protein response.  

PubMed

X-box binding protein-1 (XBP-1) is a transcription factor essential for plasma cell differentiation. XBP-1 transcripts are found at high levels in plasma cells from rheumatoid synovium and myeloma cell lines. Lymphoid chimeras deficient in XBP-1 have a profound defect in plasma cell differentiation, with few plasma cells in their periphery and severely reduced serum immunoglobulin levels. When introduced into B-lineage cells, XBP-1 initiates plasma cell differentiation. XBP-1 is also the mammalian homologue of the yeast transcription factor Hac1p, an important component of the unfolded protein response (UPR). The UPR allows cells to tolerate conditions of endoplasmic reticulum (ER) stress caused by misfolded proteins. Studies examining the relationship between plasma cell differentiation, XBP-1, and the UPR demonstrate that this novel signaling system is vital for plasma cell differentiation. Signals that induce plasma cell differentiation and the UPR cooperate via XBP-1 to induce terminal B-cell differentiation. Additionally, XBP-1 plays an important role in the regulation of interleukin-6 production, a cytokine essential for plasma cell survival. PMID:12846805

Iwakoshi, Neal N; Lee, Ann-Hwee; Glimcher, Laurie H

2003-08-01

113

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

114

Complementary cell-based high-throughput screens identify novel modulators of the unfolded protein response.  

PubMed

Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than 2 decades, indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, the authors hypothesized that high-throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small-molecule activators of the apoptotic arm of the UPR to control or kill OSCC. They have developed complementary cell-based assays using stably transfected CHO-K1 cell lines that individually assess the PERK/eIF2?/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR subpathways. An 66 K compound collection was screened at the University of Michigan Center for Chemical Genomics that included a unique library of prefractionated natural product extracts. The mycotoxin methoxycitrinin was isolated from a natural extract and found to selectively activate the CHOP-luciferase reporter at 80 µM. A series of citrinin derivatives was isolated from these extracts, including a unique congener that has not been previously described. In an effort to identify more potent compounds, the authors examined the ability of citrinin and the structurally related mycotoxins ochratoxin A and patulin to activate the UPR. Strikingly, it was found that patulin at 2.5 to 10 µM induced a terminal UPR in a panel of OSCC cells that was characterized by an increase in CHOP, GADD34, and ATF3 gene expression and XBP1 splicing. A luminescent caspase assay and the induction of several BH3-only genes indicated that patulin could induce apoptosis in OSCC cells. These data support the use of this complementary HTS strategy to identify novel modulators of UPR signaling and tumor cell death. PMID:21844328

Fribley, Andrew M; Cruz, Patricia G; Miller, Justin R; Callaghan, Michael U; Cai, Peter; Narula, Neha; Neubig, Richard R; Showalter, Hollis D; Larsen, Scott D; Kirchhoff, Paul D; Larsen, Martha J; Burr, Douglas A; Schultz, Pamela J; Jacobs, Renju R; Tamayo-Castillo, Giselle; Ron, David; Sherman, David H; Kaufman, Randal J

2011-08-15

115

Complementary Cell-Based High Throughput Screens Identify Novel Modulators of the Unfolded Protein Response  

PubMed Central

Despite advances toward understanding the prevention and treatment of many cancers, patients who suffer from oral squamous cell carcinoma (OSCC) confront a survival rate that has remained unimproved for more than two decades indicating our ability to treat them pharmacologically has reached a plateau. In an ongoing effort to improve the clinical outlook for this disease, we previously reported that an essential component of the mechanism by which the proteasome inhibitor bortezomib (PS-341, Velcade) induced apoptosis in OSCC required the activation of a terminal unfolded protein response (UPR). Predicated on these studies, we hypothesized that high throughput screening (HTS) of large diverse chemical libraries might identify more potent or selective small molecule activators of the apoptotic arm of the UPR to control or kill OSCC. We have developed complementary cell-based assays using stably transfected CHO-K1 cell lines that individually assess the PERK/eIF2?/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR sub-pathways. A ~66K compound collection was screened at the University of Michigan Center for Chemical Genomics that included a unique library of pre-fractionated natural product extracts. The mycotoxin methoxycitrinin was isolated from a natural extract and found to selectively activate the CHOP-luciferase reporter at 80?M. A series of citrinin derivatives were isolated from these extracts, including a unique congener that has not been previously described. In an effort to identify more potent compounds we examined the ability of citrinin and the structurally related mycotoxins ochratoxin A and patulin to activate the UPR. Strikingly, we found that patulin at 2.5 – 10?M induced a terminal UPR in a panel of OSCC cells that was characterized by an increase in CHOP, GADD34 and ATF3 gene expression and XBP1 splicing. A luminescent caspase assay and the induction of several BH3-only genes indicated that patulin could induce apoptosis in OSCC cells. These data support the use of this complementary HTS strategy to identify novel modulators of UPR signaling and tumor cell death.

Fribley, Andrew M.; Cruz, Patricia G.; Miller, Justin R.; Callaghan, Michael U.; Cai, Peter; Narula, Neha; Neubig, Richard R.; Showalter, Hollis D.; Larsen, Scott D.; Kirchhoff, Paul D.; Larsen, Martha J.; Burr, Douglas A.; Schultz, Pamela J.; Jacobs, Renju R.; Tamayo-Castillo, Giselle; Ron, David; Sherman, David H.; Kaufman, Randal J.

2012-01-01

116

Uterine endoplasmic reticulum stress and its unfolded protein response may regulate caspase 3 activation in the pregnant mouse uterus.  

PubMed

We have previously proposed that uterine caspase-3 may modulate uterine contractility in a gestationally regulated fashion. The objective of this study was to determine the mechanism by which uterine caspase-3 is activated and consequently controlled in the pregnant uterus across gestation. Utilizing the mouse uterus as our gestational model we examined the intrinsic and extrinsic apoptotic signaling pathways and the endoplasmic reticulum stress response as potential activators of uterine caspase-3 at the transcriptional and translational level. Our study revealed robust activation of the uterine myocyte endoplasmic reticulum stress response and its adaptive unfolded protein response during pregnancy coinciding respectively with increased uterine caspase-3 activity and its withdrawal to term. In contrast the intrinsic and extrinsic apoptotic signaling pathways remained inactive across gestation. We speculate that physiological stimuli experienced by the pregnant uterus likely potentiates the uterine myocyte endoplasmic reticulum stress response resulting in elevated caspase-3 activation, which is isolated to the pregnant mouse myometrium. However as term approaches, activation of an elevated adaptive unfolded protein response acts to limit the endoplasmic reticulum stress response inhibiting caspase-3 resulting in its decline towards term. We speculate that these events have the capacity to regulate gestational length in a caspase-3 dependent manner. PMID:24058658

Suresh, Arvind; Subedi, Kalpana; Kyathanahalli, Chandrashekara; Jeyasuria, Pancharatnam; Condon, Jennifer C

2013-09-13

117

Uterine Endoplasmic Reticulum Stress and Its Unfolded Protein Response May Regulate Caspase 3 Activation in the Pregnant Mouse Uterus  

PubMed Central

We have previously proposed that uterine caspase-3 may modulate uterine contractility in a gestationally regulated fashion. The objective of this study was to determine the mechanism by which uterine caspase-3 is activated and consequently controlled in the pregnant uterus across gestation. Utilizing the mouse uterus as our gestational model we examined the intrinsic and extrinsic apoptotic signaling pathways and the endoplasmic reticulum stress response as potential activators of uterine caspase-3 at the transcriptional and translational level. Our study revealed robust activation of the uterine myocyte endoplasmic reticulum stress response and its adaptive unfolded protein response during pregnancy coinciding respectively with increased uterine caspase-3 activity and its withdrawal to term. In contrast the intrinsic and extrinsic apoptotic signaling pathways remained inactive across gestation. We speculate that physiological stimuli experienced by the pregnant uterus likely potentiates the uterine myocyte endoplasmic reticulum stress response resulting in elevated caspase-3 activation, which is isolated to the pregnant mouse myometrium. However as term approaches, activation of an elevated adaptive unfolded protein response acts to limit the endoplasmic reticulum stress response inhibiting caspase-3 resulting in its decline towards term. We speculate that these events have the capacity to regulate gestational length in a caspase-3 dependent manner.

Suresh, Arvind; Subedi, Kalpana; Kyathanahalli, Chandrashekara; Jeyasuria, Pancharatnam; Condon, Jennifer C.

2013-01-01

118

Enhanced sensitivity to DSS colitis caused by a hypomorphic Mbtps1 mutation disrupting the ATF6-driven unfolded protein response  

PubMed Central

Here, we describe an N-ethyl-N-nitrosourea (ENU)-induced missense error in the membrane-bound transcription factor peptidase site 1 (S1P)-encoding gene (Mbtps1) that causes enhanced susceptibility to dextran sodium sulfate (DSS)-induced colitis. S1P cleaves and activates cAMP response element binding protein/ATF transcription factors, the sterol regulatory element-binding proteins (SREBPs), and other proteins of both endogenous and viral origin. Because S1P has a nonredundant function in the ATF6-dependent unfolded protein response (UPR), woodrat mice show diminished levels of major endoplasmic reticulum chaperones GRP78 (BiP) and GRP94 in the colon upon DSS administration. Experiments with bone marrow chimeric mice reveal a requirement for S1P in nonhematopoietic cells, without which a diminished UPR and colitis develop.

Brandl, Katharina; Rutschmann, Sophie; Li, Xiaohong; Du, Xin; Xiao, Nengming; Schnabl, Bernd; Brenner, David A.; Beutler, Bruce

2009-01-01

119

Protein Folding in the Endoplasmic Reticulum and the Unfolded Protein Response  

Microsoft Academic Search

In all eukaryotic cells, the endoplasmic reticulum (ER) is an intracellular organelle where folding and assembly occurs for proteins destined to the extracellular space, plasma membrane, and the exo\\/endocytic compartments (Kaufman 1999). As a protein-folding compartment, the ER is exquisitely sensitive to alterations in homeostasis, and provides stringent quality control systems to ensure that only correctly folded proteins transit to

K. Zhang; R. J. Kaufman

120

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

121

IRE1/bZIP60-Mediated Unfolded Protein Response Plays Distinct Roles in Plant Immunity and Abiotic Stress Responses  

PubMed Central

Endoplasmic reticulum (ER)-mediated protein secretion and quality control have been shown to play an important role in immune responses in both animals and plants. In mammals, the ER membrane-located IRE1 kinase/endoribonuclease, a key regulator of unfolded protein response (UPR), is required for plasma cell development to accommodate massive secretion of immunoglobulins. Plant cells can secrete the so-called pathogenesis-related (PR) proteins with antimicrobial activities upon pathogen challenge. However, whether IRE1 plays any role in plant immunity is not known. Arabidopsis thaliana has two copies of IRE1, IRE1a and IRE1b. Here, we show that both IRE1a and IRE1b are transcriptionally induced during chemically-induced ER stress, bacterial pathogen infection and treatment with the immune signal salicylic acid (SA). However, we found that IRE1a plays a predominant role in the secretion of PR proteins upon SA treatment. Consequently, the ire1a mutant plants show enhanced susceptibility to a bacterial pathogen and are deficient in establishing systemic acquired resistance (SAR), whereas ire1b is unaffected in these responses. We further demonstrate that the immune deficiency in ire1a is due to a defect in SA- and pathogen-triggered, IRE1-mediated cytoplasmic splicing of the bZIP60 mRNA, which encodes a transcription factor involved in the expression of UPR-responsive genes. Consistently, IRE1a is preferentially required for bZIP60 splicing upon pathogen infection, while IRE1b plays a major role in bZIP60 processing upon Tunicamycin (Tm)-induced stress. We also show that SA-dependent induction of UPR-responsive genes is altered in the bzip60 mutant resulting in a moderate susceptibility to a bacterial pathogen. These results indicate that the IRE1/bZIP60 branch of UPR is a part of the plant response to pathogens for which the two Arabidopsis IRE1 isoforms play only partially overlapping roles and that IRE1 has both bZIP60-dependent and bZIP60-independent functions in plant immunity.

Blanco, Francisca; Boatwright, Jon Lucas; Moreno, Ignacio; Jordan, Melissa R.; Chen, Yani; Brandizzi, Federica; Dong, Xinnian

2012-01-01

122

Discovery of Genes Activated by the Mitochondrial Unfolded Protein Response (mtUPR) and Cognate Promoter Elements  

PubMed Central

In an accompanying paper, we show that the mitochondrial Unfolded Protein Response or mtUPR is initiated by the activation of transcription of chop through an AP-1 element in the chop promoter. Further, we show that the c/ebp? gene is similarly activated and CHOP and C/EBP? subsequently hetero-dimerise to activate transcription of mtUPR responsive genes. Here, we report the discovery of six additional mtUPR responsive genes. We found that these genes encoding mitochondrial proteases YME1L1 and MPP?, import component Tim17A and enzymes NDUFB2, endonuclease G and thioredoxin 2, all contain a CHOP element in their promoters. In contrast, genes encoding mitochondrial proteins Afg3L2, Paraplegin, Lon and SAM 50, which do not have a CHOP element, were not up-regulated. Conversely, genes with CHOP elements encoding cytosolic proteins were not induced by the accumulation of unfolded proteins in mitochondria. These results indicate that mtUPR responsive genes appear to share a requirement for a CHOP element, but that this is not sufficient for the regulation of the mtUPR. A more detailed analysis of promoters of mtUPR responsive genes revealed at least two additional highly conserved, putative regulatory sites either side of the CHOP element, one a motif of 12 bp which lies 14 bp upstream of the CHOP site and another 9 bp element, 2 bp downstream of the CHOP site. Both of these additional elements are conserved in the promoters of 9 of the ten mtUPR responsive genes we have identified so far, the exception being the Cpn60/10 bidirectional promoter. Mutation of each of these elements substantially reduced the mtUPR responsiveness of the promoters suggesting that these elements coordinately regulate mtUPR.

Aldridge, Jonathan E.; Horibe, Tomohisa; Hoogenraad, Nicholas J.

2007-01-01

123

Brain trauma induces X-box protein 1 processing indicative of activation of the endoplasmic reticulum unfolded protein response.  

PubMed

Brain trauma was induced in mice using a closed head injury (CHI) model. At 1, 6 or 24 h after trauma, brains were dissected into the cortex, striatum and hippocampus. Changes in levels of processed X-box protein 1 (xbp1), glucose-regulated protein 78 (grp78), growth arrest and DNA damage-inducible gene 153 (gadd153) and heat-shock protein 70 (hsp70) mRNA, indicating impaired endoplasmic reticulum (ER) and cytoplasmic functioning, were evaluated by quantitative PCR. In the cortex, processed xbp1 mRNA levels rose to 2000% of control 1 h after CHI, and stayed high throughout the experiments. In the hippocampus and striatum, processed xbp1 mRNA levels rose in a delayed fashion, peaking at 6 h (1000% of control) and 24 h after CHI (1500% of control) respectively. Levels of grp78 mRNA were only slightly increased in the cortex 24 h after CHI (150% of control), and were unchanged or transiently decreased in the hippocampus and striatum. Levels of gadd153 mRNA did not change significantly after trauma. A transient rise in hsp70 mRNA levels was observed only in the cortex, peaking at 1 h after CHI (600% of control). Processing of xbp1 mRNA is a sign of activation of the unfolded protein response indicative of ER dysfunction. The results suggest that brain trauma induces ER dysfunction, which spreads from the ipsilateral cortex to the hippocampus and striatum. These observations may have clinical implications and should therefore be considered for future investigations on therapeutic intervention of brain injury caused by contusion-induced neurotrauma. PMID:14756820

Paschen, Wulf; Yatsiv, Ido; Shoham, Shai; Shohami, Esther

2004-02-01

124

Yeast Bax inhibitor, Bxi1p, is an ER-localized protein that links the unfolded protein response and programmed cell death in Saccharomyces cerevisiae.  

PubMed

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. PMID:21673967

Cebulski, James; Malouin, Joshua; Pinches, Nathan; Cascio, Vincent; Austriaco, Nicanor

2011-06-06

125

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

126

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

PubMed

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. PMID:23950728

Nagy, Péter; Varga, Agnes; Pircs, Karolina; Heged?s, Krisztina; Juhász, Gábor

2013-08-08

127

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

128

Nitrosative-stress induced S-glutathionylation of PDI leads to activation of the unfolded protein response  

PubMed Central

The rapid proliferation of cancer cells mandates a high protein turnover. The endoplasmic reticulum (ER) is intimately involved in protein processing. An accumulation of unfolded or misfolded proteins in the ER leads to a cascade of transcriptional and translational events collectively referred to as the unfolded protein response (UPR). Protein disulfide isomerase (PDI) is one of the most abundant ER proteins and maintains a sentinel function in organizing accurate protein folding. Treatment of cells with PABA/NO (O2- [2,4-dinitro-5- (N-methyl-N-4-carboxyphenylamino) phenyl] 1-N, N-dimethylamino) diazen-1-ium-1, 2-diolate) resulted in a dose dependent increase in intracellular NO that caused S-glutathionylation of various proteins. Within 4h, PABA/NO activated the UPR and led to translational attenuation as measured by the phosphorylation and activation of the ER transmembrane kinase, PERK, and its downstream effector eIF2 in human leukemia (HL60) and ovarian cancer cells (SKOV3). Cleavage of the transcription factor, XBP-1 and transcriptional activation of the ER resident proteins, BiP, PDI, GRP94 and ERO1 (5-10 fold induction) also occurred. Immunoprecipitation of PDI showed that while nitrosylation was undetectable, PABA/NO treatment caused S-glutathionylation of PDI. Mass spectroscopy analysis showed that single cysteine residues within each of the catalytic sites of PDI had a mass increase [+305.3 Da] consistent with S-glutathionylation. Circular dichroism confirmed that S-glutathionylation of PDI results in alterations in the alpha-helix content of PDI and is concurrent with inhibition of its isomerase activity. Thus, it appears that S-glutathionylation of PDI is an upstream signaling event in the UPR and may be linked with the cytotoxic potential of PABA/NO.

Townsend, Danyelle M.; Manevich, Yefim; He, Lin; Xiong, Ying; Bowers, Robert R.; Hutchens, Steven; Tew, Kenneth D.

2009-01-01

129

Plasma cell differentiation initiates a limited ER stress response by specifically suppressing the PERK-dependent branch of the unfolded protein response  

Microsoft Academic Search

In response to terminal differentiation signals that enable B cells to produce vast quantities of antibodies, a dramatic expansion\\u000a of the secretory pathway and a corresponding increase in the molecular chaperones and folding enzymes that aid and monitor\\u000a immunoglobulin synthesis occurs. Recent studies reveal that the unfolded protein response (UPR), which is normally activated\\u000a by endoplasmic reticulum (ER) stress, plays

Yanjun Ma; Yuichiro Shimizu; Melissa J. Mann; Yi Jin; Linda M. Hendershot

2010-01-01

130

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

PubMed Central

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.

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

2012-01-01

131

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

132

The FKB2 gene of Saccharomyces cerevisiae, encoding the immunosuppressant-binding protein FKBP-13, is regulated in response to accumulation of unfolded proteins in the endoplasmic reticulum.  

PubMed Central

The FKB2 gene of Saccharomyces cerevisiae encodes a homolog of mammalian FKBP-13, an FK506/rapamycin-binding protein that localizes to the lumen of the endoplasmic reticulum (ER). We have found that FKB2 mRNA levels increase in response to the accumulation of unfolded precursor proteins in the ER. FKB2 mRNA levels are elevated in cells blocked in N-glycosylation--i.e., in wild-type cells treated with tunicamycin and in the sec53-6 mutant grown at the nonpermissive temperature. Mutations that block other steps in secretion have no effect on FKB2 mRNA levels, indicating that increases in FKB2 mRNA are not the consequence of a general block in secretion. The increase in FKB2 mRNA in response to unfolded proteins in the ER is mediated through a 21-bp unfolded-protein response (UPR) element located in the 5' noncoding region of FKB2. UPR elements present in other ER chaperone genes, such as yeast KAR2 (BiP), mammalian GRP78 (BiP), and GRP94, function in an analogous manner to that in FKB2. As with KAR2, FKB2 mRNA levels are also elevated by heat shock. The similarities in the regulation of FKB2 and other ER chaperone genes suggest that FKBP-13 may play a role in protein trafficking in the ER. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4

Partaledis, J A; Berlin, V

1993-01-01

133

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

134

SCFCdc4-mediated Degradation of the Hac1p Transcription Factor Regulates the Unfolded Protein Response in Saccharomyces cerevisiae  

PubMed Central

The Saccharomyces cerevisiae basic leucine zipper transcription factor Hac1p is synthesized in response to the accumulation of unfolded polypeptides in the lumen of the endoplasmic reticulum (ER), and it is responsible for up-regulation of ?5% of all yeast genes, including ER-resident chaperones and protein-folding catalysts. Hac1p is one of the most short-lived yeast proteins, having a half-life of ?1.5 min. Here, we have shown that Hac1p harbors a functional PEST degron and that degradation of Hac1p by the proteasome involves the E2 ubiquitin-conjugating enzyme Ubc3/Cdc34p and the SCFCdc4 E3 complex. Consistent with the known nuclear localization of Cdc4p, rapid degradation of Hac1p requires the presence of a functional nuclear localization sequence, which we demonstrated to involve basic residues in the sequence 29RKRAKTK35. Two-hybrid analysis demonstrated that the PEST-dependent interaction of Hac1p with Cdc4p requires Ser146 and Ser149. Turnover of Hac1p may be dependent on transcription because it is inhibited in cell mutants lacking Srb10 kinase, a component of the SRB/mediator module of the RNA polymerase II holoenzyme. Stabilization of Hac1p by point mutation or deletion, or as the consequence of defects in components of the degradation pathway, results in increased unfolded protein response element-dependent transcription and improved cell viability under ER stress conditions.

Pal, Bhupinder; Chan, Nickie C.; Helfenbaum, Leon; Tan, Kaeling; Tansey, William P.

2007-01-01

135

Dolichol Biosynthesis and Its Effects on the Unfolded Protein Response and Abiotic Stress Resistance in Arabidopsis[W][OA  

PubMed Central

Dolichols are long-chain unsaturated polyisoprenoids with multiple cellular functions, such as serving as lipid carriers of sugars used for protein glycosylation, which affects protein trafficking in the endoplasmic reticulum. The biological functions of dolichols in plants are largely unknown. We isolated an Arabidopsis thaliana mutant, lew1 (for leaf wilting1), that showed a leaf-wilting phenotype under normal growth conditions. LEW1 encoded a cis-prenyltransferase, which when expressed in Escherichia coli catalyzed the formation of dolichol with a chain length around C80 in an in vitro assay. The lew1 mutation reduced the total plant content of main dolichols by ?85% and caused protein glycosylation defects. The mutation also impaired plasma membrane integrity, causing electrolyte leakage, lower turgor, reduced stomatal conductance, and increased drought resistance. Interestingly, drought stress in the lew1 mutant induced higher expression of the unfolded protein response pathway genes BINDING PROTEIN and BASIC DOMAIN/LEUCINE ZIPPER60 as well as earlier expression of the stress-responsive genes RD29A and COR47. The lew1 mutant was more sensitive to dark treatment, but this dark sensitivity was suppressed by drought treatment. Our data suggest that LEW1 catalyzes dolichol biosynthesis and that dolichol is important for plant responses to endoplasmic reticulum stress, drought, and dark-induced senescence in Arabidopsis.

Zhang, Hairong; Ohyama, Kiyoshi; Boudet, Julie; Chen, Zhizhong; Yang, Jilai; Zhang, Min; Muranaka, Toshiya; Maurel, Christophe; Zhu, Jian-Kang; Gong, Zhizhong

2008-01-01

136

Flavivirus Infection Activates the XBP1 Pathway of the Unfolded Protein Response To Cope with Endoplasmic Reticulum Stress?  

PubMed Central

The unfolded protein response (UPR) is a coordinated change in gene expression triggered by perturbations in functions of the endoplasmic reticulum (ER). XBP1, a key transcription factor of the UPR, is activated by an IRE1-mediated splicing event, which results in a frameshift and encodes a protein with transcriptional activity. Here, we report that XBP1 was activated during flaviviral infection, as evidenced by XBP1 mRNA splicing and protein expression, as well as induction of the downstream genes ERdj4, EDEM1, and p58(IPK) in Japanese encephalitis virus (JEV)- and dengue virus serotype 2 (DEN-2)-infected cells. Reporter systems based on IRE1-mediated XBP1 splicing were established, and several flaviviral proteins associated with the ER, including glycoproteins and small hydrophobic membrane-anchored proteins, were found to trigger the splicing event. Notably, nonstructural protein NS2B-3 of DEN-2, but not of JEV, was a potent inducer of XBP1 splicing through an unclear mechanism(s). Reduction of XBP1 by a small interfering RNA had no effect on cells' susceptibility to the two viruses but exacerbated the flavivirus-induced cytopathic effects. Overall, flaviviruses trigger the XBP1 signaling pathway and take advantage of this cellular response to alleviate virus-induced cytotoxicity.

Yu, Chia-Yi; Hsu, Yun-Wei; Liao, Ching-Len; Lin, Yi-Ling

2006-01-01

137

An Unfolding Story of Helical Transmembrane Proteins  

PubMed Central

Reversible unfolding of helical transmembrane proteins could provide valuable information about the free energy of interaction between transmembrane helices. Thermal unfolding experiments suggest that this process for integral membrane proteins is irreversible. Chemical unfolding has been accomplished with organic acids, but the unfolding or refolding pathways involve irreversible steps. Sodium dodecyl sulfate (SDS) has been used as a perturbant to study reversible unfolding and refolding kinetics. However, the interpretation of these experiments is not straightforward. It is shown that the results could be explained by SDS binding without substantial unfolding. Furthermore, the SDS perturbed state is unlikely to include all of the entropy terms involved in an unfolding process. Alternative directions for future research are suggested: fluorinated alcohols in homogeneous solvent systems; inverse micelles; and fragment association studies.

Renthal, Robert

2008-01-01

138

The unfolded protein response sensor IRE1? is required at 2 distinct steps in B cell lymphopoiesis  

PubMed Central

B lymphocyte differentiation is coordinated with the induction of high-level Ig secretion and expansion of the secretory pathway. Upon accumulation of unfolded proteins in the lumen of the ER, cells activate an intracellular signaling pathway termed the unfolded protein response (UPR). Two major proximal sensors of the UPR are inositol-requiring enzyme 1? (IRE1?), an ER transmembrane protein kinase/endoribonuclease, and ER-resident eukaryotic translation initiation factor 2? (eIF2?) kinase (PERK). To elucidate whether the UPR plays an important role in lymphopoiesis, we carried out reconstitution of recombinase-activating gene 2–deficient (rag2–/–) mice with hematopoietic cells defective in either IRE1?- or PERK-mediated signaling. IRE1?-deficient (ire1?–/–) HSCs can proliferate and give rise to pro–B cells that home to bone marrow. However, IRE1?, but not its catalytic activities, is required for Ig gene rearrangement and production of B cell receptors (BCRs). Analysis of rag2–/– mice transplanted with IRE1? trans-dominant-negative bone marrow cells demonstrated an additional requirement for IRE1? in B lymphopoiesis: both the IRE1? kinase and RNase catalytic activities are required to splice the mRNA encoding X-box–binding protein 1 (XBP1) for terminal differentiation of mature B cells into antibody-secreting plasma cells. Furthermore, UPR-mediated translational control through eIF2? phosphorylation is not required for B lymphocyte maturation and/or plasma cell differentiation. These results suggest specific requirements of the IRE1?-mediated UPR subpathway in the early and late stages of B lymphopoiesis.

Zhang, Kezhong; Wong, Hetty N.; Song, Benbo; Miller, Corey N.; Scheuner, Donalyn; Kaufman, Randal J.

2005-01-01

139

Protein Unfolding Energy Determined by Jarzynski's Equality  

NASA Astrophysics Data System (ADS)

The dynamic response of single protein molecules to mechanical forces and the relation of dynamics to equilibrium properties of biomolecules has been a subject of intense recent study. Characterization of the fluctuations in these small systems plays an important role in successful application of Jarzynski's equality to determine equilibrium free energies from nonequilibrium measurements. Here we used the atomic force microscope to manipulate single titin I27 molecules to unfold the protein, and we have applied Jarzynski's equality to calculate the free energy landscape for stretching this heart muscle protein.

Kiang, Ching-Hwa; Harris, Nolan; Li, Leiming; Song, Yang; Liao, Wei

2006-03-01

140

Exposure to fine airborne particulate matter induces macrophage infiltration, unfolded protein response, and lipid deposition in white adipose tissue.  

PubMed

Recent epidemiological studies have suggested a link between exposure to ambient air-pollution and susceptibility to metabolic disorders such as Type II diabetes mellitus. Previously, we provided evidence that both short- and long-term exposure to concentrated ambient particulate matter with aerodynamic diameter <2.5 ?m (PM2.5) induces multiple abnormalities associated with the pathogenesis of Type II diabetes mellitus, including insulin resistance, visceral adipose inflammation, brown adipose mitochondrial adipose changes, and hepatic endoplasmic reticulum (ER) stress. In this report, we show that chronic inhalation exposure to PM2.5 (10 months exposure) induces macrophage infiltration and Unfolded Protein Response (UPR), an intracellular stress signaling that regulates cell metabolism and survival, in mouse white adipose tissue in vivo. Gene expression studies suggested that PM2.5 exposure induces two distinct UPR signaling pathways mediated through the UPR transducer inositol-requiring 1? (IRE1?): 1) ER-associated Degradation (ERAD) of unfolded or misfolded proteins, and 2) Regulated IRE1-dependent Decay (RIDD) of mRNAs. Along with the induction of the UPR pathways and macrophage infiltration, expression of genes involved in lipogenesis, adipocyte differentiation, and lipid droplet formation was increased in the adipose tissue of the mice exposed to PM2.5. In vitro study confirmed that PM2.5 can trigger phosphorylation of the UPR transducer IRE1? and activation of macrophages. These results provide novel insights into PM2.5-triggered cell stress response in adipose tissue and increase our understanding of pathophysiological effects of particulate air pollution on the development of metabolic disorders. PMID:23573366

Mendez, Roberto; Zheng, Ze; Fan, Zhongjie; Rajagopalan, Sanjay; Sun, Qinghua; Zhang, Kezhong

2013-03-28

141

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

142

Essential role of the unfolded protein response regulator GRP78/BiP in protection from neuronal apoptosis  

PubMed Central

Neurodegenerative diseases are often associated with dysfunction in protein quality control. The endoplasmic reticulum (ER), a key site for protein synthesis, senses stressful conditions by activating the unfolded protein response (UPR). Here we report the creation of a novel mouse model where GRP78/BiP, a major ER chaperone and master regulator of UPR, is specifically eliminated in the Purkinje cells (PCs). GRP78 depleted PCs activate UPR including induction of GRP94, PDI, CHOP and GADD34, feedback suppression of eIF2? phosphorylation and apoptotic cell death. In contrast to current models of protein misfolding where abnormal accumulation of ubiquitinated protein is prominent, cytosolic ubiquitin staining is dramatically reduced in GRP78 null PCs. Ultrastructural evaluation reveals that the ER shows prominent dilatation with focal accumulation of electron-dense material within the ER. The mice show retarded growth and severe motor coordination defect by week 5 and cerebellar atrophy by week 13. Our studies uncover a novel link between GRP78 depletion and reduction in cytosolic ubiquitination and establish a novel mouse model of accelerated cerebellar degeneration with basic and clinical applications.

Wang, Miao; Ye, Risheng; Barron, Ernesto; Baumeister, Peter; Mao, Changhui; Luo, Shengzhan; Fu, Yong; Luo, Biquan; Dubeau, Louis; Hinton, David R.; Lee, Amy S.

2009-01-01

143

The effect of Zhangfei on the unfolded protein response and growth of cells derived from canine and human osteosarcomas.  

PubMed

The objective of this study was to determine whether the protein Zhangfei could suppress the unfolded protein response (UPR) and growth of osteosarcoma cells. Dog (D-17) and a human (Saos-2) osteosarcoma cells were infected with adenovirus vectors expressing either Zhangfei or the control protein beta- galactosidase. We monitored cell growth as well as levels of UPR gene transcripts and proteins. We found that Zhangfei suppressed the growth of both D-17 and Saos-2 cells. Zhangfei-expressing D-17 cells displayed large vacuoles containing culture medium and expressed phosphatidylserine on their external surface suggesting that Zhangfei induced macropinocytosis and apoptosis in these cells. While Zhangfei inhibited the growth of both D-17 and Saos-2 cells, it inhibited thapsigargin-induced UPR, as detected by a decrease in transcripts for UPR genes, and HERP and GRP78 proteins, only in D-17 cells, suggesting that the ability of Zhangfei to suppress the UPR and tumour cells growth may not be linked. PMID:22243984

Bergeron, T; Zhang, R; Elliot, K; Rapin, N; MacDonald, V; Linn, K; Simko, E; Misra, V

2012-01-15

144

Oral treatment targeting the unfolded protein response prevents neurodegeneration and clinical disease in prion-infected mice.  

PubMed

During prion disease, an increase in misfolded prion protein (PrP) generated by prion replication leads to sustained overactivation of the branch of the unfolded protein response (UPR) that controls the initiation of protein synthesis. This results in persistent repression of translation, resulting in the loss of critical proteins that leads to synaptic failure and neuronal death. We have previously reported that localized genetic manipulation of this pathway rescues shutdown of translation and prevents neurodegeneration in a mouse model of prion disease, suggesting that pharmacological inhibition of this pathway might be of therapeutic benefit. We show that oral treatment with a specific inhibitor of the kinase PERK (protein kinase RNA-like endoplasmic reticulum kinase), a key mediator of this UPR pathway, prevented UPR-mediated translational repression and abrogated development of clinical prion disease in mice, with neuroprotection observed throughout the mouse brain. This was the case for animals treated both at the preclinical stage and also later in disease when behavioral signs had emerged. Critically, the compound acts downstream and independently of the primary pathogenic process of prion replication and is effective despite continuing accumulation of misfolded PrP. These data suggest that PERK, and other members of this pathway, may be new therapeutic targets for developing drugs against prion disease or other neurodegenerative diseases where the UPR has been implicated. PMID:24107777

Moreno, Julie A; Halliday, Mark; Molloy, Colin; Radford, Helois; Verity, Nicholas; Axten, Jeffrey M; Ortori, Catharine A; Willis, Anne E; Fischer, Peter M; Barrett, David A; Mallucci, Giovanna R

2013-10-01

145

Loading device effect on protein unfolding mechanics.  

PubMed

Single-molecule mechanical manipulation has enabled quantitative understanding of not only the kinetics of both bond rupture and protein unfolding, but also the free energy landscape of chemical bond and/or protein folding. Despite recent studies reporting the role of loading device in bond rupture, a loading device effect on protein unfolding mechanics has not been well studied. In this work, we have studied the effect of loading-device stiffness on the kinetics of both bond rupture and protein unfolding mechanics using Brownian dynamics simulations. It is shown that bond rupture forces are dependent on not only loading rate but also the stiffness of loading device, and that protein unfolding mechanics is highly correlated with the stiffness of loading device. Our study sheds light on the importance of loading device effect on the mechanically induced bond ruptures and protein unfolding. PMID:22803564

Yoon, Gwonchan; Na, Sungsoo; Eom, Kilho

2012-07-14

146

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

147

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

148

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

PubMed

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 w(1118) 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. PMID:21965601

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

2011-09-29

149

Balancing life with glycoconjugates: monitoring unfolded protein response-mediated anti-angiogenic action of tunicamycin by Raman Spectroscopy  

PubMed Central

Asparagine-linked protein glycosylation is a hallmark for glycoprotein structure and function. Its impairment by tunicamycin [a competitive inhibitor of N-acetylglucosaminyl 1-phosphate transferase (GPT)] has been known to inhibit neo-vascularization (i.e., angiogenesis) in humanized breast tumor due to an induction of ER stress-mediated unfolded protein response (UPR). The studies presented here demonstrate that (i) tunicamycin (i) inhibits capillary endothelial cell proliferation in a dose dependent manner; (ii) treated cells are incapable of forming colonies upon its withdrawal; and (iii) tunicamycin treatment causes nuclear fragmentation. Tunicamycin-induced ER stress-mediated UPR event in these cells was studied with the aid of Raman spectroscopy, in particular, the interpretation of bands at 1672, 1684 and 1694 cm?1, which are characteristics of proteins and originate from C=O stretching vibrations of mono-substituted amides. In tunicamycin-treated cells these bands decreased in area as follows: at 1672 cm?1 by 41.85% at 3 h and 55.39% at 12 h; at 1684 cm?1 by 20.63% at 3 h and 40.08% at 12 h; and also at 1994 cm?1 by 33.33% at 3 h and 32.92% at 12 h, respectively. Thus, in the presence of tunicamycin, newly synthesized protein chains fail to arrange properly into their final secondary and/or tertiary structures, and the random coils they form had undergone further degradation.

Longas, Maria O.; Kotapati, Ashok; Prasad, Kilari PVRK; Banerjee, Aditi; Santiago, Jesus; Baksi, Krishna; Banerjee, Dipak K.

2012-01-01

150

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

PubMed

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. PMID:23814041

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

2013-06-28

151

p53 antagonizes the unfolded protein response and inhibits ground glass hepatocyte development during endoplasmic reticulum stress.  

PubMed

The unfolded protein response (UPR) is triggered during stress of the endoplasmic reticulum (ER) and facilitates tissue homeostasis. Considering the role of p53 tumor suppressor gene in the interpretation of stress-inducing stimuli, in this study, we explored whether p53 modulates UPR. We found that p53 ablation resulted in a profound sensitivity to tunicamycin that was associated with liver dysfunction, ground glass hepatocyte (GGH) development and nuclear atypia/dysplasia. Binding immunoglobulin protein (BiP)/glucose-regulated protein 78 (GRP78) chaperone was readily detected in the cytoplasm of GGHs, confirming ER expansion. Tunicamycin administration induced BiP/GRP78 and GRP94 expression more potently in the p53-deficient mice than in controls and elevated phosphatidylcholine, the major lipid of ER, by a p53-dependent mechanism. Furthermore, alternative splicing of XBP1, the transcription factor that executes the UPR, was more efficient in cells which do not express p53. The cytoprotective effects of p53 were confirmed by cell viability studies, indicating that p53 deficiency conferred sensitivity against tunicamycin. Our findings show that p53 protects from the hepatotoxic effects of chronic ER stress. Stimulation of p53 activity when intense UPR is undesirable may possess therapeutic implications. PMID:23038705

Dioufa, Nikolina; Chatzistamou, Ioulia; Farmaki, Elena; Papavassiliou, Athanasios G; Kiaris, Hippokratis

2012-10-04

152

The LMP1 oncogene of EBV activates PERK and the unfolded protein response to drive its own synthesis  

PubMed Central

The oncogene latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) without a ligand drives proliferation of EBV-infected B cells. Its levels vary in cells of clonal populations by more than 100-fold, which leads to multiple distinct activities of the oncogene. At intermediate levels it drives proliferation, and at high levels it inhibits general protein synthesis by inducing phosphorylation of eukaryotic initiation factor 2? (eIF2?). We have found that LMP1 activates PERK to induce phosphorylation of eIF2?, which upregulates activating transcription factor 4 (ATF4) expression. ATF4, in turn, transactivates LMP1's own promoter. LMP1 activates not only PERK but also inositol requiring kinase 1 (IRE1) and ATF6, 3 pathways of the unfolded protein response (UPR). Increasing expression levels of LMP1 induced a dose-dependent increase in IRE1 activity, as measured by its “splicing” of XBP-1. These infected B cells secrete immunoglobins independent of the levels of LMP1, indicating that only a threshold level of XBP-1 is required for the secretion. These findings indicate that LMP1's activation of the UPR is a normal event in a continuum of LMP1's expression that leads both to stimulatory and inhibitory functions and regulates the physiology of EBV-infected B cells in multiple, unexpected modes.

Lee, Dong Yun

2008-01-01

153

GLP-1 Analogs Reduce Hepatocyte Steatosis and Improve Survival by Enhancing the Unfolded Protein Response and Promoting Macroautophagy  

PubMed Central

Background Nonalcoholic fatty liver disease (NAFLD) is a known outcome of hepatosteatosis. Free fatty acids (FFA) induce the unfolded protein response (UPR) or endoplasmic reticulum (ER) stress that may induce apoptosis. Recent data indicate ER stress to be a major player in the progression of fatty liver to more aggressive lesions. Autophagy on the other hand has been demonstrated to be protective against ER stress- induced cell death. We hypothesized that exendin-4 (GLP-1 analog) treatment of fat loaded hepatocytes can reduce steatosis by autophagy which leads to reduced ER stress-related hepatocyte apoptosis. Methodology/Principal Findings Primary human hepatocytes were loaded with saturated, cis- and trans-unsaturated fatty acids (palmitic, oleic and elaidic acid respectively). Steatosis, induced with all three fatty acids, was significantly resolved after exendin-4 treatment. Exendin-4 sustained levels of GRP78 expression in fat-loaded cells when compared to untreated fat-loaded cells alone. In contrast, CHOP (C/EBP homologous protein); the penultimate protein that leads to ER stress-related cell death was significantly decreased by exendin-4 in hepatocytes loaded with fatty acids. Finally, exendin-4 in fat loaded hepatocytes clearly promoted gene products associated with macroautophagy as measured by enhanced production of both Beclin-1 and LC3B-II, markers for autophagy; and visualized by transmission electron microscopy (TEM). Similar observations were made in mouse liver lysates after mice were fed with high fat high fructose diet and treated with a long acting GLP-1 receptor agonist, liraglutide. Conclusions/Significance GLP-1 proteins appear to protect hepatocytes from fatty acid-related death by prohibition of a dysfunctional ER stress response; and reduce fatty acid accumulation, by activation of both macro-and chaperone-mediated autophagy. These findings provide a novel role for GLP-1 proteins in halting the progression of more aggressive lesions from underlying steatosis in humans afflicted with NAFLD.

Sharma, Shvetank; Mells, Jamie E.; Fu, Ping P.; Saxena, Neeraj K.; Anania, Frank A.

2011-01-01

154

Activation of the unfolded protein response and autophagy after hepatitis C virus infection suppresses innate antiviral immunity in vitro  

PubMed Central

Autophagy, a process for catabolizing cytoplasmic components, has been implicated in the modulation of interactions between RNA viruses and their host. However, the mechanism underlying the functional role of autophagy in the viral life cycle still remains unclear. Hepatitis C virus (HCV) is a single-stranded, positive-sense, membrane-enveloped RNA virus that can cause chronic liver disease. Here we report that HCV induces the unfolded protein response (UPR), which in turn activates the autophagic pathway to promote HCV RNA replication in human hepatoma cells. Further analysis revealed that the entire autophagic process through to complete autolysosome maturation was required to promote HCV RNA replication and that it did so by suppressing innate antiviral immunity. Gene silencing or activation of the UPR-autophagy pathway activated or repressed, respectively, IFN-? activation mediated by an HCV-derived pathogen-associated molecular pattern (PAMP). Similar results were achieved with a PAMP derived from Dengue virus (DEV), indicating that HCV and DEV may both exploit the UPR-autophagy pathway to escape the innate immune response. Taken together, these results not only define the physiological significance of HCV-induced autophagy, but also shed light on the knowledge of host cellular responses upon HCV infection as well as on exploration of therapeutic targets for controlling HCV infection.

Ke, Po-Yuan; Chen, Steve S.-L.

2010-01-01

155

A Peptidic Unconjugated GRP78/BiP Ligand Modulates the Unfolded Protein Response and Induces Prostate Cancer Cell Death  

PubMed Central

The molecular chaperone GRP78/BiP is a key regulator of protein folding in the endoplasmic reticulum, and it plays a pivotal role in cancer cell survival and chemoresistance. Inhibition of its function has therefore been an important strategy for inhibiting tumor cell growth in cancer therapy. Previous efforts to achieve this goal have used peptides that bind to GRP78/BiP conjugated to pro-drugs or cell-death-inducing sequences. Here, we describe a peptide that induces prostate tumor cell death without the need of any conjugating sequences. This peptide is a sequence derived from the cochaperone Bag-1. We have shown that this sequence interacts with and inhibits the refolding activity of GRP78/BiP. Furthermore, we have demonstrated that it modulates the unfolded protein response in ER stress resulting in PARP and caspase-4 cleavage. Prostate cancer cells stably expressing this peptide showed reduced growth and increased apoptosis in in vivo xenograft tumor models. Amino acid substitutions that destroyed binding of the Bag-1 peptide to GRP78/BiP or downregulation of the expression of GRP78 compromised the inhibitory effect of this peptide. This sequence therefore represents a candidate lead peptide for anti-tumor therapy.

Maddalo, Danilo; Neeb, Antje; Jehle, Katja; Schmitz, Katja; Muhle-Goll, Claudia; Shatkina, Liubov; Walther, Tamara Vanessa; Bruchmann, Anja; Gopal, Srinivasa M.; Wenzel, Wolfgang; Ulrich, Anne S.; Cato, Andrew C. B.

2012-01-01

156

Loss of Oca2 disrupts the unfolded protein response and increases resistance to endoplasmic reticulum stress in melanocytes.  

PubMed

Accumulation of proteins in the endoplasmic reticulum (ER) typically induces stress and initiates the unfolded protein response (UPR) to facilitate recovery. If homeostasis is not restored, apoptosis is induced. However, adaptation to chronic UPR activation can increase resistance to subsequent acute ER stress. We therefore investigated adaptive mechanisms in Oculocutaneous albinism type 2 (Oca2)-null melanocytes where UPR signaling is arrested despite continued tyrosinase accumulation leading to resistance to the chemical ER stressor thapsigargin. Although thapsigargin triggers UPR activation, instead of Perk-mediated phosphorylation of eIF2?, in Oca2-null melanocytes, eIF2? was rapidly dephosphorylated upon treatment. Dephosphorylation was mediated by the Gadd34-PP1? phosphatase complex. Gadd34-complex inhibition blocked eIF2? dephosphorylation and significantly increased Oca2-null melanocyte sensitivity to thapsigargin. Thus, Oca2-null melanocytes adapt to acute ER stress by disruption of pro-apoptotic Perk signaling, which promotes cell survival. This is the first study to demonstrate rapid eIF2? dephosphorylation as an adaptive mechanism to ER stress. PMID:23962237

Cheng, Tsing; Orlow, Seth J; Manga, Prashiela

2013-09-09

157

BiP-bound and nonclustered mode of Ire1 evokes a weak but sustained unfolded protein response.  

PubMed

In eukaryotic cells under nonstressed conditions, the endoplasmic reticulum (ER)-located molecular chaperone BiP is associated with an ER-membrane protein Ire1 to inhibit its self-association. While ER stress leads Ire1 to form transiently BiP-unbound clusters, which strongly evoke the unfolded protein response (UPR), here we propose an alternative activation status of Ire1. When yeast cells are physiologically ER-stressed by inositol depletion for a prolonged time, the UPR is weakly activated in a sustained manner after a transient peak of activation. During persistent stress, Ire1 foci disappear, while Ire1 continues to be self-associated. Under these conditions, Ire1 may be activated as a homo-dimer, as it shows considerable activity even when carrying the W426A mutation, which allows Ire1 to form homo-dimers but not clusters. Unlike the Ire1 clusters, the nonclustered active form seems to be associated with BiP. An Ire1 mutant not carrying the BiP-association site continued to form clusters and to be activated strongly even after long-term stress. Similar observations were obtained when cells were ER-stressed by dithiothreitol. We thus propose that upon persistent ER stress, Ire1 is weakly and continuously activated in a nonclustered form through its (re)association with BiP, which disperses the Ire1 clusters. PMID:23387983

Ishiwata-Kimata, Yuki; Promlek, Thanyarat; Kohno, Kenji; Kimata, Yukio

2013-02-06

158

Exposure to inhaled particulate matter activates early markers of oxidative stress, inflammation and unfolded protein response in rat striatum.  

PubMed

To study central nervous system airborne PM related subchronic toxicity, SD male rats were exposed for eight weeks to either coarse (32?g/m(3)), fine (178?g/m(3)) or ultrafine (107?g/m(3)) concentrated PM or filtered air. Different brain regions (olfactory bulb, frontal cortex, striatum and hippocampus), were harvested from the rats following exposure to airborne PM. Subsequently, prooxidant (HO-1 and SOD-2), and inflammatory markers (IL-1? and TNF?), apoptotic (caspase 3), and unfolded protein response (UPR) markers (XBP-1S and BiP), were also measured using real-time PCR. Activation of nuclear transcription factors Nrf-2 and NF-?B, associated with antioxidant and inflammation processes, respectively, were also analyzed by GSMA. Ultrafine PM increased HO-1 and SOD-2 mRNA levels in the striatum and hippocampus, in the presence of Nrf-2 activation. Also, ultrafine PM activated NF-?B and increased IL-1? and TNF? in the striatum. Activation of UPR was observed after exposure to coarse PM through the increment of XBP-1S and BiP in the striatum, accompanied by an increase in antioxidant response markers HO-1 and SOD-2. Our results indicate that exposure to different size fractions of PM may induce physiological changes (in a neuroanatomical manner) in the central nervous system (CNS), specifically within the striatum, where inflammation, oxidative stress and UPR signals were effectively activated. PMID:23892126

Guerra, R; Vera-Aguilar, E; Uribe-Ramirez, M; Gookin, G; Camacho, J; Osornio-Vargas, A R; Mugica-Alvarez, V; Angulo-Olais, R; Campbell, A; Froines, J; Kleinman, T M; De Vizcaya-Ruiz, A

2013-07-26

159

Adaptive Suppression of the ATF4-CHOP Branch of the Unfolded Protein Response by Toll-Like Receptor Signaling  

PubMed Central

The endoplasmic reticulum (ER) Unfolded Protein Response (UPR) restores equilibrium to the ER, but prolonged expression of the UPR effector CHOP (GADD153) is cytotoxic. We found that ER stress-induced CHOP expression was suppressed by prior engagement of toll-like receptor (TLR) 3 or 4 through a TRIF-dependent pathway. TLR engagement did not suppress phosphorylation of PERK or eIF-2?, which are upstream of CHOP, but phospho-eIF-2? failed to promote translation of the CHOP activator ATF4. In mice subjected to systemic ER stress, pre-treatment with low-dose lipopolysaccharide (LPS), a TLR4 ligand, suppressed CHOP expression and apoptosis in splenic macrophages, renal tubule cells, and hepatocytes, and prevented renal dysfunction and hepatosteatosis. This protective effect of LPS did not occur in Trif?/? mice nor in wild-type mice in which CHOP expression was genetically restored. Thus, TRIF-mediated signals from TLRs selectively attenuate translational activation of ATF4 and its downstream target gene CHOP. We speculate that this mechanism evolved to promote survival of TLR-expressing cells that experience prolonged levels of physiologic ER stress in the course of the host response to invading pathogens.

Woo, Connie W.; Cui, Dongying; Arellano, Jerry; Dorweiler, Bernhard; Harding, Heather; Fitzgerald, Katherine A.; Ron, David; Tabas, Ira

2009-01-01

160

Model study of protein unfolding by interfaces  

NASA Astrophysics Data System (ADS)

We study interface-induced protein unfolding on hydrophobic and polar interfaces by means of a two-dimensional lattice model and an exhaustive enumeration ground-state structure search, for a set of model proteins of length 20 residues. We compare the effects of the two types of interfaces, and search for criteria that influence the retention of a protein’s native-state structure upon adsorption. We find that the unfolding proceeds by a large, sudden loss of native contacts. The unfolding at polar interfaces exhibits similar behavior to that at hydrophobic interfaces but with a much weaker interface coupling strength. Further, we find that the resistance of proteins to unfolding in our model is positively correlated with the magnitude of the folding energy in the native-state structure, the thermal stability (or energy gap) for that structure, and the interface energy for native-state adsorption. We find these factors to be of roughly equal importance.

Chakarova, S. D.; Carlsson, A. E.

2004-02-01

161

Loss of the Tuberous Sclerosis Complex Tumor Suppressors Triggers the Unfolded Protein Response to Regulate Insulin Signaling and Apoptosis  

PubMed Central

SUMMARY Mammalian target of rapamycin, mTOR, is a major sensor of nutrient and energy availability in the cell and regulates a variety of cellular processes including growth, proliferation and metabolism. Loss of the tuberous sclerosis complex genes (TSC1 or TSC2) leads to constitutive activation of mTOR and downstream signaling elements, resulting in the development of tumors, neurological disorders, and at the cellular level, severe insulin/IGF-1 resistance. Here, we show that loss of TSC1 or TSC2 in cell lines and mouse or human tumors causes endoplasmic reticulum (ER) stress and activates the unfolded protein response (UPR). The resulting ER stress plays a significant role in the mTOR-mediated negative-feedback inhibition of insulin action and increases the vulnerability to apoptosis. These results demonstrate ER stress as a critical component of the pathologies associated with dysregulated mTOR activity and offer the possibility to exploit this mechanism for new therapeutic opportunities.

Ozcan, Umut; Ozcan, Lale; Yilmaz, Erkan; Duvel, Katrin; Sahin, Mustafa; Manning, Brendan D.; Hotamisligil, Gokhan S.

2008-01-01

162

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

163

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

PubMed

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. PMID:23798569

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

2013-06-20

164

DNA unzipping and protein unfolding using nanopores.  

PubMed

We present here an overview on unfolding of biomolecular structures as DNA double strands or protein folds. After some theoretical considerations giving orders of magnitude about transport timescales through pores, forces involved in unzipping processes … we present our experiments on DNA unzipping or protein unfolding using a nanopore. We point out the difficulties that can be encountered during these experiments, such as the signal analysis problems, noise issues, or experimental limitations of such system. PMID:22528258

Merstorf, Céline; Cressiot, Benjamin; Pastoriza-Gallego, Manuela; Oukhaled, Abdel Ghani; Bacri, Laurent; Gierak, Jacques; Pelta, Juan; Auvray, Loïc; Mathé, Jérôme

2012-01-01

165

Cigarette smoke induces endoplasmic reticulum stress and the unfolded protein response in normal and malignant human lung cells  

PubMed Central

Background Although lung cancer is among the few malignancies for which we know the primary etiological agent (i.e., cigarette smoke), a precise understanding of the temporal sequence of events that drive tumor progression remains elusive. In addition to finding that cigarette smoke (CS) impacts the functioning of key pathways with significant roles in redox homeostasis, xenobiotic detoxification, cell cycle control, and endoplasmic reticulum (ER) functioning, our data highlighted a defensive role for the unfolded protein response (UPR) program. The UPR promotes cell survival by reducing the accumulation of aberrantly folded proteins through translation arrest, production of chaperone proteins, and increased degradation. Importance of the UPR in maintaining tissue health is evidenced by the fact that a chronic increase in defective protein structures plays a pathogenic role in diabetes, cardiovascular disease, Alzheimer's and Parkinson's syndromes, and cancer. Methods Gene and protein expression changes in CS exposed human cell cultures were monitored by high-density microarrays and Western blot analysis. Tissue arrays containing samples from 110 lung cancers were probed with antibodies to proteins of interest using immunohistochemistry. Results We show that: 1) CS induces ER stress and activates components of the UPR; 2) reactive species in CS that promote oxidative stress are primarily responsible for UPR activation; 3) CS exposure results in increased expression of several genes with significant roles in attenuating oxidative stress; and 4) several major UPR regulators are increased either in expression (i.e., BiP and eIF2?) or phosphorylation (i.e., phospho-eIF2?) in a majority of human lung cancers. Conclusion These data indicate that chronic ER stress and recruitment of one or more UPR effector arms upon exposure to CS may play a pivotal role in the etiology or progression of lung cancers, and that phospho-eIF2? and BiP may have diagnostic and/or therapeutic potential. Furthermore, we speculate that upregulation of UPR regulators (in particular BiP) may provide a pro-survival advantage by increasing resistance to cytotoxic stresses such as hypoxia and chemotherapeutic drugs, and that UPR induction is a potential mechanism that could be attenuated or reversed resulting in a more efficacious treatment strategy for lung cancer.

Jorgensen, Ellen; Stinson, Andy; Shan, Lin; Yang, Jin; Gietl, Diana; Albino, Anthony P

2008-01-01

166

Probing Membrane Protein Unfolding with Pulse Proteolysis  

PubMed Central

Technical challenges have greatly impeded the investigation of membrane protein folding and unfolding. To develop a new tool that facilitates the study of membrane proteins, we tested pulse proteolysis as a probe for membrane protein unfolding. Pulse proteolysis is a method to monitor protein folding and unfolding, which exploits the significant difference in proteolytic susceptibility between folded and unfolded proteins. This method requires only a small amount of protein and in many cases may be used with unpurified proteins in cell lysates. To evaluate the effectiveness of pulse proteolysis as a probe for membrane protein unfolding, we chose H. halobium bacteriorhodopsin (bR) as a model system. The denaturation of bR in SDS has been investigated extensively by monitoring the change in the absorbance at 560 nm (A560). In this work we demonstrate that denaturation of bR by SDS results in a significant increase in its susceptibility to proteolysis by subtilisin. When pulse proteolysis was applied to bR incubated in varying concentrations of SDS, the remaining intact protein determined by electrophoresis shows a cooperative transition. The midpoint of the cooperative transition (Cm) shows excellent agreement with that determined by A560. The Cm values determined by pulse proteolysis for M56A and Y57A bR are also consistent with the measurements made by A560. Our results suggest that pulse proteolysis is a quantitative tool to probe membrane protein unfolding. Combining pulse proteolysis with western blotting may allow the investigation of membrane protein unfolding in situ without over-expression or purification.

Schlebach, Jonathan P.; Kim, Moon-Soo; Joh, Nathan H.; Bowie, James U.; Park, Chiwook

2011-01-01

167

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

168

Identification of ERSE-II, a new cis-acting element responsible for the ATF6-dependent mammalian unfolded protein response.  

PubMed

Herp is a 54-kDa membrane protein in the endoplasmic reticulum (ER). The mRNA expression level of Herp is increased by the accumulation of unfolded proteins in the ER. Transcriptional changes designed to deal with this type of ER stress is called the unfolded protein response (UPR). Most mammalian UPR-target genes encode ER-resident molecular chaperones: GRP78, GRP94, and calreticulin. The promoter regions of these genes contain a cis-acting ER stress response element, ERSE, with the consensus sequence of CCAAT-N(9)-CCACG. Under conditions of ER stress, p50ATF6 (the active form of the transcription factor, ATF6) binds to CCACG when CCAAT is bound by the general transcription factor, NF-Y/CBF. Here, we report the genomic structure of human Herp and the presence of a new ER stress response element, ERSE-II, in its promoter region. The gene for Herp consists of eight exons, localized to chromosome 16q12.2-13. The promoter region contains a single ERSE-like sequence. In reporter gene assays, disruption of this cis-element resulted in a partial reduction of the transcriptional response to ER stress, suggesting that the element is functional for the UPR. These results also suggest the involvement of additional elements in the UPR. Further analysis, using an optimized plasmid containing an mRNA-destabilizing sequence, revealed ERSE-II (ATTGG-N-CCACG) as the second ER stress response element. Interestingly, ERSE-II was also dependent on p50ATF6, in a manner similar to that of ERSE, despite the disparate structure. The strong induction of Herp mRNA by ER stress would be achieved by the cooperation of ERSE and ERSE-II. PMID:11112790

Kokame, K; Kato, H; Miyata, T

2000-12-08

169

Endocellular polyamine availability modulates epithelial-to-mesenchymal transition and unfolded protein response in MDCK cells.  

PubMed

Epithelial-to-mesenchymal transition (EMT) is involved in embryonic development as well as in several pathological conditions. Literature indicates that polyamine availability may affect transcription of c-myc, matrix metalloproteinase (MMP)1, MMP2, TGFbeta(1), and collagen type I mRNA. The aim of this study was to elucidate polyamines role in EMT in vitro. Madin-Darby canine kidney (MDCK) cells were subjected to experimental manipulation of intracellular levels of polyamines. Acquisition of mesenchymal phenotype was evaluated by means of immunofluorescence, western blots, and zymograms. MDCK cells were then subjected to 2D gel proteomic study and incorporation of a biotinilated polyamine (BPA). Polyamine endocellular availability modulated EMT process. Polyamine-depleted cells treated with TGFbeta(1) showed enhanced EMT with a marked decrease of E-cadherin expression at plasma membrane level and an increased expression of mesenchymal markers such as fibronectin and alpha-smooth muscle actin. Polyamine-depleted cells showed a twofold increased expression of the rough endoplasmic reticulum (ER)-stress proteins GRP78, GRP94, and HSP90 alpha/beta in 2D gels. The latter data were confirmed by western blot analysis. Administration of BPA showed that polyamines are covalently linked, within the cell, to ER-stress proteins. Intracellular polyamine availability affects EMT in MDCK cells possibly through the modulation of ER-stress protein homeostasis. PMID:20212449

Prunotto, Marco; Compagnone, Alessandra; Bruschi, Maurizio; Candiano, Giovanni; Colombatto, Sebastiano; Bandino, Andrea; Petretto, Andrea; Moll, Solange; Bochaton-Piallat, Marie Luce; Gabbiani, Giulio; Dimuccio, Veronica; Parola, Maurizio; Citti, Lorenzo; Ghiggeri, Gianmarco

2010-03-08

170

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

171

Shutdown of Translation: Lethal or Protective? Unfolded Protein Response Versus Apoptosis  

Microsoft Academic Search

Shutdown of translation is a highly conserved response of cells to a severe form of metabolic, thermal, or physical stress. After the metabolic stress induced by transient cerebral ischemia, translational recovery is observed only in cells that withstand the transient interruption of blood supply, implying that restoration of translation critically determines the final outcome. On the other hand, apoptosis is

Wulf Paschen

2003-01-01

172

BAX inhibitor-1 regulates autophagy by controlling the IRE1? branch of the unfolded protein response  

PubMed Central

Both autophagy and apoptosis are tightly regulated processes playing a central role in tissue homeostasis. Bax inhibitor 1 (BI-1) is a highly conserved protein with a dual role in apoptosis and endoplasmic reticulum (ER) stress signalling through the regulation of the ER stress sensor inositol requiring kinase 1 ? (IRE1?). Here, we describe a novel function of BI-1 in the modulation of autophagy. BI-1-deficient cells presented a faster and stronger induction of autophagy, increasing LC3 flux and autophagosome formation. These effects were associated with enhanced cell survival under nutrient deprivation. Repression of autophagy by BI-1 was dependent on cJun-N terminal kinase (JNK) and IRE1? expression, possibly due to a displacement of TNF-receptor associated factor-2 (TRAF2) from IRE1?. Targeting BI-1 expression in flies altered autophagy fluxes and salivary gland degradation. BI-1 deficiency increased flies survival under fasting conditions. Increased expression of autophagy indicators was observed in the liver and kidney of bi-1-deficient mice. In summary, we identify a novel function of BI-1 in multicellular organisms, and suggest a critical role of BI-1 as a stress integrator that modulates autophagy levels and other interconnected homeostatic processes.

Castillo, Karen; Rojas-Rivera, Diego; Lisbona, Fernanda; Caballero, Benjamin; Nassif, Melissa; Court, Felipe A; Schuck, Sebastian; Ibar, Consuelo; Walter, Peter; Sierralta, Jimena; Glavic, Alvaro; Hetz, Claudio

2011-01-01

173

Pleiotropic potential of dehydroxymethylepoxyquinomicin for NF-?B suppression via reactive oxygen species and unfolded protein response.  

PubMed

Dehydroxymethylepoxyquinomicin (DHMEQ) is a low-m.w. compound that strongly inhibits NF-?B. Previous reports showed that DHMEQ directly binds to specific cysteine residues of NF-?B subunits and thereby inhibits their nuclear translocation and DNA binding. In this work, we describe novel mechanisms by which DHMEQ suppresses cytokine-triggered activation of NF-?B. We found that sustained exposure of renal tubular cells to DHMEQ blocked TNF-?- and IL-1?-induced TGF-?-activated kinase 1 (TAK1) phosphorylation, a crucial event for NF-?B activation upstream of I?B kinase. This inhibition was mediated by reactive oxygen species (ROS), because of the following: 1) DHMEQ caused generation of ROS; 2) pretreatment with ROS generator inhibited cytokine-induced TAK1 phosphorylation and NF-?B activation; and 3) scavenging of ROS attenuated the suppressive effects of DHMEQ on TAK1 and NF-?B. We also found that DHMEQ caused the unfolded protein response (UPR) through generation of ROS. Alleviation of the UPR by chemical and genetic chaperones partially attenuated the suppressive effect of DHMEQ on NF-?B. The UPR-mediated inhibition of NF-?B occurred downstream of degradation of I?B? and phosphorylation of p65. Subsequent experiments revealed the following: 1) DHMEQ caused selective induction of C/EBP? through the UPR; 2) overexpression of C/EBP? suppressed activation of NF-?B; 3) knockdown of C/EBP? attenuated the inhibitory effect of DHMEQ; and 4) DHMEQ-induced expression of C/EBP? did not affect TNF-?-triggered degradation of I?B? and phosphorylation of p65. These results suggest that, in addition to its known effect on nuclear translocation of NF-?B, DHMEQ interferes with the cytokine-induced NF-?B signaling via generation of ROS at both upstream and downstream of the I?B kinase-I?B level. PMID:23690471

Nakajima, Shotaro; Kato, Hironori; Gu, Liubao; Takahashi, Shuhei; Johno, Hisashi; Umezawa, Kazuo; Kitamura, Masanori

2013-05-20

174

Co-operative unfolding of protein domains  

NASA Astrophysics Data System (ADS)

How well does the worm-like chain force extension curve fit single-molecule protein unfolding data? Careful analysis of dynamic force spectroscopy data for different proteins[1] suggests that the compliance of a protein is generically larger than that predicted by the worm-like chain model. We propose that the observed excess compliance is due to pre-transitional conformational rearrangements within the protein domain that occur before the more dramatic failure of the domain as a whole. Using a generalization of the formalism introduced by Evans and Ritchie[2], we study protein--unfolding kinetics in our model where these internal conformational rearrangements are represented by a number of interacting Ising-type variables, which cooperatively escape over a barrier to the unfolded state. From this model, we predict a relation between the statistics of the fluctuations of the peak domain--unfolding force and the deviations of the force extension curves from the worm-like chain prediction. We suggest that, by using this approach, one can extract further details on the domain--unfolding pathway from extant force spectroscopy data. [1] D. J. Brockwell (private communication). [2] E. Evans, and K. Ritchie, Biophys. J., 72 1541 (1997).

Chakrabarti, Buddhapriya; Liverpool, Tanniemola B.; Levine, Alex J.

2006-03-01

175

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

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

Kinetics of protein unfolding at interfaces  

NASA Astrophysics Data System (ADS)

The conformation of protein molecules is determined by a balance of various forces, including van der Waals attraction, electrostatic interaction, hydrogen bonding, and conformational entropy. When protein molecules encounter an interface, they are often adsorbed on the interface. The conformation of an adsorbed protein molecule strongly depends on the interaction between the protein and the interface. Recent time-resolved investigations have revealed that protein conformation changes during the adsorption process due to the protein-protein interaction increasing with increasing interface coverage. External conditions also affect the protein conformation. This review considers recent dynamic observations of protein adsorption at various interfaces and their implications for the kinetics of protein unfolding at interfaces.

Yano, Yohko F.

2012-12-01

178

Identification and characterization of Inositol-requiring enzyme-1 and X-box binding protein 1, two proteins involved in the unfolded protein response of Litopenaeus vannamei.  

PubMed

The inositol-requiring enzyme-1 (IRE1)-X-box binding protein 1 (IRE1-XBP1) pathway is the key branch of the unfolded protein response (UPR). To investigate the role of the IRE1-XBP1 pathway in reducing environmental stress and increasing anti-viral immunity in Litopenaeus vannamei, homologues of IRE1 (designated as LvIRE1) and XBP1 (designated as LvXBP1) were identified and characterized. The full-length cDNA of LvIRE1 is 4908bp long, with an open reading frame (ORF) that encodies a putative 1174 amino acid protein. The full-length cDNA of LvXBP1 is 1746bp long. It contains two ORFs that encode putative 278 amino acid and 157 amino acid proteins, respectively. LvXBP1 mRNA has the predicted IRE1 splicing motifs CNG'CNGN located within the loop regions of two short hairpins. Sequencing of the splicing fragment induced by endoplasmic reticulum (ER)-stress showed a 3bp or 4bp frame shift from the predicted sites. The spliced form LvXBP1 (LvXBP1s) contained an ORF encodes a putative 463 amino acid protein. The reporter gene assays indicated that LvXBP1s activates the promoter of L. vannamei immunoglobulin heavy chain binding protein (LvBip), an important UPR effector. RT-PCR showed that LvXBP1 was spliced during the experiments. For heat shock treatment, the total LvXBP1 expression was increased and peaked at about 36h, whereas the percentages of the two isoforms were relatively stable. For the WSSV challenge, LvXBP1 was upregulated during the experiment and the percentage of the spliced form continuously declined after 18h of infection. Knock-down of LvXBP1 by RNA interference resulted in a lower cumulative mortality of L. vannamei under WSSV infection. Furthermore, the expression profiles of LvIRE1 and LvXBP1 in the gills, hemocytes, intestines, and hepatopancreas of the WSSV-challenged shrimp were detected using real-time RT-PCR. Taken together, these results confirm that the IRE1-XBP1 pathway is important for L. vannamei environmental stress resistance, suggest that L. vannamei IRE1-XBP1 may activated by WSSV and be annexed to serve the virus. PMID:22554476

Chen, Yi-Hong; Zhao, Li; Pang, Li-Ran; Li, Xiao-Yun; Weng, Shao-Ping; He, Jian-Guo

2012-04-30

179

A regulatory subunit of phosphoinositide 3-kinase increases the nuclear accumulation of X-box-binding protein-1 to modulate the unfolded protein response.  

PubMed

Class Ia phosphoinositide 3-kinase (PI3K), an essential mediator of the metabolic actions of insulin, is composed of a catalytic (p110alpha or p110beta) and regulatory (p85alphaalpha, p85betaalpha or p55alpha) subunit. Here we show that p85alphaalpha interacts with X-box-binding protein-1 (XBP-1), a transcriptional mediator of the unfolded protein response (UPR), in an endoplasmic reticulum (ER) stress-dependent manner. Cell lines with knockout or knockdown of p85alphaalpha show marked alterations in the UPR, including reduced ER stress-dependent accumulation of nuclear XBP-1, decreased induction of UPR target genes and increased rates of apoptosis. This is associated with a decreased activation of inositol-requiring protein-1alpha (IRE1alpha) and activating transcription factor-6alphaalpha (ATF6alpha). Mice with deletion of p85alpha in liver (L-Pik3r1(-/-)) show a similar attenuated UPR after tunicamycin administration, leading to an increased inflammatory response. Thus, p85alphaalpha forms a previously unrecognized link between the PI3K pathway, which is central to insulin action, and the regulation of the cellular response to ER stress, a state that when unresolved leads to insulin resistance. PMID:20348923

Winnay, Jonathon N; Boucher, Jeremie; Mori, Marcelo A; Ueki, Kohjiro; Kahn, C Ronald

2010-03-28

180

Defects in IRE1 enhance cell death and fail to degrade mRNAs encoding secretory pathway proteins in the Arabidopsis unfolded protein response  

PubMed Central

The unfolded protein response (UPR) is a cellular response highly conserved in eukaryotes to obviate accumulation of misfolded proteins in the endoplasmic reticulum (ER). Inositol-requiring enzyme 1 (IRE1) catalyzes the cytoplasmic splicing of mRNA encoding bZIP transcription factors to activate the UPR signaling pathway. Arabidopsis IRE1 was recently shown to be involved in the cytoplasmic splicing of bZIP60 mRNA. In the present study, we demonstrated that an Arabidopsis mutant with defects in two IRE1 paralogs showed enhanced cell death upon ER stress compared with a mutant with defects in bZIP60 and wild type, suggesting an alternative function of IRE1 in the UPR. Analysis of our previous microarray data and subsequent quantitative PCR indicated degradation of mRNAs encoding secretory pathway proteins by tunicamycin, DTT, and heat in an IRE1-dependent manner. The degradation of mRNAs localized to the ER during the UPR was considered analogous to a molecular mechanism referred to as the regulated IRE1-dependent decay of mRNAs reported in metazoans. Another microarray analysis conducted in the condition repressing transcription with actinomycin D and a subsequent Gene Set Enrichment Analysis revealed the regulated IRE1-dependent decay of mRNAs-mediated degradation of a significant portion of mRNAs encoding the secretory pathway proteins. In the mutant with defects in IRE1, genes involved in the cytosolic protein response such as heat shock factor A2 were up-regulated by tunicamycin, indicating the connection between the UPR and the cytosolic protein response.

Mishiba, Kei-ichiro; Nagashima, Yukihiro; Suzuki, Eiji; Hayashi, Noriko; Ogata, Yoshiyuki; Shimada, Yukihisa; Koizumi, Nozomu

2013-01-01

181

Defects in IRE1 enhance cell death and fail to degrade mRNAs encoding secretory pathway proteins in the Arabidopsis unfolded protein response.  

PubMed

The unfolded protein response (UPR) is a cellular response highly conserved in eukaryotes to obviate accumulation of misfolded proteins in the endoplasmic reticulum (ER). Inositol-requiring enzyme 1 (IRE1) catalyzes the cytoplasmic splicing of mRNA encoding bZIP transcription factors to activate the UPR signaling pathway. Arabidopsis IRE1 was recently shown to be involved in the cytoplasmic splicing of bZIP60 mRNA. In the present study, we demonstrated that an Arabidopsis mutant with defects in two IRE1 paralogs showed enhanced cell death upon ER stress compared with a mutant with defects in bZIP60 and wild type, suggesting an alternative function of IRE1 in the UPR. Analysis of our previous microarray data and subsequent quantitative PCR indicated degradation of mRNAs encoding secretory pathway proteins by tunicamycin, DTT, and heat in an IRE1-dependent manner. The degradation of mRNAs localized to the ER during the UPR was considered analogous to a molecular mechanism referred to as the regulated IRE1-dependent decay of mRNAs reported in metazoans. Another microarray analysis conducted in the condition repressing transcription with actinomycin D and a subsequent Gene Set Enrichment Analysis revealed the regulated IRE1-dependent decay of mRNAs-mediated degradation of a significant portion of mRNAs encoding the secretory pathway proteins. In the mutant with defects in IRE1, genes involved in the cytosolic protein response such as heat shock factor A2 were up-regulated by tunicamycin, indicating the connection between the UPR and the cytosolic protein response. PMID:23509268

Mishiba, Kei-ichiro; Nagashima, Yukihiro; Suzuki, Eiji; Hayashi, Noriko; Ogata, Yoshiyuki; Shimada, Yukihisa; Koizumi, Nozomu

2013-03-18

182

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

183

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

184

Endoplasmic reticulum stress and the unfolded protein response are linked to synergistic IFN-beta induction via X-box binding protein 1.  

PubMed

Type I IFN are strongly induced upon engagement of certain pattern recognition receptors by microbial products, and play key roles in regulating innate and adaptive immunity. It has become apparent that the endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR), in addition to restoring ER homeostasis, also influences the expression of certain inflammatory cytokines. However, the extent to which UPR signaling regulates type I IFN remains unclear. Here we show that cells undergoing a UPR respond to TLR4 and TLR3 ligands, and intracellular dsRNA, with log-fold greater IFN-beta induction. This synergy is not dependent on autocrine type I IFN signaling, but unexpectedly requires the UPR transcription factor X-box binding protein 1 (XBP-1). Synergistic IFN-beta induction also occurs in HLA-B27/human beta(2)m-transgenic rat macrophages exhibiting a UPR as a consequence of HLA-B27 up-regulation, where it correlates with activation of XBP-1 splicing. Together these findings indicate that the cellular response to endogenous 'danger' that disrupts ER homeostasis is coupled to IFN-beta induction by XBP-1, which has implications for the immune response and the pathogenesis of diseases involving the UPR. PMID:18412159

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

2008-05-01

185

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

186

Sep15, a Thioredoxin-like Selenoprotein, is Involved in the Unfolded Protein Response and Differentially Regulated by Adaptive and Acute ER Stresses†  

PubMed Central

The accumulation of misfolded proteins in the endoplasmic reticulum (ER) results in activation of sinaling pathways collectively known as the unfolded protein response (UPR). The UPR promotes adaptation of cells to ER stress by transient inhibition of protein translation and transcriptional up-regulation of genes encoding chaperones, oxidoreductases and ER-associated degradation components. But it may also trigger apoptosis in response to persistent ER stress. Recently, a novel selenocysteine-containing oxidoreductase, Sep15, has been reported to reside in the ER lumen. It has been proposed that this oxidoreductase may assist oxidative folding and structural maturation of N-glycosylated proteins targeted by UDP-glucose:glycoprotein glucosyltransferase, a chaperone implicated in quality control in the ER that forms a 1:1 complex with Sep15. To address the role of Sep15 in protein folding, we analyzed changes in Sep15 expression in murine fibroblast NIH3T3 cells in response to tunicamycin, brefeldin A (brefA), thapsigargin and DTT that lead to accumulation of unfolded proteins within the ER. We show that expression of this protein is transcriptionally up-regulated in response to adaptive UPR caused by tunicamycin and brefA, whereas acute ER stress caused by DTT and thapsigargin leads to rapid and specific degradation of Sep15 by proteasomes. However, Sep15 deficiency did not result in detectable ER stress, consistent with the idea that Sep15 assists in the maturation of a restricted group of N-glycosylated proteins and/or that its function may be compensated by other mechanisms.

Labunskyy, Vyacheslav M.; Yoo, Min-Hyuk; Hatfield, Dolph L.; Gladyshev, Vadim N.

2009-01-01

187

Blunted activation of NF-{kappa}B and NF-{kappa}B-dependent gene expression by geranylgeranylacetone: Involvement of unfolded protein response  

SciTech Connect

Geranylgeranylacetone (GGA), an anti-ulcer agent, has anti-inflammatory potential against experimental colitis and ischemia-induced renal inflammation. However, molecular mechanisms involved in its anti-inflammatory effects are largely unknown. We found that, in glomerular mesangial cells, GGA blocked activation of nuclear factor-{kappa}B and consequent induction of monocyte chemoattractant protein 1 (MCP-1) by inflammatory cytokines. It was inversely correlated with induction of unfolded protein response (UPR) evidenced by expression of 78 kDa glucose-regulated protein (GRP78) and suppression of endoplasmic reticulum stress-responsive alkaline phosphatase. Various inducers of UPR including tunicamycin, thapsigargin, A23187, 2-deoxyglucose, dithiothreitol, and AB{sub 5} subtilase cytotoxin reproduced the suppressive effects of GGA. Furthermore, attenuation of UPR by stable transfection with GRP78 diminished the anti-inflammatory effects of GGA. These results disclosed a novel, UPR-dependent mechanism underlying the anti-inflammatory potential of GGA.

Hayakawa, Kunihiro; Hiramatsu, Nobuhiko; Okamura, Maro; Yao, Jian [Department of Molecular Signaling, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi 409-3898 (Japan); Paton, Adrienne W.; Paton, James C. [School of Molecular and Biomedical Science, University of Adelaide, SA 5005 (Australia); Kitamura, Masanori [Department of Molecular Signaling, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi 409-3898 (Japan)], E-mail: masanori@yamanashi.ac.jp

2008-01-04

188

Active unfolding of precursor proteins during mitochondrial protein import.  

PubMed

Precursor proteins made in the cytoplasm must be in an unfolded conformation during import into mitochondria. Some precursor proteins have tightly folded domains but are imported faster than they unfold spontaneously, implying that mitochondria can unfold proteins. We measured the import rates of artificial precursors containing presequences of varying length fused to either mouse dihydrofolate reductase or bacterial barnase, and found that unfolding of a precursor at the mitochondrial surface is dramatically accelerated when its presequence is long enough to span both membranes and to interact with mhsp70 in the mitochondrial matrix. If the presequence is too short, import is slow but can be strongly accelerated by urea-induced unfolding, suggesting that import of these 'short' precursors is limited by spontaneous unfolding at the mitochondrial surface. With precursors that have sufficiently long presequences, unfolding by the inner membrane import machinery can be orders of magnitude faster than spontaneous unfolding, suggesting that mhsp70 can act as an ATP-driven force-generating motor during protein import. PMID:9362487

Matouschek, A; Azem, A; Ratliff, K; Glick, B S; Schmid, K; Schatz, G

1997-11-17

189

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

190

The crystal structure of human IRE1 luminal domain reveals a conserved dimerization interface required for activation of the unfolded protein response  

SciTech Connect

The unfolded protein response (UPR) is an evolutionarily conserved mechanism by which all eukaryotic cells adapt to the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Inositol-requiring kinase 1 (IRE1) and PKR-related ER kinase (PERK) are two type I transmembrane ER-localized protein kinase receptors that signal the UPR through a process that involves homodimerization and autophosphorylation. To elucidate the molecular basis of the ER transmembrane signaling event, we determined the x-ray crystal structure of the luminal domain of human IRE1{alpha}. The monomer of the luminal domain comprises a unique fold of a triangular assembly of {beta}-sheet clusters. Structural analysis identified an extensive dimerization interface stabilized by hydrogen bonds and hydrophobic interactions. Dimerization creates an MHC-like groove at the interface. However, because this groove is too narrow for peptide binding and the purified luminal domain forms high-affinity dimers in vitro, peptide binding to this groove is not required for dimerization. Consistent with our structural observations, mutations that disrupt the dimerization interface produced IRE1{alpha} molecules that failed to either dimerize or activate the UPR upon ER stress. In addition, mutations in a structurally homologous region within PERK also prevented dimerization. Our structural, biochemical, and functional studies in vivo altogether demonstrate that IRE1 and PERK have conserved a common molecular interface necessary and sufficient for dimerization and UPR signaling.

Zhou, Jiahai; Liu, Chuan Yin; Back, Sung Hoon; Clark, Robert L.; Peisach, Daniel; Xu, Zhaohui; Kaufman, Randal J. (Michigan)

2010-03-08

191

Analyzing Forced Unfolding of Protein Tandems by Ordered Variates, 1: Independent Unfolding Times  

PubMed Central

Most of the mechanically active proteins are organized into tandems of identical repeats, (D)N, or heterogeneous tandems, D1–D2–…–DN. In current atomic force microscopy experiments, conformational transitions of protein tandems can be accessed by employing constant stretching force f (force-clamp) and by analyzing the recorded unfolding times of individual domains. Analysis of unfolding data for homogeneous tandems relies on the assumption that unfolding times are independent and identically distributed, and involves inference of the (parent) probability density of unfolding times from the histogram of the combined unfolding times. This procedure cannot be used to describe tandems characterized by interdomain interactions, or heteregoneous tandems. In this article, we introduce an alternative approach that is based on recognizing that the observed data are ordered, i.e., first, second, third, etc., unfolding times. The approach is exemplified through the analysis of unfolding times for a computer model of the homogeneous and heterogeneous tandems, subjected to constant force. We show that, in the experimentally accessible range of stretching forces, the independent and identically distributed assumption may not hold. Specifically, the uncorrelated unfolding transitions of individual domains at lower force may become correlated (dependent) at elevated force levels. The proposed formalism can be used in atomic force microscopy experiments to infer the unfolding time distributions of individual domains from experimental histograms of ordered unfolding times, and it can be extended to analyzing protein tandems that exhibit interdomain interactions.

Bura, E.; Klimov, D. K.; Barsegov, V.

2007-01-01

192

Glucose starvation induces cell death in K-ras-transformed cells by interfering with the hexosamine biosynthesis pathway and activating the unfolded protein response.  

PubMed

Cancer cells, which use more glucose than normal cells and accumulate extracellular lactate even under normoxic conditions (Warburg effect), have been reported to undergo cell death under glucose deprivation, whereas normal cells remain viable. As it may be relevant to exploit the molecular mechanisms underlying this biological response to achieve new cancer therapies, in this paper we sought to identify them by using transcriptome and proteome analysis applied to an established glucose-addicted cellular model of transformation, namely, murine NIH-3T3 fibroblasts harboring an oncogenic K-RAS gene, compared with parental cells. Noteworthy is that the analyses performed in high- and low-glucose cultures indicate that reduction of glucose availability induces, especially in transformed cells, a significant increase in the expression of several unfolded protein response (UPR) hallmark genes. We show that this response is strictly associated with transformed cell death, given that its attenuation, by reducing protein translation or by increasing cell protein folding capacity, preserves the survival of transformed cells. Such an effect is also observed by inhibiting c-Jun NH2-terminal kinase, a pro-apoptotic signaling mediator set downstream of UPR. Strikingly, addition of N-acetyl-D-glucosamine, a specific substrate for the hexosamine biosynthesis pathway (HBP), to glucose-depleted cells completely prevents transformed cell death, stressing the important role of glucose in HBP fuelling to ensure UPR attenuation and increased cell survival. Interestingly, these results have been fully recognized in a human model of breast cancer, MDA-MB-231 cells. In conclusion, we show that glucose deprivation, leading to harmful accumulation of unfolded proteins in consequence of a reduction of protein glycosylation, induces a UPR-dependent cell death mechanism. These findings may open the way for new therapeutic strategies to specifically kill glycolytic cancer cells. PMID:23868065

Palorini, R; Cammarata, F; Balestrieri, C; Monestiroli, A; Vasso, M; Gelfi, C; Alberghina, L; Chiaradonna, F

2013-07-18

193

Glucose starvation induces cell death in K-ras-transformed cells by interfering with the hexosamine biosynthesis pathway and activating the unfolded protein response  

PubMed Central

Cancer cells, which use more glucose than normal cells and accumulate extracellular lactate even under normoxic conditions (Warburg effect), have been reported to undergo cell death under glucose deprivation, whereas normal cells remain viable. As it may be relevant to exploit the molecular mechanisms underlying this biological response to achieve new cancer therapies, in this paper we sought to identify them by using transcriptome and proteome analysis applied to an established glucose-addicted cellular model of transformation, namely, murine NIH-3T3 fibroblasts harboring an oncogenic K-RAS gene, compared with parental cells. Noteworthy is that the analyses performed in high- and low-glucose cultures indicate that reduction of glucose availability induces, especially in transformed cells, a significant increase in the expression of several unfolded protein response (UPR) hallmark genes. We show that this response is strictly associated with transformed cell death, given that its attenuation, by reducing protein translation or by increasing cell protein folding capacity, preserves the survival of transformed cells. Such an effect is also observed by inhibiting c-Jun NH2-terminal kinase, a pro-apoptotic signaling mediator set downstream of UPR. Strikingly, addition of N-acetyl-𝒟-glucosamine, a specific substrate for the hexosamine biosynthesis pathway (HBP), to glucose-depleted cells completely prevents transformed cell death, stressing the important role of glucose in HBP fuelling to ensure UPR attenuation and increased cell survival. Interestingly, these results have been fully recognized in a human model of breast cancer, MDA-MB-231 cells. In conclusion, we show that glucose deprivation, leading to harmful accumulation of unfolded proteins in consequence of a reduction of protein glycosylation, induces a UPR-dependent cell death mechanism. These findings may open the way for new therapeutic strategies to specifically kill glycolytic cancer cells.

Palorini, R; Cammarata, F; Balestrieri, C; Monestiroli, A; Vasso, M; Gelfi, C; Alberghina, L; Chiaradonna, F

2013-01-01

194

The role of endoplasmic reticulum stress-related unfolded protein response in the radiocontrast medium-induced renal tubular cell injury.  

PubMed

Contrast medium (CM) induces a direct toxic effect on renal tubular cells. This toxic effect may have a role in the pathophysiology of CM-induced nephropathy. CM has been shown to affect the endoplasmic reticulum (ER)-related capacity. Unfolded protein response (UPR) is known as a prosurvival response to reduce the accumulation of unfolded proteins and restore normal ER function. However, the role of ER stress-related UPR in the CM-induced renal cell injury still remains unclear. In this study, we examined whether UPR participates in urografin (an ionic CM)-induced renal tubular cells apoptosis. Treatment with urografin in normal rat renal tubular cell line (NRK52E) markedly increased cell apoptosis and decreased cell viability with a dose- and time-dependent manner. The cell necrosis was not increased in urografin-treated cells. Urografin also enhance the induction of ER stress-related markers in NRK52E cells, including glucose-regulated protein (GRP)78 and GRP94 expressions, procaspase-12 cleavage, phosphorylation of PERK (PKR [double-stranded RNA-activated protein kinase]-like ER kinase), and eukaryotic initiation factor 2alpha (eIF2alpha). Salubrinal, a selective inhibitor of eIF2alpha dephosphorylation, effectively decreased urografin-induced cell apoptosis. Furthermore, transfection of GRP78-small interfering RNA in NRK52E cells significantly enhanced urografin-induced cell apoptosis. These results suggest that GRP78/eIF2alpha-related signals play a protective role during UPR, and the activation of ER stress-related UPR may play an important regulative role in urografin-induced renal tubular injury. PMID:20071420

Wu, Cheng T; Sheu, Meei L; Tsai, Keh S; Weng, Te I; Chiang, Chih K; Liu, Shing H

2010-01-13

195

Chronic ethanol exposure increases cytochrome P-450 and decreases activated in blocked unfolded protein response gene family transcripts in caenorhabditis elegans.  

PubMed

Ethanol is a widely consumed and rapidly absorbed toxin. While the physiological effects of ethanol consumption are well known, the underlying biochemical and molecular changes at the gene expression level in whole animals remain obscure. We exposed the model organism Caenorhabditis elegans to 0.2 M ethanol from the embryo to L4 larva stage and assayed gene expression changes in whole animals using RNA-Seq and quantitative real-time PCR. We observed gene expression changes in 1122 genes (411 up, 711 down). Cytochrome P-450 (CYP) gene family members (12 of 78) were upregulated, whereas activated in blocked unfolded protein response (ABU) (7 of 15) were downregulated. Other detoxification gene family members were also regulated including four glutathione-S-transferases and three flavin monooxygenases. The results presented show specific gene expression changes following chronic ethanol exposure in C. elegans that indicate both persistent upregulation of detoxification response genes and downregulation of endoplasmic reticulum stress pathway genes. PMID:23381935

Peltonen, Juhani; Aarnio, Vuokko; Heikkinen, Liisa; Lakso, Merja; Wong, Garry

2013-02-04

196

Identification of the G13 (cAMP-response-element-binding protein-related protein) gene product related to activating transcription factor 6 as a transcriptional activator of the mammalian unfolded protein response.  

PubMed Central

Eukaryotic cells control the levels of molecular chaperones and folding enzymes in the endoplasmic reticulum (ER) by a transcriptional induction process termed the unfolded protein response (UPR). The mammalian UPR is mediated by the cis-acting ER stress response element consisting of 19 nt (CCAATN(9)CCACG), the CCACG part of which is considered to provide specificity. We recently identified the basic leucine zipper (bZIP) protein ATF6 as a mammalian UPR-specific transcription factor; ATF6 is activated by ER stress-induced proteolysis and binds directly to CCACG. Here we report that eukaryotic cells express another bZIP protein closely related to ATF6 in both structure and function. This protein encoded by the G13 (cAMP response element binding protein-related protein) gene is constitutively synthesized as a type II transmembrane glycoprotein anchored in the ER membrane and processed into a soluble form upon ER stress as occurs with ATF6. The proteolytic processing of ATF6 and the G13 gene product is accompanied by their relocation from the ER to the nucleus; their basic regions seem to function as a nuclear localization signal. Overexpression of the soluble form of the G13 product constitutively activates the UPR, whereas overexpression of a mutant lacking the activation domain exhibits a strong dominant-negative effect. Furthermore, the soluble forms of ATF6 and the G13 gene product are unable to bind to several point mutants of the cis-acting ER stress response element in vitro that hardly respond to ER stress in vivo. We thus concluded that the two related bZIP proteins are crucial transcriptional regulators of the mammalian UPR, and propose calling the ATF6 gene product ATF6alpha and the G13 gene product ATF6beta.

Haze, K; Okada, T; Yoshida, H; Yanagi, H; Yura, T; Negishi, M; Mori, K

2001-01-01

197

Electrospray Ionization-Induced Protein Unfolding  

NASA Astrophysics Data System (ADS)

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 < 80 mM) revealed evidence, based on the measured charge state distribution, of 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.

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

2012-12-01

198

Differential unfolded protein response during Chikungunya and Sindbis virus infection: CHIKV nsP4 suppresses eIF2? phosphorylation.  

PubMed

Chikungunya (CHIKV) and Sindbis (SINV) are arboviruses belonging to the alphavirus genus within the Togaviridae family. They cause frequent epidemics of febrile illness and long-term arthralgic sequelae that affect millions of people each year. Both viruses replicate prodigiously in infected patients and in vitro in mammalian cells, suggesting some level of control over the host cellular translational machinery that senses and appropriately directs the cell's fate through the unfolded protein response (UPR). The mammalian UPR involves BIP (or GRP78), the master sensor in the endoplasmic reticulum (ER) together with the three downstream effector branches: inositol-requiring ser/thr protein kinase/endonuclease (IRE-1), PKR-like ER resident kinase (PERK) and activating transcription factor 6 (ATF-6). Through careful analysis of CHIKV and SINV infections in cell culture we found that the former selectively activates ATF-6 and IRE-1 branches of UPR and suppresses the PERK pathway. By separately expressing each of the CHIKV proteins as GFP-fusion proteins, we found that non-structural protein 4 (nsP4), which is a RNA-dependent-RNA polymerase, suppresses the serine-51 phosphorylation of eukaryotic translation initiation factor, alpha subunit (eIF2?), which in turn regulates the PERK pathway. This study provides insight into a mechanism by which CHIKV replication responds to overcome the host UPR machinery. PMID:23356742

Rathore, Abhay P S; Ng, Mah-Lee; Vasudevan, Subhash G

2013-01-28

199

The Graded Unfolding Model: A Unidimensional Item Response Model for Unfolding Graded Responses.  

ERIC Educational Resources Information Center

Binary or graded disagree-agree responses to attitude items are often collected for the purpose of attitude measurement. Although such data are sometimes analyzed with cumulative measurement models, recent investigations suggest that unfolding models are more appropriate (J. S. Roberts, 1995; W. H. Van Schuur and H. A. L. Kiers, 1994). Advances in…

Roberts, James S.; Laughlin, James E.

200

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

2013-07-12

201

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

2012-08-27

202

Activation of the Unfolded Protein Response by 2-Deoxy-d-Glucose Inhibits Kaposi's Sarcoma-Associated Herpesvirus Replication and Gene Expression  

PubMed Central

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.

Leung, Howard J.; Duran, Elda M.; Kurtoglu, Metin; Andreansky, Samita

2012-01-01

203

Trans-Golgi network and endosome dynamics connect ceramide homeostasis with regulation of the unfolded protein response and TOR signaling in yeast.  

PubMed

Synthetic genetic array analyses identify powerful genetic interactions between a thermosensitive allele (sec14-1(ts)) of the structural gene for the major yeast phosphatidylinositol transfer protein (SEC14) and a structural gene deletion allele (tlg2Delta) for the Tlg2 target membrane-soluble N-ethylmaleimide-sensitive factor attachment protein receptor. The data further demonstrate Sec14 is required for proper trans-Golgi network (TGN)/endosomal dynamics in yeast. Paradoxically, combinatorial depletion of Sec14 and Tlg2 activities elicits trafficking defects from the endoplasmic reticulum, and these defects are accompanied by compromise of the unfolded protein response (UPR). UPR failure occurs downstream of Hac1 mRNA splicing, and it is further accompanied by defects in TOR signaling. The data link TGN/endosomal dynamics with ceramide homeostasis, UPR activity, and TOR signaling in yeast, and they identify the Sit4 protein phosphatase as a primary conduit through which ceramides link to the UPR. We suggest combinatorial Sec14/Tlg2 dysfunction evokes inappropriate turnover of complex sphingolipids in endosomes. One result of this turnover is potentiation of ceramide-activated phosphatase-mediated down-regulation of the UPR. These results provide new insight into Sec14 function, and they emphasize the TGN/endosomal system as a central hub for homeostatic regulation in eukaryotes. PMID:18753406

Mousley, Carl J; Tyeryar, Kimberly; Ile, Kristina E; Schaaf, Gabriel; Brost, Renee L; Boone, Charles; Guan, Xueli; Wenk, Markus R; Bankaitis, Vytas A

2008-08-27

204

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

205

Melatonin treatment reduces endoplasmic reticulum stress and modulates the unfolded protein response in rabbits with lethal fulminant hepatitis of viral origin.  

PubMed

Hepatocyte apoptosis plays an important role in the development of fulminant hepatic failure (FHF). The objective of this study was to investigate whether endoplasmic reticulum (ER) stress and unfolded protein response (UPR) inhibition is an underlying mechanism of melatonin anti-apoptotic effects in an animal model of FHF of viral origin induced by the rabbit hemorrhagic disease virus (RHDV). Rabbits were experimentally infected with 2 × 10(4) hemagglutination units of a RHDV isolate and received melatonin at two concentrations of 10 mg/kg and 20 mg/kg at 0 hr, 12 hr and 24 hr postinfection. RHDV infection induced increased expression of CCAAT/enhancer-binding protein homologous protein (CHOP), immunoglobulin heavy chain binding protein (BiP/GRP78), glucose-regulated protein 94 (GRP94), phospho-c-Jun N-terminal kinase (JNK) and caspase-12. These effects were attenuated by melatonin. Double immunofluorescence staining showed colocalization of CHOP and cleaved caspase-3 in liver sections of RHDV-infected rabbits, while immunostaining decreased markedly with melatonin treatment. RHDV infection resulted in significant increases in the mRNA levels of activating transcription factor 6 (ATF6), ATF4, inositol-requiring enzyme 1 (IRE1), spliced X-box binding protein-1 (XBP1s) and tumor necrosis factor receptor-associated factor 2 (TRAF2). Melatonin attenuated the extent of the changes. Data obtained provide evidence that in rabbits with experimental infection by RHDV, reduction in apoptotic liver damage by melatonin is associated with attenuation of ER stress through a modulation of the three arms of UPR signaling and further support a potential hepatoprotective role of melatonin in FHF. PMID:23679826

Tuñón, María J; San-Miguel, Beatriz; Crespo, Irene; Laliena, Almudena; Vallejo, Daniela; Alvarez, Marcelino; Prieto, Jesús; González-Gallego, Javier

2013-05-16

206

Protein unfolding in drug-RNase complexes.  

PubMed

Bovine pancreatic ribonuclease A (RNase A) catalyzes the cleavage of P-O5' bonds in RNA on the 3' side of pyrimidine to form cyclic 2', 5'-phosphates. It has several high affinity binding sites that make it possible target for many organic and inorganic molecules. Ligand binding to RNase A can alter protein secondary structure and its catalytic activity. In this review, the effects of several drugs such as AZT (anti-AIDS), cis-Pt (antitumor), aspirin (anti-inflammatory), and vitamin C (antioxidant) on the stability and conformation of RNase A in vitro are compared. The results of UV-visible, FTIR, and CD spectroscopic analysis of RNase complexes with aspirin, AZT, cis-Pt, and vitamin C at physiological conditions are discussed here. Spectroscopic results showed one major binding for each drug-RNase adduct with KAZT=5.29 (+/-1.6)x10(4) M(-1), Kaspirin=3.57 (+/-1.4)x10(4) M(-1), Kcis-Pt=5.66 (+/-1.9)x10(3) M(-1), and Kascorbate=3.50 (+/-1.5)x10(3) M(-1). Major protein unfolding occurred with reduction of alpha-helix from 29% (free protein) to 20% and increase of beta-sheet from 39% (free protein) to 45% in the aspirin-, ascorbate-, and cis-Pt-RNase complexes, while minor increase of alpha-helix was observed for AZT-RNase adduct. PMID:18092833

Neault, J F; Diamantoglou, S; Beauregard, M; Nafisi, Sh; Tajmir-Riahi, H A

2008-02-01

207

Treadmill exercise represses neuronal cell death and inflammation during A?-induced ER stress by regulating unfolded protein response in aged presenilin 2 mutant mice.  

PubMed

Alzheimer's disease (AD) is characterized by the deposition of aggregated amyloid-beta (A?), which triggers a cellular stress response called the unfolded protein response (UPR). The UPR signaling pathway is a cellular defense system for dealing with the accumulation of misfolded proteins but switches to apoptosis when endoplasmic reticulum (ER) stress is prolonged. ER stress is involved in neurodegenerative diseases including AD, but the molecular mechanisms of neuronal apoptosis and inflammation by A?-induced ER stress to exercise training are not fully understood. Here, we demonstrated that treadmill exercise (TE) prevented PS2 mutation-induced memory impairment and reduced A?-42 deposition through the inhibition of ?-secretase (BACE-1) and its product, C-99 in cortex and/or hippocampus of aged PS2 mutant mice. We also found that TE down-regulated the expression of GRP78/Bip and PDI proteins and inhibited activation of PERK, eIF2?, ATF6?, sXBP1 and JNK-p38 MAPK as well as activation of CHOP, caspase-12 and caspase-3. Moreover, TE up-regulated the expression of Bcl-2 and down-regulated the expressions of Bax in the hippocampus of aged PS2 mutant mice. Finally, the generation of TNF? and IL-1? and the number of TUNEL-positive cells in the hippocampus of aged PS2 mutant mice was also prevented or decreased by TE. These results showed that TE suppressed the activation of UPR signaling pathways as well as inhibited the apoptotic pathways of the UPR and inflammatory response following A?-induced ER stress. Thus, therapeutic strategies that modulate A?-induced ER stress through TE could represent a promising approach for the prevention or treatment of AD. PMID:23907580

Kang, Eun-Bum; Kwon, In-Su; Koo, Jung-Hoon; Kim, Eung-Joon; Kim, Chul-Hyun; Lee, Jin; Yang, Choon-Ho; Lee, Young-Il; Cho, In-Ho; Cho, Joon-Yong

2013-11-01

208

Methylmercury exposure increases lipocalin related (lpr) and decreases activated in blocked unfolded protein response (abu) genes and specific miRNAs in Caenorhabditis elegans.  

PubMed

Methylmercury (MeHg) is a persistent environmental and dietary contaminant that causes serious adverse developmental and physiologic effects at multiple cellular levels. In order to understand more fully the consequences of MeHg exposure at the molecular level, we profiled gene and miRNA transcripts from the model organism Caenorhabditis elegans. Animals were exposed to MeHg (10?M) from embryo to larval 4 (L4) stage and RNAs were isolated. RNA-seq analysis on the Illumina platform revealed 541 genes up- and 261 genes down-regulated at a cutoff of 2-fold change and false discovery rate-corrected significance q<0.05. Among the up-regulated genes were those previously shown to increase under oxidative stress conditions including hsp-16.11 (2.5-fold), gst-35 (10.1-fold), and fmo-2 (58.5-fold). In addition, we observed up-regulation of 6 out of 7 lipocalin related (lpr) family genes and down regulation of 7 out of 15 activated in blocked unfolded protein response (abu) genes. Gene Ontology enrichment analysis highlighted the effect of genes related to development and organism growth. miRNA-seq analysis revealed 6-8 fold down regulation of mir-37-3p, mir-41-5p, mir-70-3p, and mir-75-3p. Our results demonstrate the effects of MeHg on specific transcripts encoding proteins in oxidative stress responses and in ER stress pathways. Pending confirmation of these transcript changes at protein levels, their association and dissociation characteristics with interaction partners, and integration of these signals, these findings indicate broad and dynamic mechanisms by which MeHg exerts its harmful effects. PMID:23872261

Rudgalvyte, Martina; Vanduyn, Natalia; Aarnio, Vuokko; Heikkinen, Liisa; Peltonen, Juhani; Lakso, Merja; Nass, Richard; Wong, Garry

2013-07-18

209

Thermodynamics of the complex protein unfolding reaction of barstar.  

PubMed

The complex unfolding reaction of barstar has been characterized by studying the apparent rate of unfolding, monitored by intrinsic Trp fluorescence, as a function of temperature and guanidine hydrochloride (GdnHCl) concentration. The kinetics of unfolding and folding of wild-type (wt) barstar at 5 degrees C were first studied in detail. It is shown that when unfolding is carried out using concentrations of GdnHCl in the posttransition zone of unfolding, the change in fluorescence that accompanies unfolding occurs in two phases: 30% of the change occurs in a burst phase that is complete within 4 ms, and 70% of the change occurs in a fast phase that is complete within 2 s. In contrast, when the protein is unfolded at 25 degrees C, no burst-phase change in fluorescence is observed. To confirm that a burst-phase change in fluorescence indeed accompanies unfolding at low temperature, unfolding studies were also carried out on a marginally destabilized mutant form of barstar for which the burst-phase change in fluorescence is shown to be as high as 70%. These results confirm a previous report [Nath et al., (1996), Nat. Struct. Biol. 3, 920-923], in which the detection of a burst-phase change in circular dichroism at 222 nm during unfolding at 25 degrees C led to the inclusion of a rapidly formed kinetic intermediate, IU, on the unfolding pathway. To characterize thermodynamically the unfolding pathway, apparent unfolding rates were then measured at six different concentrations of GdnHCl in the range 2.6 to 5.0 M, at five different temperatures from 5 to 46 degrees C. The subsequent analysis was done on the basis of the observation that a preequilibrium between the fully folded state (F) and IU gets established rapidly before further unfolding to the completely unfolded state (U). The results indicate that IU has a specific heat capacity similar to that of F and therefore suggest that IU is as compact as F, with practically no exposure of the hydrophobic core. On the other hand, the transition state of unfolding has a 45% greater heat capacity than F, indicating that significant hydration of the hydrophobic core occurs only after the rate-limiting step of unfolding. PMID:9315868

Agashe, V R; Schmid, F X; Udgaonkar, J B

1997-10-01

210

The next generation proteasome inhibitors carfilzomib and oprozomib activate prosurvival autophagy via induction of the unfolded protein response and ATF4.  

PubMed

The proteasome inhibitor bortezomib has shown remarkable clinical success in the treatment of multiple myeloma. However, the efficacy and mechanism of action of bortezomib in solid tumor malignancies is less well understood. In addition, the use of this first-in-class proteasome inhibitor is limited by several factors, including off-target effects that lead to adverse toxicities. We recently reported the impact and mechanisms of carfilzomib and oprozomib, second-in-class proteasome inhibitors with higher specificities and reduced toxicities, against head and neck squamous cell carcinoma (HNSCC). Carfilzomib and oprozomib potently inhibit HNSCC cell survival and the growth of HNSCC tumors. Both compounds promote upregulation of proapoptotic BIK and antiapoptotic MCL1, which serves to mediate and attenuate, respectively, the killing activities of these proteasome inhibitors. Both compounds also induce complete autophagic flux that is partially dependent on activation of the unfolded protein response (UPR) and upregulation of ATF4. Carfilzomib- and oprozomib-induced autophagy acts to promote HNSCC cell survival. Our study indicates that the therapeutic benefit of these promising proteasome inhibitors may be improved by inhibiting MCL1 expression or autophagy. PMID:22995770

Zang, Yan; Thomas, Sufi M; Chan, Elena T; Kirk, Christopher J; Freilino, Maria L; DeLancey, Hannah M; Grandis, Jennifer R; Li, Changyou; Johnson, Daniel E

2012-09-20

211

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

212

Multimolecule test-tube simulations of protein unfolding and aggregation.  

PubMed

Molecular dynamics simulations of protein folding or unfolding, unlike most in vitro experimental methods, are performed on a single molecule. The effects of neighboring molecules on the unfolding/folding pathway are largely ignored experimentally and simply not modeled computationally. Here, we present two all-atom, explicit solvent molecular dynamics simulations of 32 copies of the Engrailed homeodomain (EnHD), an ultrafast-folding and -unfolding protein for which the folding/unfolding pathway is well-characterized. These multimolecule simulations, in comparison with single-molecule simulations and experimental data, show that intermolecular interactions have little effect on the folding/unfolding pathway. EnHD unfolded by the same mechanism whether it was simulated in only water or also in the presence of other EnHD molecules. It populated the same native state, transition state, and folding intermediate in both simulation systems, and was in good agreement with experimental data available for each of the three states. Unfolding was slowed slightly by interactions with neighboring proteins, which were mostly hydrophobic in nature and ultimately caused the proteins to aggregate. Protein-water hydrogen bonds were also replaced with protein-protein hydrogen bonds, additionally contributing to aggregation. Despite the increase in protein-protein interactions, the protein aggregates formed in simulation did not do so at the total exclusion of water. These simulations support the use of single-molecule techniques to study protein unfolding and also provide insight into the types of interactions that occur as proteins aggregate at high temperature at an atomic level. PMID:23091038

McCully, Michelle E; Beck, David A C; Daggett, Valerie

2012-10-22

213

Multimolecule test-tube simulations of protein unfolding and aggregation  

PubMed Central

Molecular dynamics simulations of protein folding or unfolding, unlike most in vitro experimental methods, are performed on a single molecule. The effects of neighboring molecules on the unfolding/folding pathway are largely ignored experimentally and simply not modeled computationally. Here, we present two all-atom, explicit solvent molecular dynamics simulations of 32 copies of the Engrailed homeodomain (EnHD), an ultrafast-folding and -unfolding protein for which the folding/unfolding pathway is well-characterized. These multimolecule simulations, in comparison with single-molecule simulations and experimental data, show that intermolecular interactions have little effect on the folding/unfolding pathway. EnHD unfolded by the same mechanism whether it was simulated in only water or also in the presence of other EnHD molecules. It populated the same native state, transition state, and folding intermediate in both simulation systems, and was in good agreement with experimental data available for each of the three states. Unfolding was slowed slightly by interactions with neighboring proteins, which were mostly hydrophobic in nature and ultimately caused the proteins to aggregate. Protein–water hydrogen bonds were also replaced with protein–protein hydrogen bonds, additionally contributing to aggregation. Despite the increase in protein–protein interactions, the protein aggregates formed in simulation did not do so at the total exclusion of water. These simulations support the use of single-molecule techniques to study protein unfolding and also provide insight into the types of interactions that occur as proteins aggregate at high temperature at an atomic level.

McCully, Michelle E.; Beck, David A. C.; Daggett, Valerie

2012-01-01

214

Targeting the IRE1?/XBP1 and ATF6 arms of the unfolded protein response enhances VEGF blockade to prevent retinal and choroidal neovascularization.  

PubMed

Although anti-vascular endothelial growth factor (VEGF) treatments reduce pathological neovascularization in the eye and in tumors, the regression is often not sustainable or is incomplete. We investigated whether vascular endothelial cells circumvent anti-VEGF therapies by activating the unfolded protein response (UPR) to override the classic extracellular VEGF pathway. Exposure of endothelial cells to VEGF, high glucose, or H2O2 up-regulated the X-box binding protein-1/inositol-requiring protein-1 (IRE1) ? and activating transcription factor 6 (ATF6) arms of the UPR compared with untreated cells. This was associated with increased expression in ?-basic crystallin (CRYAB), which has previously bound VEGF. siRNA knockdown or pharmacological blockade of IRE1?, ATF6, or CRYAB increased intracellular VEGF degradation and decreased full-length intracellular VEGF. Inhibition of IRE1?, ATF6, or CRYAB resulted in an approximately 40% reduction of in vitro angiogenesis, which was further reduced in combination with a neutralizing antibody against extracellular VEGF. Blockade of IRE1? or ATF6 in the oxygen-induced retinopathy or choroidal neovascularization mouse models caused an approximately 35% reduction in angiogenesis. However, combination therapy of VEGF neutralizing antibody with UPR inhibitors or siRNAs reduced retinal/choroidal neovascularization by a further 25% to 40%, and this inhibition was significantly greater than either treatment alone. In conclusion, activation of the UPR sustains angiogenesis by preventing degradation of intracellular VEGF. The IRE1?/ATF6 arms of the UPR offer a potential therapeutic target in the treatment of pathological angiogenesis. PMID:23395094

Liu, Li; Qi, Xiaoping; Chen, Zhijuan; Shaw, Lynn; Cai, Jun; Smith, Layton H; Grant, Maria B; Boulton, Michael E

2013-02-08

215

Chaperone machines for protein folding, unfolding and disaggregation.  

PubMed

Molecular chaperones are diverse families of multidomain proteins that have evolved to assist nascent proteins to reach their native fold, protect subunits from heat shock during the assembly of complexes, prevent protein aggregation or mediate targeted unfolding and disassembly. Their increased expression in response to stress is a key factor in the health of the cell and longevity of an organism. Unlike enzymes with their precise and finely tuned active sites, chaperones are heavy-duty molecular machines that operate on a wide range of substrates. The structural basis of their mechanism of action is being unravelled (in particular for the heat shock proteins HSP60, HSP70, HSP90 and HSP100) and typically involves massive displacements of 20-30 kDa domains over distances of 20-50 Å and rotations of up to 100°. PMID:24026055

Saibil, Helen

2013-09-12

216

Retention, Unfolding, and Deformation of Soluble Proteins on Solids  

SciTech Connect

The behavior of proteins on solids has been studied over several decades. Recent developments have expanded the variety of methods that can be used to examine protein-surface interactions. This paper summarizes the adsorptive behavior of proteins on various surfaces including cotton and synthetic polymers. In general, it is postulated that surfaces act like catalysts for protein unfolding. We give specific examples of the unfolding of cytochrome c and show a lowering of the unfolding temperature from solution to an anionic surface. Additionally, we show how chromatography can be used to screen adsorbents, such as cotton for possible commercial applications. The ability of each surface chemistry to act as a catalyst for unfolding is discussed.

Goheen, Steven C.; Gibbins, Betty M.; Hilsenbeck, Jacqueline L.; Edwards, J V.; J. Vincent Edwards and Tyrone L. Vigo

2001-03-01

217

Hypoxia stimulates migration of breast cancer cells via the PERK/ATF4/LAMP3-arm of the unfolded protein response  

PubMed Central

Introduction The hypoxia-inducible factor (HIF)-1 pathway can stimulate tumor cell migration and metastasis. Furthermore, hypoxic tumors are associated with a poor prognosis. Besides the HIF-1 pathway, the unfolded protein response (UPR) is also induced by hypoxic conditions. The PKR-like ER kinase (PERK)/activating transcription factor 4 (ATF4)-arm of the UPR induces expression of lysosomal-associated membrane protein 3 (LAMP3), a factor that has been linked to metastasis and poor prognosis in solid tumors. In this study the role of UPR-induced LAMP3 in hypoxia-mediated migration of breast cancer cells was examined. Methods A number of in vitro metastasis models were used to study the migration and invasion of MDA-MB-231 breast cancer cells under hypoxic conditions. PERK, ATF4 and their downstream factor LAMP3 were knocked down to examine their role in cell migration. In addition, multicellular tumor spheroids were used to study the involvement of the tumor microenvironment in invasion. Results Using transwell assays, migration of different breast cancer cell lines was assessed. A direct correlation was found between cell migration and baseline LAMP3 expression. Furthermore, moderate hypoxia (1% O2) was found to be optimal in stimulating migration of MDA-MB-231 cells. siRNA mediated knockdown of PERK, ATF4 and LAMP3 reduced migration of cells under these conditions. Using gap closure assays, similar results were found. In a three-dimensional invasion assay into collagen, LAMP3 knockdown cells showed a diminished capacity to invade compared to control cells when collectively grown in multicellular spheroids. Conclusions Thus, the PERK/ATF4/LAMP3-arm of the UPR is an additional pathway mediating hypoxia-induced breast cancer cell migration.

2013-01-01

218

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

219

Unfolded protein ensembles, folding trajectories, and refolding rate prediction.  

PubMed

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. PMID:24089737

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

2013-09-28

220

Protein folding and unfolding under force.  

PubMed

The recent revolution in optics and instrumentation has enabled the study of protein folding using extremely low mechanical forces as the denaturant. This exciting development has led to the observation of the protein folding process at single molecule resolution and its response to mechanical force. Here, we describe the principles and experimental details of force spectroscopy on proteins, with a focus on the optical tweezers instrument. Several recent results will be discussed to highlight the importance of this technique in addressing a variety of questions in the protein folding field. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 860-869, 2013. PMID:23784721

Jagannathan, Bharat; Marqusee, Susan

2013-11-01

221

Temperature and Pressure effects on folding/unfolding of proteins  

NASA Astrophysics Data System (ADS)

High hydrostatic pressures change the energy landscape of proteins, affecting the thermodynamics and kinetics of folding. Proteins denature at high hydrostatic pressures, implying that the unfolded proteins in aqueous solution have lower volume than the folded state. A model that explains pressure unfolding requires water to penetrate the protein interior and disrupt the protein hydrophobic core. I will explore the energetics of water penetration and the effect of pressure on hydrophobic interactions. I will also describe molecular simulations of the reversible folding/unfolding equilibrium as a function of density and temperature of solvated peptides that can form alpha helices (the AK peptide) and beta hairpins (the C terminal domain of protein G). I will characterize the structural, thermodynamic and hydration changes as a function of temperature and pressure. To study protein folding equilibrium thermodynamics we use an extension of the replica exchange molecular dynamics (REMD) method that allows for density and temperature Monte Carlo exchange moves.

Garcia, Angel

2006-03-01

222

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

223

Evidence that autophagy, but not the unfolded protein response, regulates the expression of IL-23 in the gut of patients with ankylosing spondylitis and subclinical gut inflammation.  

PubMed

OBJECTIVES: Interleukin (IL)-23 has been implicated in the pathogenesis of ankylosing spondylitis (AS). The aim of the study was to clarify the mechanisms underlying the increased IL-23 expression in the gut of AS patients. METHODS: Consecutive gut biopsies from 30 HLA-B27(+) AS patients, 15 Crohn's disease (CD) patients and 10 normal subjects were obtained. Evidence for HLA-B27 misfolding was studied. Unfolded protein response (UPR) and autophagy were assessed by RT-PCR and immunohistochemistry. The contribution of UPR and autophagy in the regulation of IL-23 expression was evaluated in in vitro experiments on isolated lamina propria mononuclear cells (LPMCs). RESULTS: Intracellular colocalisation of SYVN1 and FHCs but not a significant overexpression of UPR genes was observed in the gut of AS patients. Conversely, upregulation of the genes involved in the autophagy pathway was observed in the gut of AS and CD patients. Immunohistochemistry showed an increased expression of LC3II, ATG5 and ATG12 but not of SQSTM1 in the ileum of AS and CD patients. LC3II was expressed among infiltrating mononuclear cells and epithelial cells resembling Paneth cells (PC) and colocalised with ATG5 in AS and CD. Autophagy but not UPR was required to modulate the expression of IL-23 in isolated LPMCs of AS patients with chronic gut inflammation, CD patients and controls. CONCLUSIONS: Our data suggest that HLA-B27 misfolding occurs in the gut of AS patients and is accompanied by activation of autophagy rather than a UPR. Autophagy appears to be associated with intestinal modulation of IL-23 in AS. PMID:23740229

Ciccia, Francesco; Accardo-Palumbo, Antonina; Rizzo, Aroldo; Guggino, Giuliana; Raimondo, Stefania; Giardina, Annarita; Cannizzaro, Alessandra; Colbert, Robert A; Alessandro, Riccardo; Triolo, Giovanni

2013-06-01

224

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

225

Dual regulation of cadmium-induced apoptosis by mTORC1 through selective induction of IRE1 branches in unfolded protein response.  

PubMed

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. PMID:23696882

Kato, Hironori; Katoh, Ryohei; Kitamura, Masanori

2013-05-16

226

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

227

TNF potentiates anticancer activity of bortezomib (Velcade) through reduced expression of proteasome subunits and dysregulation of unfolded protein response.  

PubMed

Bortezomib (Velcade) exploits proteasome inhibition as a unique mechanism of anticancer activity. The effectiveness of bortezomib is, however, limited, therefore, the search for therapeutic regimens combining bortezomib with other agents. In the present work we demonstrate enhanced anticancer activity of bortezomib by its combination with tumor necrosis factor (TNF) in the experimental model of C-26 colon carcinoma in mice. This interaction likely relies on the induction of a dysregulated response to ER stress, leading to apoptosis of cancer cells, evidenced by caspase-3 cleavage, p53 accumulation as well as increased SAPK/JNK phosphorylation. ER stress induced by the combination of TNF and bortezomib is corroborated by upregulation of BiP, PDI and calnexin as well as cleavage of caspase-12; however, in contrast to the classic pathway, it is also associated with decreased phosphorylation of eIF2 alpha and prevention of XBP-1 splicing. TNF prevented the upregulation of Hsp27 induced by bortezomib, which may contribute to enhanced ER stress. Moreover, TNF interfered with bortezomib-induced upregulation of distinct subunits of the 26S proteasome. Bortezomib concentration used in this study was not sufficient to prevent TNF from inducing nuclear translocation of p65/RelA; however, the combination of both agents reduced total p65/RelA levels. Combined treatment of tumor-bearing mice with bortezomib and TNF not only inhibited tumor growth but also significantly prolonged animal survival. Therefore, combination of bortezomib with TNF is an attractive option for further clinical studies. PMID:17373661

Nowis, Dominika; McConnell, Elizabeth J; Dierlam, Lindsey; Palamarchuk, Alla; Lass, Agnieszka; Wójcik, Cezary

2007-07-15

228

Expansion and internal friction in unfolded protein chain.  

PubMed

Similarities in global properties of homopolymers and unfolded proteins provide approaches to mechanistic description of protein folding. Here, hydrodynamic properties and relaxation rates of the unfolded state of carbonmonoxide-liganded cytochrome c (cyt-CO) have been measured using nuclear magnetic resonance and laser photolysis methods. Hydrodynamic radius of the unfolded chain gradually increases as the solvent turns increasingly better, consistent with theory. Curiously, however, the rate of intrachain contact formation also increases with an increasing denaturant concentration, which, by Szabo, Schulten, and Schulten theory for the rate of intramolecular contact formation in a Gaussian polymer, indicates growing intramolecular diffusion. It is argued that diminishing nonbonded atom interactions with increasing denaturant reduces internal friction and, thus, increases the rate of polypeptide relaxation. Qualitative scaling of the extent of unfolding with nonbonded repulsions allows for description of internal friction by a phenomenological model. The degree of nonbonded atom interactions largely determines the extent of internal friction. PMID:24044733

Yasin, U Mahammad; Sashi, Pulikallu; Bhuyan, Abani K

2013-09-30

229

Analysis and Interpretation of Single Molecule Protein Unfolding Kinetics  

NASA Astrophysics Data System (ADS)

The kinetics of protein unfolding under a stretching force has been extensively studied by atomic force microscopy (AFM) over the past decade [1]. Experimental artifacts at the single molecule level introduce uncertainties in the data analysis that have led to several competing physical models for the unfolding process. For example, the unfolding dynamics of the protein ubiquitin under constant force has been described by probability distributions as diverse as exponential [2,3], a sum of exponentials, log-normal [4], and more recently a function describing static disorder in the Arrhenius model [5]. A new method for data analysis is presented that utilizes maximum likelihood estimation (MLE) combined with other traditional statistical tests to unambiguously rank the consistency of these and other models with the experimental data. These techniques applied to the ubiquitin unfolding data shows that the probability of unfolding is best fit with a stretched exponential distribution, with important implications on the complexity of the mechanism of protein unfolding. [4pt] [1] Carrion-Vazquez, et. al. Springer Series in Biophys. 2006 [0pt] [2] Fernandez et. al. Science 2004 [0pt] [3] Brujic et. al. Nat. Phys 2006 [0pt] [4] Garcia-Manyes et. al. Biophys. J. 2007 [0pt] [5] Kuo et. al. PNAS 2010

Lannon, Herbert; Brujic, Jasna

2012-02-01

230

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

231

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

2012-11-20

232

Prefoldin, a Chaperone that Delivers Unfolded Proteins to Cytosolic Chaperonin  

Microsoft Academic Search

We describe the discovery of a heterohexameric chaperone protein, prefoldin, based on its ability to capture unfolded actin. Prefoldin binds specifically to cytosolic chaperonin (c-cpn) and transfers target proteins to it. Deletion of the gene encoding a prefoldin subunit in S. cerevisiae results in a phenotype similar to those found when c-cpn is mutated, namely impaired functions of the actin

Irina E Vainberg; Sally A Lewis; Heidi Rommelaere; Christophe Ampe; Joel Vandekerckhove; Hannah L Klein; Nicholas J Cowan

1998-01-01

233

Direct Association of Unfolded Proteins with Mammalian ER Stress Sensor, IRE1?  

PubMed Central

IRE1, an ER-localized transmembrane protein, plays a central role in the unfolded protein response (UPR). IRE1 senses the accumulation of unfolded proteins in its luminal domain and transmits a signal to the cytosolic side through its kinase and RNase domains. Although the downstream pathways mediated by two mammalian IRE1s, IRE1? and IRE1?, are well documented, their luminal events have not been fully elucidated. In particular, there have been no reports on how IRE1? senses the unfolded proteins. In this study, we performed a comparative analysis to clarify the luminal event mediated by the mammalian IRE1s. Confocal fluorescent microscopy using GFP-fused IRE1s revealed that IRE1? clustered into discrete foci upon ER stress. Also, fluorescence correlation spectroscopy (FCS) analysis in living cells indicated that the size of the IRE1? complex is robustly increased upon ER stress. Moreover, unlike IRE1?, the luminal domain of IRE1? showed anti-aggregation activity in vitro, and IRE1? was coprecipitated with the model unfolded proteins in cells. Strikingly, association with BiP was drastically reduced in IRE1?, while IRE1? was associated with BiP and dissociated upon ER stress. This is the first report indicating that, differently from IRE1?, the luminal event mediated by IRE1? involves direct interaction with unfolded proteins rather than association/dissociation with BiP, implying an intrinsic diversity in the sensing mechanism of mammalian sensors.

Oikawa, Daisuke; Kitamura, Akira; Kinjo, Masataka; Iwawaki, Takao

2012-01-01

234

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

235

The effect of pulsed electromagnetic fields on protein unfolding  

Microsoft Academic Search

This thesis describes a spectroscopic investigation of the effects of pulsed electromagnetic radiation on the conformation, unfolding, aggregation and precipitation of a variety of proteins. Initial experiments required the calibration of a microwave exposure system thus the temperature of different buffer solutions was studied in detail. The exposure system comprised of an incubator and a rebuilt domestic microwave oven that

Yoke Berry

2005-01-01

236

Protein's unfolding and the glass transition: a common thermodynamic signature.  

NASA Astrophysics Data System (ADS)

Recently, it has been recognized that protein's folding and unfolding mechanisms exhibit a wide range of common features with the glass transition observed in supercooled organic and inorganic liquids. Such similarities range from pure thermodynamic aspects such an anomalous ?Cp and a substantial entropy decrease ?S<0, to strictly kinetic aspects as the existence of an excess of vibrational modes at low frequencies (bosonic peak) revealed by Raman and neutron scattering experiments. In this work, we discuss both the experimental and theoretical facts that might enable an extrapolation of the Adam-Gibbs scheme for the standard glass transition to describe the relaxation time ? as function of temperature T in biological macromolecules' unfolding.

Olivares-Quiroz, L.; Garcia-Colin, L. S.

2008-02-01

237

Do proteins really unfold in a shear flow?  

NASA Astrophysics Data System (ADS)

Many protein structures unfold (denature) when subjected to extremes of heat, cold, pH, solvent composition, or mechanical stress. One might expect that shearing forces induced by a nonuniform fluid flow would also destabilize proteins, as when a protein solution flows rapidly through a narrow channel. However, although we find many references to shear denaturation in the protein literature, we find no quantitative demonstration of the phenomenon. Therefore we have investigated whether a high shear can destabilize a protein to any measureable extent. We study a small globular protein (horse cytochrome c, 104 amino acids) whose fluorescence increases sharply upon unfolding. We pump the sample through a silica capillary (180 ?m ID) at speeds ˜ 10 m/s to create a simple shear dvz/dx ˜ 5 x 10^5 s-1, under UV laser illumination. We can detect unfolding of as little as 1% of the sample, or (under favorable conditions) a reduction of ˜0.05 kJ/mol in the protein's stability. We will discuss preliminary results along with a simple theoretical perspective on shear denaturation.

Jaspe, Juan

2005-11-01

238

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

239

A General Item Response Theory Model for Unfolding Unidimensional Polytomous Responses.  

ERIC Educational Resources Information Center

Develops a generalized graded unfolding model that allows for either binary or graded responses and generalizes previous item response models for unfolding in two useful ways. It implements a discrimination parameter that varies across items and permits response category threshold parameters to vary across items. (Author/SLD)

Roberts, James S.; Donoghue, John R.; Laughlin, James E.

2000-01-01

240

Protein Unfolding in Drug-RNase Complexes  

Microsoft Academic Search

Bovine pancreatic ribonuclease A (RNase A) catalyzes the cleavage of P-O5? bonds in RNA on the 3? side of pyrimidine to form cyclic 2?, 5?-phosphates. It has several high affinity binding sites that make it possible target for many organic and inorganic molecules. Ligand binding to RNase A can alter protein secondary structure and its catalytic activity. In this review,

J. F. Neault; S. Diamantoglou; M. Beauregard; Sh. Nafisi; H. A. Tajmir-Riahi

2008-01-01

241

Unfolding times for proteins in a force clamp  

NASA Astrophysics Data System (ADS)

The escape process from the native valley for proteins subjected to a constant stretching force is examined using a model for a ? barrel. For a wide range of forces, the unfolding dynamics can be treated as one-dimensional diffusion, parametrized in terms of the end-to-end distance. In particular, the escape times can be evaluated as first passage times for a Brownian particle moving on the protein free-energy landscape, using the Smoluchowski equation. At strong forces, the unfolding process can be viewed as a diffusive drift away from the native state, while at weak forces thermal activation is the relevant mechanism. An escape-time analysis within this approach reveals a crossover from an exponential to an inverse Gaussian escape-time distribution upon passing from weak to strong forces. Moreover, a single expression valid at weak and strong forces can be devised both for the average unfolding time as well as for the corresponding variance. The analysis offers a possible explanation of recent experimental findings for the proteins ddFLN4 and ubiquitin.

Luccioli, Stefano; Imparato, Alberto; Mitternacht, Simon; Irbäck, Anders; Torcini, Alessandro

2010-01-01

242

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

2013-01-15

243

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 Central

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.

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

2013-01-01

244

Differential stability of the bovine prion protein upon urea unfolding  

PubMed Central

Prion diseases, or transmissible spongiform encephalopathies, are a group of infectious neurological diseases associated with the structural conversion of an endogenous protein (PrP) in the central nervous system. There are two major forms of this protein: the native and noninfectious cellular form, PrPC; and the misfolded, infectious, and proteinase K-resistant form, PrPSc. The C-terminal domain of PrPC is mainly ?-helical in structure, whereas PrPSc in known to aggregate into an assembly of ?-sheets, forming amyloid fibrils. To identify the regions of PrPC potentially involved in the initial steps of the conversion to the infectious conformation, we have used high-resolution NMR spectroscopy to characterize the stability and structure of bovine recombinant PrPC (residues 121 to 230) during unfolding with the denaturant urea. Analysis of the 800 MHz 1H NMR spectra reveals region-specific information about the structural changes occurring upon unfolding. Our data suggest that the dissociation of the native ?-sheet of PrPC is a primary step in the urea-induced unfolding process, while strong hydrophobic interactions between helices ?1 and ?3, and between ?2 and ?3, stabilize these regions even at very high concentrations of urea.

Julien, Olivier; Chatterjee, Subhrangsu; Thiessen, Angela; Graether, Steffen P; Sykes, Brian D

2009-01-01

245

Complete all-atom hydrodynamics of protein unfolding in uniform flow  

NASA Astrophysics Data System (ADS)

The unfolding dynamics of a protein, ubiquitin, pinned in several uniform flows, was studied at low and high flow rates in an all-atom style through a non-equilibrium molecular dynamics approach with explicit water molecules included. Atomic hydrodynamic force components on individual amino acids, as a function of time, due to the collisional interactions with the flowing water molecules were calculated explicitly. The protein conformational change in response to those time-varying forces was computed completely at the high flow rate up to nanosecond until the fully stretched state was reached. The end-to-end length of the single ubiquitin protein molecule at high flow rate is smoothly increasing. The step-like jumps between metastable states that describe the µm ms - 1 scale force pulling experiments conducted on polyubiquitins at low flow rates, are not seen at the high flow speeds necessary to computationally probe the ns nm - 1 scale regime. No unfolding was observed in the low flow rate atomic computations at nanosecond scale while partial and complete unfolding was observed in the coarse-grained low flow rate computations at microsecond scale. Examination of the all-atom computation of the time variation of the hydrodynamic forces on, and the velocity components of, the protein molecule unveiled to some extent the details of the complexity of the hydrodynamic friction variation in the nm ns - 1 regime of high rate flow-driven protein unfolding. This demonstrates quantitatively that all-atom computations are more suitable than the Langevin equation or Brownian dynamics methods for probing the interaction dynamics and resulting conformational dynamics of protein unfolding in strong flows on nm ns - 1 time/length scales while the reverse is true for investigation of slow, diffusively driven systems.

Wang, Guan M.; Sandberg, William C.

2010-06-01

246

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

247

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

248

Size of Unfolded and Dissociated Subunits versus that of Native Multimeric Proteins  

Microsoft Academic Search

Two factors, unfolding and dissociation, act in opposition indetermining the size of the unfolded state of multimeric proteins. Ananalysis has been presented to correlate relative expansion of the unfoldedmonomers in absence of disulfide bridges over the native state of differenthomomultimeric proteins of varying molecular weights. The Stoke's radii ofabout 70 proteins of Mw between 6 kDa to 4000 kDa and

Samrajnee Dutta; Debasish Bhattacharyya

2001-01-01

249

Me22 chanical and chemical unfolding of a single protein: A comparison  

Microsoft Academic Search

Is the mechanical unraveling of protein do- mains by atomic force microscopy (AFM) just a technological feat or a true measurement of their unfolding? By engineering a protein made of tandem repeats of identical Ig modules, we were able to get explicit AFM data on the unfolding rate of a single protein domain that can be accurately extrapolated to zero

Mariano Carrion-vazquez; ANDRES F. OBERHAUSER; SUSAN B. FOWLER; Piotr Marszalek; Sheldon Broedel; Jane Clarke; Julio Fernandez

1999-01-01

250

Direct Observation of Markovian Behavior of the Mechanical Unfolding of Individual Proteins  

PubMed Central

Single-molecule force-clamp spectroscopy is a valuable tool to analyze unfolding kinetics of proteins. Previous force-clamp spectroscopy experiments have demonstrated that the mechanical unfolding of ubiquitin deviates from the generally assumed Markovian behavior and involves the features of glassy dynamics. Here we use single molecule force-clamp spectroscopy to study the unfolding kinetics of a computationally designed fast-folding mutant of the small protein GB1, which shares a similar ?-grasp fold as ubiquitin. By treating the mechanical unfolding of polyproteins as the superposition of multiple identical Poisson processes, we developed a simple stochastic analysis approach to analyze the dwell time distribution of individual unfolding events in polyprotein unfolding trajectories. Our results unambiguously demonstrate that the mechanical unfolding of NuG2 fulfills all criteria of a memoryless Markovian process. This result, in contrast with the complex mechanical unfolding behaviors observed for ubiquitin, serves as a direct experimental demonstration of the Markovian behavior for the mechanical unfolding of a protein and reveals the complexity of the unfolding dynamics among structurally similar proteins. Furthermore, we extended our method into a robust and efficient pseudo-dwell-time analysis method, which allows one to make full use of all the unfolding events obtained in force-clamp experiments without categorizing the unfolding events. This method enabled us to measure the key parameters characterizing the mechanical unfolding energy landscape of NuG2 with improved precision. We anticipate that the methods demonstrated here will find broad applications in single-molecule force-clamp spectroscopy studies for a wide range of proteins.

Cao, Yi; Kuske, Rachel; Li, Hongbin

2008-01-01

251

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

252

The plant-specific transcription factor gene NAC103 is induced by bZIP60 through a new cis-regulatory element to modulate the unfolded protein response in Arabidopsis.  

PubMed

The unfolded protein response (UPR) plays important roles in plant development and plant-pathogen interactions, as well as in plant adaptation to adverse environmental stresses. Previously bZIP28 and bZIP60 have been identified as important UPR regulators for mitigating the endoplasmic reticulum (ER) stress in Arabidopsis thaliana. Here we report the biological function of NAC103 in a novel transcriptional regulatory cascade, connecting bZIP60 to the UPR downstream genes in Arabidopsis. Expression of NAC103 was induced by ER stress, and was completely abolished in the bZIP60 null mutant. A new ER stress-responsive cis-element UPRE-III (TCATCG) on the NAC103 promoter was identified, and trans-activation of UPRE-III by bZIP60 was confirmed in both yeast cells and Arabidopsis protoplasts. The direct binding of bZIP60 to UPRE-III-containing DNA was also demonstrated in an electrophoretic mobility shift assay. NAC103 formed homodimers in yeast two-hybrid and bimolecular fluorescence complementation assays. It had transcriptional activation activity and was localized in the nucleus. Over-expression of NAC103 had pleiotropic effects on plant growth, and induced expression of several UPR downstream genes in Arabidopsis under normal growth conditions. The activation of UPR gene promoters by NAC103 was also confirmed in effector/reporter protoplast assays. Thus, our study demonstrates a transcriptional regulatory cascade in which NAC103 relays ER stress signals from bZIP60 to UPR downstream genes through a newly identified ER stress cis-element (UPRE-III) and transcriptional activation activity of its encoded protein NAC103. PMID:23869562

Sun, Ling; Yang, Zheng-Ting; Song, Ze-Ting; Wang, Mei-Jing; Sun, Le; Lu, Sun-Jie; Liu, Jian-Xiang

2013-08-12

253

Experimental study of single protein mechanics and protein rates of unfolding  

NASA Astrophysics Data System (ADS)

This dissertation is a multidisciplinary approach to the understanding of the mechanical properties of proteins and their kinetics of unfolding, through the use of an atomic force microscope in force-clamp mode to perform single-molecule force spectroscopy. It begins with the development of a new experimental framework, which includes the design and construction of a new faster atomic force microscopy instrumentation, the definition of new bias-free experimental protocols, and the application of modern statistical tools for data analysis, all of these optimized for the use in force spectroscopy at the single molecule level. Through this new framework new insight was gained on the laws governing the mechanics of a protein in solution. The kinetics of unfolding of the protein ubiquitin were experimentally observed to feature a broad power-law distribution of unfolding rates, quality characteristic of glassy kinetics, which challenges the commonly accepted simplest single rate two-state model models for protein unfolding. Also, the transition between two highly stretched structural configurations of a protein was directly observed to be rate-limited, revealing the magnitude of the intrinsic friction limiting the speed of the first stage of protein folding. This document is presented with detailed technical and mathematical considerations with the intention to serve as handbook for the experimentalist interested in the study of polymers using force-clamp single molecule force spectroscopy.

Hermans, Rodolfo I.

254

Combining predictors of natively unfolded proteins to detect a twilight zone between order and disorder in generic datasets  

Microsoft Academic Search

Natively unfolded proteins lack a well defined three dimensional structure but have important biological functions, suggesting a re-assignment of the structure-function paradigm. Many proteins have amino acidic compositions compatible both with the folded and unfolded status, and belong to a twilight zone between order and disorder. This makes difficult a dichotomic classification of protein sequences into folded and natively unfolded

Antonio Deiana; Andrea Giansanti

2009-01-01

255

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

PubMed

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. PMID:24050368

Vitarelli, Michael J; Talaga, David S

2013-09-14

256

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

257

Identifying protein folding cores from the evolution of flexible regions during unfolding  

Microsoft Academic Search

The unfolding of a protein can be described as a transition from a predominantly rigid, folded structure to an ensemble of denatured states. During unfolding, the hydrogen bonds and salt bridges break, destabilizing the secondary and tertiary structure. Our previous work shows that the network of covalent bonds, salt bridges, hydrogen bonds, and hydrophobic interactions forms constraints that define which

Brandon M. Hespenheide; A. J. Rader; M. F. Thorpe; Leslie A. Kuhn

2002-01-01

258

Low-cost equilibrium unfolding of heme proteins using 2 ?l samples.  

PubMed

Equilibrium unfolding experiments provide access to protein thermodynamic stability revealing basic aspects of protein structure-function relationships. A limitation of these experiments stands on the availability of large amounts of protein samples. Here we present the use of the NanoDrop for monitoring guanidinium chloride-induced unfolding by Soret absorbance of monomeric heme proteins. Unfolding experiments using 2?l of reactant are validated by fluorescence and circular dichroism spectroscopy and supported with five heme proteins including neuroglobin, cytochrome b5, and cyanoglobin. This work guarantees 2 orders of magnitude reduction in protein expense. Promising low-cost protein unfolding experiments following other chromophores and high-throughput screenings are discussed. PMID:23958270

Guca, Ewelina; Roumestand, Christian; Vallone, Beatrice; Royer, Catherine A; Dellarole, Mariano

2013-08-16

259

Mechanical unfolding of proteins L and G with constant force: Similarities and differences  

NASA Astrophysics Data System (ADS)

Mechanical unfolding of proteins L and G, which have similar structures, is considered in this work, and the question arises what changes happen in the unfolding pathways under the action of mechanical force. Molecular dynamics simulations with explicit water (134 trajectories) demonstrate that the mechanical unfolding with constant force occurs through at least two pathways in both proteins. These pathways practically coincide for both proteins and under different constant extensional forces (600, 700, 800, 900, and 1050 pN) and at different temperatures (320, 350, and 400 K at F=1050 pN). Go-like modeling of forced unfolding of proteins L and G does not agree with experimental results that protein G is more mechanically resistant than protein L. At the same time, molecular dynamics simulations of forced unfolding of proteins L and G with explicit water demonstrate that protein G is more mechanically resistant than protein L. Our investigation demonstrates that mechanical stable elements are the same for both proteins, namely, the N-terminal ?-hairpin. This result agrees with experimental data on denaturant unfolding for protein L but not for protein G.

Glyakina, A. V.; Balabaev, N. K.; Galzitskaya, O. V.

2009-07-01

260

Sequential unfolding events in proteins monitored by 2D correlation analysis of FTIR spectra  

NASA Astrophysics Data System (ADS)

The Cro-V55C (cysteine cross-linked) dimer of the ? Cro repressor protein undergoes thermal unfolding in two discrete steps. The secondary structure of the stable equilibrium intermediate exhibits partial unfolding and reorganization at the N-terminal ends while other parts of the structure (some of the ?-sheets) remain intact. To test whether the transition from the native to the intermediate state involves sequential events, we used a 2D-IR approach capable of detecting small differences of individual spectral features in response to external factors. The 2D-IR analysis shows that the intermediate state is formed in closely related sequential steps. To interpret the experimental 2D-IR data, 2D correlation plots for single and multiple sequential events were simulated. These plots were compared with the experimental data and translated into structural changes occurring within Cro-V55C. They reveal that the formation of the stable intermediate starts with the unfolding of the short N-terminal ?-strand, followed by that of the three ?-helices, and ends with the rearrangement of the remaining major ?-sheet. .

Fabian, Heinz; Mantsch, Henry H.; Schultz, Christian P.

2000-03-01

261

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

262

Worm-Like Ising Model for Protein Mechanical Unfolding under the Effect of Osmolytes  

PubMed Central

We show via single-molecule mechanical unfolding experiments that the osmolyte glycerol stabilizes the native state of the human cardiac I27 titin module against unfolding without shifting its unfolding transition state on the mechanical reaction coordinate. Taken together with similar findings on the immunoglobulin-binding domain of streptococcal protein G (GB1), these experimental results suggest that osmolytes act on proteins through a common mechanism that does not entail a shift of their unfolding transition state. We investigate the above common mechanism via an Ising-like model for protein mechanical unfolding that adds worm-like-chain behavior to a recent generalization of the Wako-Saitô-Muñoz-Eaton model with support for group-transfer free energies. The thermodynamics of the model are exactly solvable, while protein kinetics under mechanical tension can be simulated via Monte Carlo algorithms. Notably, our force-clamp and velocity-clamp simulations exhibit no shift in the position of the unfolding transition state of GB1 and I27 under the effect of various osmolytes. The excellent agreement between experiment and simulation strongly suggests that osmolytes do not assume a structural role at the mechanical unfolding transition state of proteins, acting instead by adjusting the solvent quality for the protein chain analyte.

Aioanei, Daniel; Brucale, Marco; Tessari, Isabella; Bubacco, Luigi; Samori, Bruno

2012-01-01

263

Unfolding the response of a Ge detector used for in-situ gamma-ray spectrometry  

Microsoft Academic Search

In environmental radiation protection portable Ge detectors are used for in-situ gamma-ray spectrometry. In order to determine the complete photon fluence rate spectra including the continuum component due to photons scattered in the environment from measured pulse height distributions one needs to apply unfolding methods. A prerequisite of the unfolding is the knowledge of the response functions of the detector

Georg Fehrenbacher; Reinhard Meckbach; Peter Jacob

1996-01-01

264

Experimental study of single protein mechanics and protein rates of unfolding  

Microsoft Academic Search

This dissertation is a multidisciplinary approach to the understanding of the mechanical properties of proteins and their kinetics of unfolding, through the use of an atomic force microscope in force-clamp mode to perform single-molecule force spectroscopy. It begins with the development of a new experimental framework, which includes the design and construction of a new faster atomic force microscopy instrumentation,

Rodolfo I. Hermans

2010-01-01

265

Reversible dissociation and unfolding of the dimeric protein thymidylate synthase.  

PubMed Central

Conditions for in vitro unfolding and refolding of dimeric thymidylate synthase from Lactobacillus casei were found. Ultraviolet difference and circular dichroism spectra showed that the enzyme was completely unfolded at concentrations of urea over 5.5 M. As measured by restoration of enzyme activity, refolding was accomplished when 0.5 M potassium chloride was included in the refolding mixture. Recombination of subunits from catalytically inactive mutant homodimers to form an active hybrid dimer was achieved under these unfolding-refolding conditions, demonstrating a monomer to dimer association step.

Perry, K. M.; Pookanjanatavip, M.; Zhao, J.; Santi, D. V.; Stroud, R. M.

1992-01-01

266

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

267

Thermal unfolding of proteins probed at the single molecule level using nanopores.  

PubMed

The nanopore technique has great potential to discriminate conformations of proteins. It is a very interesting system to mimic and understand the process of translocation of biomacromolecules through a cellular membrane. In particular, the unfolding and folding of proteins before and after going through the nanopore are not well understood. We study the thermal unfolding of a protein, probed by two protein nanopores: aerolysin and ?-hemolysin. At room temperature, the native folded protein does not enter into the pore. When we increase the temperature from 25 to 50 °C, the molecules unfold and the event frequency of current blockade increases. A similar sigmoid function fits the normalized event frequency evolution for both nanopores, thus the unfolding curve does not depend on the structure and the net charge of the nanopore. We performed also a circular dichroism bulk experiment. We obtain the same melting temperature (around 45 °C) using the bulk and single molecule techniques. PMID:22486207

Payet, Linda; Martinho, Marlène; Pastoriza-Gallego, Manuela; Betton, Jean-Michel; Auvray, Loïc; Pelta, Juan; Mathé, Jérôme

2012-04-18

268

Stepwise unfolding of a ?-barrel protein by the AAA+ ClpXP protease  

PubMed Central

In the AAA+ ClpXP protease, ClpX uses the energy of ATP binding and hydrolysis to unfold proteins before translocating them into ClpP for degradation. For proteins with C-terminal ssrA tags, ClpXP pulls on the tag to initiate unfolding and subsequent degradation. Here, we demonstrate that an initial step in ClpXP unfolding of the 11-stranded ? barrel of superfolder GFP-ssrA involves extraction of the C-terminal ? strand. The resulting 10-stranded intermediate is populated at low ATP concentrations, which stall ClpXP unfolding, and at high ATP concentrations, which support robust degradation. To determine if stable unfolding intermediates cause low-ATP stalling, we designed and characterized circularly permuted GFP variants. Notably, stalling was observed for a variant that formed a stable 10-stranded intermediate but not for one in which this intermediate was unstable. A stepwise degradation model in which the rates of terminal-strand extraction, strand refolding or recapture, and unfolding of the 10-stranded intermediate all depend on the rate of ATP hydrolysis by ClpXP accounts for the observed changes in degradation kinetics over a broad range of ATP concentrations. Our results suggest that the presence or absence of unfolding intermediates will play important roles in determining whether forced enzymatic unfolding requires a minimum rate of ATP hydrolysis.

Nager, Andrew R.; Baker, Tania A.; Sauer, Robert T.

2011-01-01

269

Nonparametric density estimation and optimal bandwidth selection for protein unfolding and unbinding data  

NASA Astrophysics Data System (ADS)

Dynamic force spectroscopy and steered molecular simulations have become powerful tools for analyzing the mechanical properties of proteins, and the strength of protein-protein complexes and aggregates. Probability density functions of the unfolding forces and unfolding times for proteins, and rupture forces and bond lifetimes for protein-protein complexes allow quantification of the forced unfolding and unbinding transitions, and mapping the biomolecular free energy landscape. The inference of the unknown probability distribution functions from the experimental and simulated forced unfolding and unbinding data, as well as the assessment of analytically tractable models of the protein unfolding and unbinding requires the use of a bandwidth. The choice of this quantity is typically subjective as it draws heavily on the investigator's intuition and past experience. We describe several approaches for selecting the ``optimal bandwidth'' for nonparametric density estimators, such as the traditionally used histogram and the more advanced kernel density estimators. The performance of these methods is tested on unimodal and multimodal skewed, long-tailed distributed data, as typically observed in force spectroscopy experiments and in molecular pulling simulations. The results of these studies can serve as a guideline for selecting the optimal bandwidth to resolve the underlying distributions from the forced unfolding and unbinding data for proteins.

Bura, E.; Zhmurov, A.; Barsegov, V.

2009-01-01

270

The use of fluorescence methods to monitor unfolding transitions in proteins.  

PubMed Central

This article discusses several strategies for the use steady-state and time-resolved fluorescence methods to monitor unfolding transitions in proteins. The assumptions and limitations of several methods are discussed. Simulations are presented to show that certain fluorescence observables directly track the population of states in an unfolding transition, whereas other observables skew the transition toward the dominant fluorescing species. Several examples are given, involving the unfolding of Staphylococcal aureus nuclease A, in which thermodynamic information is obtained for the temperature and denaturant induced transitions in this protein.

Eftink, M R

1994-01-01

271

A comprehensive library of blocked dipeptides reveals intrinsic backbone conformational propensities of unfolded proteins.  

PubMed

Despite prolonged scientific efforts to elucidate the intrinsic peptide backbone preferences of amino-acids based on understanding of intermolecular forces, many open questions remain, particularly concerning neighboring peptide interaction effects on the backbone conformational distribution of short peptides and unfolded proteins. Here, we show that spectroscopic studies of a complete library of 400 dipeptides reveal that, irrespective of side-chain properties, the backbone conformation distribution is narrow and they adopt polyproline II and ?-strand, indicating the importance of backbone peptide solvation and electronic effects. By directly comparing the dipeptide circular dichroism and NMR results with those of unfolded proteins, the comprehensive dipeptides form a complete set of structural motifs of unfolded proteins. We thus anticipate that the present dipeptide library with spectroscopic data can serve as a useful database for understanding the nature of unfolded protein structures and for further refinements of molecular mechanical parameters. PMID:22223291

Oh, Kwang-Im; Lee, Kyung-Koo; Park, Eun-Kyung; Jung, Youngae; Hwang, Geum-Sook; Cho, Minhaeng

2012-01-04

272

A Measure of Conformational Entropy Change during Thermal Protein Unfolding Using Neutron Spectroscopy  

PubMed Central

Thermal unfolding of proteins at high temperatures is caused by a strong increase of the entropy change which lowers Gibbs free energy change of the unfolding transition (?Gunf = ?H ? T?S). The main contributions to entropy are the conformational entropy of the polypeptide chain itself and ordering of water molecules around hydrophobic side chains of the protein. To elucidate the role of conformational entropy upon thermal unfolding in more detail, conformational dynamics in the time regime of picoseconds was investigated with neutron spectroscopy. Confined internal structural fluctuations were analyzed for ?-amylase in the folded and the unfolded state as a function of temperature. A strong difference in structural fluctuations between the folded and the unfolded state was observed at 30°C, which increased even more with rising temperatures. A simple analytical model was used to quantify the differences of the conformational space explored by the observed protein dynamics for the folded and unfolded state. Conformational entropy changes, calculated on the basis of the applied model, show a significant increase upon heating. In contrast to indirect estimates, which proposed a temperature independent conformational entropy change, the measurements presented here, demonstrated that the conformational entropy change increases with rising temperature and therefore contributes to thermal unfolding.

Fitter, Jorg

2003-01-01

273

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

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. PMID:23478138

Leiria, Luiz O; Sollon, Carolina; Báu, Fernando R; Mónica, Fabíola Z; D'Ancona, Carlos L; De Nucci, Gilberto; Grant, Andrew D; Anhê, Gabriel F; Antunes, Edson

2013-03-11

274

The Unfolded Protein Response (UPR)-activated Transcription Factor X-box-binding Protein 1 (XBP1) Induces MicroRNA-346 Expression That Targets the Human Antigen Peptide Transporter 1 (TAP1) mRNA and Governs Immune Regulatory Genes*  

PubMed Central

To identify endoplasmic reticulum (ER) stress-induced microRNAs (miRNA) that govern ER protein influx during the adaptive phase of unfolded protein response, we performed miRNA microarray profiling and analysis in human airway epithelial cells following ER stress induction using proteasome inhibition or tunicamycin treatment. We identified miR-346 as the most significantly induced miRNA by both classic stressors. miR-346 is encoded within an intron of the glutamate receptor ionotropic delta-1 gene (GRID1), but its ER stress-associated expression is independent of GRID1. We demonstrated that the spliced X-box-binding protein-1 (sXBP1) is necessary and sufficient for ER stress-associated miR-346 induction, revealing a novel role for this unfolded protein response-activated transcription factor. In mRNA profiling arrays, we identified 21 mRNAs that were reduced by both ER stress and miR-346. The target genes of miR-346 regulate immune responses and include the major histocompatibility complex (MHC) class I gene products, interferon-induced genes, and the ER antigen peptide transporter 1 (TAP1). Although most of the repressed mRNAs appear to be indirect targets because they lack specific seeding sites for miR-346, we demonstrate that the human TAP1 mRNA is a direct target of miR-346. The human TAP1 mRNA 3?-UTR contains a 6-mer canonical seeding site for miR-346. Importantly, the ER stress-associated reduction in human TAP1 mRNA and protein levels could be reversed with an miR-346 antagomir. Because TAP function is necessary for proper MHC class I-associated antigen presentation, our results provide a novel mechanistic explanation for reduced MHC class I-associated antigen presentation that was observed during ER stress.

Bartoszewski, Rafal; Brewer, Joseph W.; Rab, Andras; Crossman, David K.; Bartoszewska, Sylwia; Kapoor, Niren; Fuller, Cathy; Collawn, James F.; Bebok, Zsuzsa

2011-01-01

275

The unfolded protein response (UPR)-activated transcription factor X-box-binding protein 1 (XBP1) induces microRNA-346 expression that targets the human antigen peptide transporter 1 (TAP1) mRNA and governs immune regulatory genes.  

PubMed

To identify endoplasmic reticulum (ER) stress-induced microRNAs (miRNA) that govern ER protein influx during the adaptive phase of unfolded protein response, we performed miRNA microarray profiling and analysis in human airway epithelial cells following ER stress induction using proteasome inhibition or tunicamycin treatment. We identified miR-346 as the most significantly induced miRNA by both classic stressors. miR-346 is encoded within an intron of the glutamate receptor ionotropic delta-1 gene (GRID1), but its ER stress-associated expression is independent of GRID1. We demonstrated that the spliced X-box-binding protein-1 (sXBP1) is necessary and sufficient for ER stress-associated miR-346 induction, revealing a novel role for this unfolded protein response-activated transcription factor. In mRNA profiling arrays, we identified 21 mRNAs that were reduced by both ER stress and miR-346. The target genes of miR-346 regulate immune responses and include the major histocompatibility complex (MHC) class I gene products, interferon-induced genes, and the ER antigen peptide transporter 1 (TAP1). Although most of the repressed mRNAs appear to be indirect targets because they lack specific seeding sites for miR-346, we demonstrate that the human TAP1 mRNA is a direct target of miR-346. The human TAP1 mRNA 3'-UTR contains a 6-mer canonical seeding site for miR-346. Importantly, the ER stress-associated reduction in human TAP1 mRNA and protein levels could be reversed with an miR-346 antagomir. Because TAP function is necessary for proper MHC class I-associated antigen presentation, our results provide a novel mechanistic explanation for reduced MHC class I-associated antigen presentation that was observed during ER stress. PMID:22002058

Bartoszewski, Rafal; Brewer, Joseph W; Rab, Andras; Crossman, David K; Bartoszewska, Sylwia; Kapoor, Niren; Fuller, Cathy; Collawn, James F; Bebok, Zsuzsa

2011-10-14

276

Protein Thermal Aggregation Involves Distinct Regions: Sequential Events in the Heat-Induced Unfolding and Aggregation of Hemoglobin  

PubMed Central

Protein thermal aggregation plays a crucial role in protein science and engineering. Despite its biological importance, little is known about the mechanism and pathway(s) involved in the formation of aggregates. In this report, the sequential events occurring during thermal unfolding and aggregation process of hemoglobin were studied by two-dimensional infrared correlation spectroscopy. Analysis of the infrared spectra recorded at different temperatures suggested that hemoglobin denatured by a two-stage thermal transition. At the initial structural perturbation stage (30–44°C), the fast red shift of the band from ?-helix indicated that the native helical structures became more and more solvent-exposed as temperature increased. At the thermal unfolding stage (44–54°C), the unfolding of solvent-exposed helical structures dominated the transition and was supposed to be responsible to the start of aggregation. At the thermal aggregation stage (54–70°C), the transition was dominated by the formation of aggregates and the further unfolding of the buried structures. A close inspection of the sequential events occurring at different stages suggested that protein thermal aggregation involves distinct regions.

Yan, Yong-Bin; Wang, Qi; He, Hua-Wei; Zhou, Hai-Meng

2004-01-01

277

JAMP optimizes ERAD to protect cells from unfolded proteins.  

PubMed

Clearance of misfolded proteins from the ER is central for maintenance of cellular homeostasis. This process requires coordinated recognition, ER-cytosol translocation, and finally ubiquitination-dependent proteasomal degradation. Here, we identify an ER resident seven-transmembrane protein (JAMP) that links ER chaperones, channel proteins, ubiquitin ligases, and 26S proteasome subunits, thereby optimizing degradation of misfolded proteins. Elevated JAMP expression promotes localization of proteasomes at the ER, with a concomitant effect on degradation of specific ER-resident misfolded proteins, whereas inhibiting JAMP promotes the opposite response. Correspondingly, a jamp-1 deleted Caenorhabditis elegans strain exhibits hypersensitivity to ER stress and increased UPR. Using biochemical and genetic approaches, we identify JAMP as important component for coordinated clearance of misfolded proteins from the ER. PMID:18784250

Tcherpakov, Marianna; Broday, Limor; Delaunay, Agnes; Kadoya, Takayuki; Khurana, Ashwani; Erdjument-Bromage, Hediye; Tempst, Paul; Qiu, Xiao-Bo; DeMartino, George N; Ronai, Ze'ev

2008-09-10

278

JAMP Optimizes ERAD to Protect Cells from Unfolded Proteins  

PubMed Central

Clearance of misfolded proteins from the ER is central for maintenance of cellular homeostasis. This process requires coordinated recognition, ER-cytosol translocation, and finally ubiquitination-dependent proteasomal degradation. Here, we identify an ER resident seven-transmembrane protein (JAMP) that links ER chaperones, channel proteins, ubiquitin ligases, and 26S proteasome subunits, thereby optimizing degradation of misfolded proteins. Elevated JAMP expression promotes localization of proteasomes at the ER, with a concomitant effect on degradation of specific ER-resident misfolded proteins, whereas inhibiting JAMP promotes the opposite response. Correspondingly, a jamp-1 deleted Caenorhabditis elegans strain exhibits hypersensitivity to ER stress and increased UPR. Using biochemical and genetic approaches, we identify JAMP as important component for coordinated clearance of misfolded proteins from the ER.

Tcherpakov, Marianna; Broday, Limor; Delaunay, Agnes; Kadoya, Takayuki; Khurana, Ashwani; Erdjument-Bromage, Hediye; Tempst, Paul; Qiu, Xiao-Bo; DeMartino, George N.

2008-01-01

279

Thermodynamics of protein self-association and unfolding. The case of apolipoprotein A-I.  

PubMed

Protein self-association and protein unfolding are two temperature-dependent processes whose understanding is of utmost importance for the development of biological pharmaceuticals because protein association may stabilize or destabilize protein structure and function. Here we present new theoretical and experimental methods for analyzing the thermodynamics of self-association and unfolding. We used isothermal dilution calorimetry and analytical ultracentrifugation to measure protein self-association and introduced binding partition functions to analyze the cooperative association equilibria. In a second type of experiment, we monitored thermal protein unfolding with differential scanning calorimetry and circular dichroism spectroscopy and used the Zimm?Bragg theory to analyze the unfolding process. For ?-helical proteins, the cooperative Zimm?Bragg theory appears to be a powerful alternative to the classical two-state model. As a model protein, we chose highly purified human recombinant apolipoprotein A-I. Self-association of Apo A-I showed a maximum at 21 °C with an association constant Ka of 5.6 × 10(5) M(?1), a cooperativity parameter ? of 0.003, and a maximal association number n of 8. The association enthalpy was linearly dependent on temperature and changed from endothermic at low temperatures to exothermic above 21 °C with a molar heat capacity ?C(p)° of ?2.76 kJ mol(?1) K(?1). Above 45 °C, the association could no longer be measured because of the onset of unfolding. Unfolding occurred between 45 and 65 °C and was reversible and independent of protein concentration up to 160 ?M. The midpoint of unfolding (T(0)) as measured by DSC was 52?53 °C; the enthalpy of unfolding (?H(N)(U)) was 420 kJ/mol. The molar heat capacity (?(N)(U)C(p)) increased by 5.0 ± 0.5 kJ mol(?1) K(?1) upon unfolding corresponding to a loss of 80?85 helical segments, which was confirmed by circular dichroism spectroscopy. Unfolding was highly cooperative with a nucleation parameter ? of 4.4 × 10(?5). PMID:22320308

Zehender, F; Ziegler, A; Schönfeld, H-J; Seelig, J

2012-02-03

280

Direct observation of protein unfolded state compaction in the presence of macromolecular crowding.  

PubMed

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. PMID:23442920

Mikaelsson, Therese; Adén, Jörgen; Johansson, Lennart B-Å; Wittung-Stafshede, Pernilla

2013-02-01

281

Conformational dynamics of a protein in the folded and the unfolded state  

NASA Astrophysics Data System (ADS)

In a quasielastic neutron scattering experiment, the picosecond dynamics of ?-amylase was investigated for the folded and the unfolded state of the protein. In order to ensure a reasonable interpretation of the internal protein dynamics, the protein was measured in D 2O-buffer solution. The much higher structural flexibility of the pH induced unfolded state as compared to the native folded state was quantified using a simple analytical model, describing a local diffusion inside a sphere. In terms of this model the conformational volume, which is explored mainly by confined protein side-chain movements, is parameterized by the radius of a sphere (folded state, r=1.2 Å; unfolded state, 1.8 Å). Differences in conformational dynamics between the folded and the unfolded state of a protein are of fundamental interest in the field of protein science, because they are assumed to play an important role for the thermodynamics of folding/unfolding transition and for protein stability.

Fitter, Jörg

2003-08-01

282

Quantifying internal friction in unfolded and intrinsically disordered proteins with single-molecule spectroscopy.  

PubMed

Internal friction, which reflects the "roughness" of the energy landscape, plays an important role for proteins by modulating the dynamics of their folding and other conformational changes. However, the experimental quantification of internal friction and its contribution to folding dynamics has remained challenging. Here we use the combination of single-molecule Förster resonance energy transfer, nanosecond fluorescence correlation spectroscopy, and microfluidic mixing to determine the reconfiguration times of unfolded proteins and investigate the mechanisms of internal friction contributing to their dynamics. Using concepts from polymer dynamics, we determine internal friction with three complementary, largely independent, and consistent approaches as an additive contribution to the reconfiguration time of the unfolded state. We find that the magnitude of internal friction correlates with the compactness of the unfolded protein: its contribution dominates the reconfiguration time of approximately 100 ns of the compact unfolded state of a small cold shock protein under native conditions, but decreases for more expanded chains, and approaches zero both at high denaturant concentrations and in intrinsically disordered proteins that are expanded due to intramolecular charge repulsion. Our results suggest that internal friction in the unfolded state will be particularly relevant for the kinetics of proteins that fold in the microsecond range or faster. The low internal friction in expanded intrinsically disordered proteins may have implications for the dynamics of their interactions with cellular binding partners. PMID:22492978

Soranno, Andrea; Buchli, Brigitte; Nettels, Daniel; Cheng, Ryan R; Müller-Späth, Sonja; Pfeil, Shawn H; Hoffmann, Armin; Lipman, Everett A; Makarov, Dmitrii E; Schuler, Benjamin

2012-04-06

283

Nonkinetic Modeling of the Mechanical Unfolding of Multimodular Proteins: Theory and Experiments  

PubMed Central

We introduce and discuss a novel approach called back-calculation for analyzing force spectroscopy experiments on multimodular proteins. The relationship between the histograms of the unfolding forces for different peaks, corresponding to a different number of not-yet-unfolded protein modules, is exploited in such a manner that the sole distribution of the forces for one unfolding peak can be used to predict the unfolding forces for other peaks. The scheme is based on a bootstrap prediction method and does not rely on any specific kinetic model for multimodular unfolding. It is tested and validated in both theoretical/computational contexts (based on stochastic simulations) and atomic force microscopy experiments on (GB1)8 multimodular protein constructs. The prediction accuracy is so high that the predicted average unfolding forces corresponding to each peak for the GB1 construct are within only 5 pN of the averaged directly-measured values. Experimental data are also used to illustrate how the limitations of standard kinetic models can be aptly circumvented by the proposed approach.

Benedetti, F.; Micheletti, C.; Bussi, G.; Sekatskii, S.K.; Dietler, G.

2011-01-01

284

Mechanical Unfolding of a Titin Ig Domain: Structure of Unfolding Intermediate Revealed by Combining AFM, Molecular Dynamics Simulations, NMR and Protein Engineering  

Microsoft Academic Search

The mechanical unfolding of an immunoglobulin domain from the human muscle protein titin (TI I27) has been shown to proceed via a metastable intermediate in which the A-strand is detached. The structure and properties of this intermediate are characterised in this study. A conservative destabilising mutation in the A-strand has no effect on the unfolding force, nor the dependence of

Susan B. Fowler; Robert B. Best; José L Toca Herrera; Trevor J Rutherford; Annette Steward; Emanuele Paci; Martin Karplus; Jane Clarke

2002-01-01

285

Predictors of natively unfolded proteins: unanimous consensus score to detect a twilight zone between order and disorder in generic datasets  

Microsoft Academic Search

BACKGROUND: Natively unfolded proteins lack a well defined three dimensional structure but have important biological functions, suggesting a re-assignment of the structure-function paradigm. To assess that a given protein is natively unfolded requires laborious experimental investigations, then reliable sequence-only methods for predicting whether a sequence corresponds to a folded or to an unfolded protein are of interest in fundamental and

Antonio Deiana; Andrea Giansanti

2010-01-01

286

Probing osmolyte participation in the unfolding transition state of a protein  

PubMed Central

Understanding the molecular mechanisms of osmolyte protection in protein stability has proved to be challenging. In particular, little is known about the role of osmolytes in the structure of the unfolding transition state of a protein, the main determinant of its dynamics. We have developed an experimental protocol to directly probe the transition state of a protein in a range of osmolyte environments. We use an atomic force microscope in force-clamp mode to apply mechanical forces to the protein I27 and obtain force-dependent rate constants of protein unfolding. We measure the distance to the unfolding transition state, ?xu, along a 1D reaction coordinate imposed by mechanical force. We find that for the small osmolytes, ethylene glycol, propylene glycol, and glycerol, ?xu scales with the size of the molecule, whereas for larger osmolytes, sorbitol and sucrose, ?xu remains the same as that measured in water. These results are in agreement with steered molecular dynamics simulations that show that small osmolytes act as solvent bridges in the unfolding transition state structure, whereas only water molecules act as solvent bridges in large osmolyte environments. These results demonstrate that novel force protocols combined with solvent substitution can directly probe angstrom changes in unfolding transition state structure. This approach creates new opportunities to gain molecular level understanding of the action of osmolytes in biomolecular processes.

Dougan, Lorna; Genchev, Georgi Z.; Lu, Hui; Fernandez, Julio M.

2011-01-01

287

Universality in the timescales of internal loop formation in unfolded proteins and single-stranded oligonucleotides  

NASA Astrophysics Data System (ADS)

Understanding the rate at which various parts of a molecular chain come together to facilitate the folding of a biopolymer (e.g., a protein) 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 DNA 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. Our measurements of the loop closure times in single-stranded oligonucleotides, as well the internal loop closure kinetics in unfolded proteins reported by others, are all well described by this theoretically predicted dependence. 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; Uzawa, Takanori; Plaxco, Kevin; Makarov, Dmitrii

2011-03-01

288

Theoretical study of a landscape of protein folding-unfolding pathways. Folding rates at midtransition.  

PubMed

This paper presents a new method for calculating the folding-unfolding rates of globular proteins. The method is based on solution of kinetic equations for a network of folding-unfolding pathways of the proteins. The rates are calculated in the point of thermodynamic equilibrium between the native and completely unfolded states. The method has been applied to all the proteins listed by Jackson [Jackson, S. E. (1998) Folding Des. 3, R81-R91] and some peptides. Although the studied protein chains differ by more than 1 order of magnitude in size and exhibit two- as well as three-state kinetics in water, and their folding rates cover more than 11 orders of magnitude, the theoretical estimates are reasonable close to the experimentally measured folding rates in midtransition (the correlation coefficient being as high as 0.78). This means that the presented theory (having no adjustable parameters at all) is consistent with the experimental observations. PMID:11502191

Ivankov, D N; Finkelstein, A V

2001-08-21

289

Folding and Unfolding in the Blue Copper Protein Rusticyanin: Role of the Oxidation State  

PubMed Central

The unfolding process of the blue copper protein rusticyanin has been studied from the structural and the thermodynamic points of view at two pH values (pH 2.5 and 7.0). When Rc unfolds, copper ion remains bound to the polypeptide chain. Nuclear magnetic resonance data suggest that three of the copper ligands in the folded state are bound to the metal ion in the unfolded form, while the other native ligand is detached. These structural changes are reflected in the redox potentials of the protein in both folded and unfolded forms. The affinities of the copper ion in both redox states have been also determined at the two specified pH values. The results indicate that the presence of two histidine ligands in the folded protein can compensate the change in the net charge that the copper ion receives from their ligands, while, in the unfolded protein, charges of aminoacids are completely transferred to the copper ion, altering decisively the relative stability of its two-redox states.

Alcaraz, Luis A.; Gomez, Javier; Ramirez, Pablo; Calvente, Juan J.; Andreu, Rafael; Donaire, Antonio

2007-01-01

290

Single-molecule spectroscopy of cold denaturation and the temperature-induced collapse of unfolded proteins.  

PubMed

Recent Förster resonance energy transfer (FRET) experiments show that heat-unfolded states of proteins become more compact with increasing temperature. At the same time, NMR results indicate that cold-denatured proteins are more expanded than heat-denatured proteins. To clarify the connection between these observations, we investigated the unfolded state of yeast frataxin, whose cold denaturation occurs at temperatures above 273 K, with single-molecule FRET. This method allows the unfolded state dimensions to be probed not only in the cold- and heat-denatured range but also in between, i.e., in the presence of folded protein, and can thus be used to link the two regimes directly. The results show a continuous compaction of unfolded frataxin from 274 to 320 K, with a slight re-expansion at higher temperatures. Cold- and heat-denatured states are thus essentially two sides of the same coin, and their behavior can be understood within the framework of the overall temperature dependence of the unfolded state dimensions. PMID:24010673

Aznauryan, Mikayel; Nettels, Daniel; Holla, Andrea; Hofmann, Hagen; Schuler, Benjamin

2013-09-11

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

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

293

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

294

A method for determining states in the course of protein unfolding  

Microsoft Academic Search

A simple model is presented for analyzing a set of spectra obtained from spectrophotometric study of protein titration. With this model one can determine the states of (probable) intermediates in the course of protein unfolding. The model is developed based on abundance of native state, intermediate(s) and denatured state, and their contributions to differential absorbance at selected wavelengths. The model

Mostafa Rezaei-Tavirani; Sayed-Amir Marashi; Seyed Mohammad Mahdavi

2006-01-01

295

Direct Quantification of the Attempt Frequency Determining the Mechanical Unfolding of Ubiquitin Protein*  

PubMed Central

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 × 109 s?1. Although the measured prefactor value is 3 orders of magnitude smaller than the value predicted by the transition state theory (?6 × 1012 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 (?107 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.

Popa, Ionel; Fernandez, Julio M.; Garcia-Manyes, Sergi

2011-01-01

296

Geofold: topology-based protein unfolding pathways capture the effects of engineered disulfides on kinetic stability.  

PubMed

Protein unfolding is modeled as an ensemble of pathways, where each step in each pathway is the addition of one topologically possible conformational degree of freedom. Starting with a known protein structure, GeoFold hierarchically partitions (cuts) the native structure into substructures using revolute joints and translations. The energy of each cut and its activation barrier are calculated using buried solvent accessible surface area, side chain entropy, hydrogen bonding, buried cavities, and backbone degrees of freedom. A directed acyclic graph is constructed from the cuts, representing a network of simultaneous equilibria. Finite difference simulations on this graph simulate native unfolding pathways. Experimentally observed changes in the unfolding rates for disulfide mutants of barnase, T4 lysozyme, dihydrofolate reductase, and factor for inversion stimulation were qualitatively reproduced in these simulations. Detailed unfolding pathways for each case explain the effects of changes in the chain topology on the folding energy landscape. GeoFold is a useful tool for the inference of the effects of disulfide engineering on the energy landscape of protein unfolding. PMID:22189917

Ramakrishnan, Vibin; Srinivasan, Sai Praveen; Salem, Saeed M; Matthews, Suzanne J; Colón, Wilfredo; Zaki, Mohammed; Christopher Bystroff

2011-12-21

297

Water as ligand: Preferential binding and exclusion of denaturants in protein unfolding  

Microsoft Academic Search

The denaturation of proteins by agents such as urea or guanidine hydrochloride (GdmHC1) usually requires high concentrations, 3-8 M, ofdenaturant. At theseconcentrations, denaturants destabilize the folded state of a typical globular protein by about 10-20 kcal\\/mol (Pace, 1975). This free energy contribution (6AG) is the result of additional inter-actions between the protein and the denaturant when the protein becomes unfolded.

Serge N. Timasheff

1992-01-01

298

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

299

Characterizing the unfolded states of proteins using single-molecule FRET spectroscopy and molecular simulations  

PubMed Central

To obtain quantitative information on the size and dynamics of unfolded proteins we combined single-molecule lifetime and intensity FRET measurements with molecular simulations. We compared the unfolded states of the 64-residue, ?/? protein L and the 66-residue, all-? cold-shock protein CspTm. The average radius of gyration (Rg) calculated from FRET data on freely diffusing molecules was identical for the two unfolded proteins at guanidinium chloride concentrations >3 M, and the FRET-derived Rg of protein L agreed well with the Rg previously measured by equilibrium small-angle x-ray scattering. As the denaturant concentration was lowered, the mean FRET efficiency of the unfolded subpopulation increased, signaling collapse of the polypeptide chain, with protein L being slightly more compact than CspTm. A decrease in Rg with decreasing denaturant was also observed in all-atom molecular dynamics calculations in explicit water/urea solvent, and Langevin simulations of a simplified representation of the polypeptide suggest that collapse can result from either increased interresidue attraction or decreased excluded volume. In contrast to both the FRET and simulation results, previous time-resolved small-angle x-ray scattering experiments showed no collapse for protein L. Analysis of the donor fluorescence decay of the unfolded subpopulation of both proteins gives information about the end-to-end chain distribution and suggests that chain dynamics is slow compared with the donor life-time of ?2 ns, whereas the bin-size independence of the small excess width above the shot noise for the FRET efficiency distributions may result from incomplete conformational averaging on even the 1-ms time scale.

Merchant, Kusai A.; Best, Robert B.; Louis, John M.; Gopich, Irina V.; Eaton, William A.

2007-01-01

300

Simulation of urea-induced protein unfolding: a lesson from bovine ?-lactoglobulin.  

PubMed

To investigate the molecular mechanisms involved in the very initial stages of protein unfolding, we carried out one long (1 ?s) simulation of bovine ?-lactoglobulin (BLG) together with three (500 ns) supporting MD runs, in which the unfolding conditions were produced by adding the osmolyte urea to the simulated systems and/or by increasing the thermal energy raising the temperature from 300 to 350 K. BLG was chosen, since it is a well-characterized model protein, for which structural and folding properties have been widely investigated by X-ray and NMR. MD trajectories were analyzed not only in terms of standard progress variables, such as backbone H-bonds, gyration radius width, secondary structure elements, but also through the scrutiny of interactions and dynamical behavior of specific key residues previously pointed out and investigated by NMR and belonging to a well known hydrophobic cluster. MD trajectories simulated in different unfolding conditions suggest that urea destabilizes BLG structure weakening protein::protein hydrophobic interactions and the hydrogen bond network. The early unfolding events, better observed at higher temperature, affect both secondary and tertiary structure of the protein. PMID:21724434

Eberini, Ivano; Emerson, Andrew; Sensi, Cristina; Ragona, Laura; Ricchiuto, Piero; Pedretti, Alessandro; Gianazza, Elisabetta; Tramontano, Anna

2011-06-13

301

New bonner sphere response matrix, ARK1, for neutron spectral unfolding  

SciTech Connect

Although the energy spectra of neutron radiation cannot be measured directly, knowledge of the neutron spectrum in the workplace is necessary for predictions of personnel radiation doses and shielding design. Bonner spheres consist of a central detector over which polyethylene moderating spheres are placed, permitting measurement of the counting rate for various combinations of the central detector and several moderators. The process of approximating neutron spectra from Bonner sphere count-rate data is known as spectral unfolding and requires knowledge of the energy response of each detector-moderator combination (i.e., a response function). The unfolding process may be sensitive to small changes because the response functions are usually ill conditioned making an accurate set of response functions vital to the unfolding process. Previous response function calculations have been limited to a one-dimensional model of the detector-moderator combination and to binned cross-section sets, already averaged over some representative energy spectrum. Of these available response matrices, UTA4 and SAN4 perform the best in unfolding tests. This paper focuses on the relative performance differences between previously published response functions and those we have calculated by modeling Bonner spheres in three dimensions with the Monte Carlo code MCNP4A.

Lemley, E.C.; West. L. [Univ. of Arkansas, Fayetteville, AR (United States)

1996-12-31

302

Protein folding and unfolding simulations: a new challenge for data mining.  

PubMed

One of the unsolved paradigms in molecular biology is the protein folding problem. In recent years, with the identification of several diseases as protein folding disorders and with the explosion of genome information and the need for efficient ways to predict protein structure, protein folding became a central issue in molecular sciences research. Using molecular dynamics unfolding simulations of an amyloidogenic protein--transthyretin--as an example, we put forward a series of ideas on how simulations of this type may be used to infer rules and unfolding behavior in amyloidogenic proteins, and to extrapolate rules for protein folding in different structural classes of proteins. These, in turn, could help in the development of protein structure prediction methods. The need to analyse different proteins and to run multiple simulations creates a huge amount of data which has to be stored, managed, analyzed and shared (database and Grid technology; data mining). Once the data is captured, the next challenge is to find meaningful patterns (associations, correlations, clusters, rules, relationships) among molecular properties, or their relative importance at different stages of the folding or unfolding processes. This clearly puts new and interesting challenges to the bioinformatics community. PMID:15268773

Brito, Rui M M; Dubitzky, Werner; Rodrigues, J Rui

2004-01-01

303

Protein Folding, Unfolding, and Aggregation Processes Revealed by Rapid Sampling of Time-Domain Fluorescence  

Microsoft Academic Search

\\u000a Temporal information on the structural transformations occurring during the folding and unfolding reactions of proteins is\\u000a of great importance for elucidating the mechanism of protein folding [1–6]. The rapidity of protein folding reactions precludes\\u000a the use of standard structural tools such as NMR and X-ray crystallography. Furthermore, the conformational heterogeneity\\u000a inherent in these reactions poses a further problem in using

Saswata Sankar Sarkar; Anoop Saxena; Nihav Dhawale; Jayant B. Udgaonkar; G. Krishnamoorthy

304

Maximum likelihood estimation of protein kinetic parameters under weak assumptions from unfolding force spectroscopy experiments  

NASA Astrophysics Data System (ADS)

Single molecule force spectroscopy (SMFS) is extensively used to characterize the mechanical unfolding behavior of individual protein domains under applied force by pulling chimeric polyproteins consisting of identical tandem repeats. Constant velocity unfolding SMFS data can be employed to reconstruct the protein unfolding energy landscape and kinetics. The methods applied so far require the specification of a single stretching force increase function, either theoretically derived or experimentally inferred, which must then be assumed to accurately describe the entirety of the experimental data. The very existence of a suitable optimal force model, even in the context of a single experimental data set, is still questioned. Herein, we propose a maximum likelihood (ML) framework for the estimation of protein kinetic parameters which can accommodate all the established theoretical force increase models. Our framework does not presuppose the existence of a single force characteristic function. Rather, it can be used with a heterogeneous set of functions, each describing the protein behavior in the stretching time range leading to one rupture event. We propose a simple way of constructing such a set of functions via piecewise linear approximation of the SMFS force vs time data and we prove the suitability of the approach both with synthetic data and experimentally. Additionally, when the spontaneous unfolding rate is the only unknown parameter, we find a correction factor that eliminates the bias of the ML estimator while also reducing its variance. Finally, we investigate which of several time-constrained experiment designs leads to better estimators.

Aioanei, Daniel; Samorì, Bruno; Brucale, Marco

2009-12-01

305

Measuring unfolding of proteins in the presence of denaturant using fluorescence correlation spectroscopy.  

PubMed

IFABP is a small (15 kDa) protein consisting mostly of antiparallel beta-strands that surround a large cavity into which ligands bind. We have previously used FCS to show that the native protein, labeled with fluorescein, exhibits dynamic fluctuation with a relaxation time of 35 micros. Here we report the use of FCS to study the unfolding of the protein induced by guanidine hydrochloride. Although the application of this technique to measure diffusion coefficients and molecular dynamics is straightforward, the FCS results need to be corrected for both viscosity and refractive index changes as the guanidine hydrochloride concentration increases. We present here a detailed study of the effects of viscosity and refractive index of guanidine hydrochloride solutions to calibrate FCS data. After correction, the increase in the diffusion time of IFABP corresponds well with the unfolding transition monitored by far ultraviolet circular dichroism. We also show that the magnitude of the 35 micros phase, reflecting the conformational fluctuation in the native state, decreases sharply as the concentration of denaturant increases and the protein unfolds. Although FCS experiments indicate that the unfolded state at pH 2 is rather compact and native-like, the radius in the presence of guanidine hydrochloride falls well within the range expected for a random coil. PMID:15556973

Chattopadhyay, Krishnananda; Saffarian, Saveez; Elson, Elliot L; Frieden, Carl

2004-11-19

306

Mechanically Unfolding the Small, Topologically Simple Protein L  

Microsoft Academic Search

?-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

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

2005-01-01

307

Chaperone activation by unfolding.  

PubMed

Conditionally disordered proteins can alternate between highly ordered and less ordered configurations under physiological conditions. Whereas protein function is often associated with the ordered conformation, for some of these conditionally unstructured proteins, the opposite applies: Their activation is associated with their unfolding. An example is the small periplasmic chaperone HdeA, which is critical for the ability of enteric bacterial pathogens like Escherichia coli to survive passage through extremely acidic environments, such as the human stomach. At neutral pH, HdeA is a chaperone-inactive dimer. On a shift to low pH, however, HdeA monomerizes, partially unfolds, and becomes rapidly active in preventing the aggregation of substrate proteins. By mutating two aspartic acid residues predicted to be responsible for the pH-dependent monomerization of HdeA, we have succeeded in isolating an HdeA mutant that is active at neutral pH. We find this HdeA mutant to be substantially destabilized, partially unfolded, and mainly monomeric at near-neutral pH at a concentration at which it prevents aggregation of a substrate protein. These results provide convincing evidence for direct activation of a protein by partial unfolding. PMID:23487787

Foit, Linda; George, Jenny S; Zhang, Bin W; Brooks, Charles L; Bardwell, James C A

2013-03-04

308

Assembly and function of the Photosystem II manganese stabilizing protein: lessons from its natively unfolded behavior  

Microsoft Academic Search

The Photosystem II (PS II) manganese stabilizing protein (MSP) possesses characteristics, including thermostability, ascribed to the natively unfolded class of proteins (Lydakis-Simantiris et al. (1999) Biochemistry 38: 404–414). A site-directed mutant of MSP, C28A, C51A, which lacks the -S–S- bridge, also binds to PS II at wild-type levels and reconstitutes oxygen evolution activity [Betts et al. (1996) Biochim Biophys Acta

Aaron J. Wyman; Charles F. Yocum

2005-01-01

309

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

PubMed

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. PMID:23827883

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

2013-01-01

310

On the performance of combined dichotomic predictors of natively unfolded proteins  

Microsoft Academic Search

The performance of single folding predictors and combination scores is critically evaluated. We test mean packing, mean pairwise energy and the new index gVSL2 on a dataset of 743 folded proteins and 81 natively unfolded proteins. These predictors have an individual performance comparable or even better than other proposed methods. We introduce here a strictly unanimous score S_{SU} that combines

Antonio Deiana; Andrea Giansanti

2008-01-01

311

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

312

Single-molecule force spectroscopy reveals the individual mechanical unfolding pathways of a surface layer protein.  

PubMed

Surface layers (S-layers) represent an almost universal feature of archaeal cell envelopes and are probably the most abundant bacterial cell proteins. S-layers are monomolecular crystalline structures of single protein or glycoprotein monomers that completely cover the cell surface during all stages of the cell growth cycle, thereby performing their intrinsic function under a constant intra- and intermolecular mechanical stress. In gram-positive bacteria, the individual S-layer proteins are anchored by a specific binding mechanism to polysaccharides (secondary cell wall polymers) that are linked to the underlying peptidoglycan layer. In this work, atomic force microscopy-based single-molecule force spectroscopy and a polyprotein approach are used to study the individual mechanical unfolding pathways of an S-layer protein. We uncover complex unfolding pathways involving the consecutive unfolding of structural intermediates, where a mechanical stability of 87 pN is revealed. Different initial extensibilities allow the hypothesis that S-layer proteins adapt highly stable, mechanically resilient conformations that are not extensible under the presence of a pulling force. Interestingly, a change of the unfolding pathway is observed when individual S-layer proteins interact with secondary cell wall polymers, which is a direct signature of a conformational change induced by the ligand. Moreover, the mechanical stability increases up to 110 pN. This work demonstrates that single-molecule force spectroscopy offers a powerful tool to detect subtle changes in the structure of an individual protein upon binding of a ligand and constitutes the first conformational study of surface layer proteins at the single-molecule level. PMID:21690085

Horejs, Christine; Ristl, Robin; Tscheliessnig, Rupert; Sleytr, Uwe B; Pum, Dietmar

2011-06-19

313

Universal convergence of the specific volume changes of globular proteins upon unfolding.  

PubMed

Both pressure and temperature are important environmental variables, and to obtain a complete understanding of the mechanisms of protein folding, it is necessary to determine how protein stability is dependent on these fundamental thermodynamic parameters. Although the temperature dependence of protein stability has been widely explored, the dependence of protein stability on pressure is not as well studied. In this paper, we report the results of the direct thermodynamic determination of the change in specific volume (DeltaV/V) upon protein unfolding, which defines the pressure dependence of protein stability, for five model proteins (ubiquitin, eglin c, ribonuclease A, lysozyme, and cytochrome c). We have shown that the specific volumetric changes upon unfolding for four of the proteins (ubiquitin, eglin c, ribonuclease A, and lysozyme) appear to converge to a common value at high temperatures. Analysis of various contributions to the change in volume upon protein unfolding allowed us to put forth the hypothesis that the change in volume due to hydration is very close to zero at this temperature, such that DeltaV/V is defined largely by the total volume of cavities and voids within a protein, and that this is a universal property of all small globular proteins without prosthetic groups. To test this hypothesis, additional experiments were performed with variants of eglin c that had site-directed substitutions at two buried positions, to create an additional cavity in the protein core. The results of these experiments, coupled with the structural analysis of cytochrome c showing a lower packing density compared to those of the other four proteins, provided further support for the hypothesis. Finally, we have shown that the deviation of the high-temperature DeltaV value of a given protein from the convergence value can be used to determine the size of the excess cavities in globular proteins. PMID:19877593

Schweiker, Katrina L; Fitz, Victoria W; Makhatadze, George I

2009-11-24

314

Thermal unfolding of the archaeal DNA and RNA binding protein Ssh10.  

PubMed

The reversible thermal unfolding of the archaeal histone-like protein Ssh10b from the extremophile Sulfolobus shibatae was studied using differential scanning calorimetry and circular dichroism spectroscopy. Analytical ultracentrifugation and gel filtration showed that Ssh10b is a stable dimer in the pH range 2.5-7.0. Thermal denaturation data fit into a two-state unfolding model, suggesting that the Ssh10 dimer unfolds as a single cooperative unit with a maximal melting temperature of 99.9 degrees C and an enthalpy change of 134 kcal/mol at pH 7.0. The heat capacity change upon unfolding determined from linear fits of the temperature dependence of DeltaH(cal) is 2.55 kcal/(mol K). The low specific heat capacity change of 13 cal/(mol K residue) leads to a considerable flattening of the protein stability curve (DeltaG (T)) and results in a maximal DeltaG of only 9.5 kcal/mol at 320 K and a DeltaG of only 6.0 kcal/mol at the optimal growth temperature of Sulfolobus. PMID:18571501

Wu, Xiaoqiu; Oppermann, Madalina; Berndt, Kurt D; Bergman, Tomas; Jörnvall, Hans; Knapp, Stefan; Oppermann, Udo

2008-06-20

315

A back hydrogen exchange procedure via the acid-unfolded state for a large protein.  

PubMed

A deuterated protein sample is required for nuclear magnetic resonance (NMR) measurements of a large protein because severe signal broadenings occur because of the high molecular weight. The deuterated sample expressed in (2)H(2)O should subsequently be subjected to a back hydrogen exchange at amide groups. To perform the back exchange, the protein molecule is unfolded or destabilized so that internal residues become accessible to the solvent. However, the refolding yield from the destabilized or unfolded state of a large protein is usually low, leading to a dilemma in NMR measurements of large proteins. In our previous paper [Suzuki, M., et al. (2011) Biochemistry 50, 10390-10398], we suggested that an acid-denatured microbial transglutaminase (MTG) consisting of 331 amino acid residues can be recovered effectively under low-salt conditions, escaping from the aggregation-prone intermediate. Here, we demonstrate that proMTG, the pro form of MTG consisting of 376 amino acid residues, can be refolded perfectly from the acid-unfolded state under low-salt conditions, as confirmed by circular dichroism and NMR spectroscopies. By performing the same procedure with a deuterated proMTG expressed in (2)H(2)O, we observed complete back exchanges for internal residues by NMR spectroscopy. Our procedure has potential applications to the back hydrogen exchange of large proteins for NMR measurements. PMID:22738018

Suzuki, Mototaka; Sakurai, Kazumasa; Lee, Young-Ho; Ikegami, Takahisa; Yokoyama, Keiichi; Goto, Yuji

2012-07-03

316

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

317

Folding/unfolding/refolding of proteins: present methodologies in comparison with capillary zone electrophoresis.  

PubMed

A series of techniques for monitoring protein folding/unfolding/misfolding equilibria are here assessed and compared with capillary zone electrophoresis (CZE). They include spectroscopic techniques, such as circular dichroism, intrinsic fluorescence, nuclear magnetic resonance, Fourier transform infrared and Raman spectroscopy, small-angle X-ray scattering, as well as techniques based on biological assays, such as limited proteolysis and immunochemical analysis of different conformational states. Some unusual probes, such as mass spectrometry for probing unfolding transitions, are also discussed. Size-exclusion chromatography is also evaluated in view of the fact that this technique, like all electrophoretic techniques, and unlike spectroscopic probes, which can only see an average signal in mixed populations, can indeed physically separate folded vs. unfolded macromolecules, especially in the case of slow equilibria. Particular emphasis is devoted to electrophoretic techniques, such as gel-slab electrophoresis in transverse urea or thermal gradients, and CZE. In the latter case, a number of applications are shown, demonstrating the excellent correlation of CZE with more traditional probes, such as intrinsic fluorescence monitoring. It is additionally shown that CZE can be used for measuring the deltaG degrees of unfolding over the pH scale, in good agreement with theoretical calculations on the electrostatic free energy of folding vs. pH, as calculated with a linearized Poisson-Boltzmann equation. Finally, it is demonstrated that CZE can probe also aggregate formation in the presence of helix-inducing agents, such as trifluorethanol. PMID:11519938

Righetti, P G; Verzola, B

2001-08-01

318

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

2013-03-09

319

Effect of the choice or response matrix on unfolded Bonner sphere spectra. Memorandum report  

SciTech Connect

The effect of the choice of response matrix on neutron spectra unfolded from Bonner sphere data was studied. In particular, the variation of integral parameters calculated from the unfolded spectra was determined for six matrices. It was determined that, depending on the choice of matrix, the calculated neutron fluence varies by approximately + or - 15%, the average neutron energy by + or - 40%, the dose by + or - 30%, the dose equivalent by up to a factor of 4, and the quality factor by + or - 35%. For personnel monitoring devices, the calculated response varied by up to a factor of 4 for the Navy albedo badge, a factor of 7 for a cadmium-covered albedo badge, a factor of 6 for a CR-39 dosimeter, and more than two orders of magnitude for NTA type emulsion film. Two of the response matrices gave results in good agreement with other calculated and experimental data.

Lowry, K.A.; Johnson, T.L.

1984-12-31

320

Unfolding the role of protein misfolding in neurodegenerative diseases  

Microsoft Academic Search

Recent evidence indicates that diverse neurodegenerative diseases might have a common cause and pathological mechanism — the misfolding, aggregation and accumulation of proteins in the brain, resulting in neuronal apoptosis. Studies from different disciplines strongly support this hypothesis and indicate that a common therapy for these devastating disorders might be possible. The aim of this article is to review the

Claudio Soto

2003-01-01

321

Single-molecule protein unfolding and refolding using atomic force microscopy.  

PubMed

Over the past few years, atomic force microscopy (AFM) became a prominent tool to study the mechanical properties of proteins and protein interactions on a single-molecule level. AFM together with other mechanical, single-molecule manipulating techniques (Bustamante et al., Nat Rev Mol Cell Biol 1:130-136, 2000) made it possible to probe biological molecules in a way that is complementary to single-molecule methods using chemicals or temperature as a denaturant (Borgia et al., Annu Rev Biochem 77:101-125, 2008). For example, AFM offered new insights into the process of protein folding and unfolding by probing single proteins with mechanical forces. Since many proteins fulfill mechanical function or are exerted to mechanical forces in their natural environment, AFM allows to target physiologically relevant questions. Although the number of proteins unfolded with AFM continually increases (Linke and Grutzner, Pflugers Arch 456:101-115, 2008; Zhuang and Rief, Curr Opin Struct Biol 13:88-97, 2003; Clausen-Schaumann et al., Curr Opin Chem Biol 4:524-530, 2000; Rounsevell et al., Methods 34:100-111, 2004), the total number of proteins studied so far is still relatively small (Oberhauser and Carrion-Vazquez, J Biol Chem 283:6617-6621, 2008). This chapter aims at giving protocol-like instructions for people who are actually getting started using AFM to study mechanical protein unfolding or refolding. The instruction includes different approaches to produce polyproteins or modular protein chains which are commonly used to screen for true single-molecule AFM data traces. Also, the basic principles for data collection with AFM and the basic data analysis methods are explained. For people who want to study proteins that unfold at small forces or for people who want to study protein folding which also occurs typically at small forces (<30 pN), an averaging technique is explained, allowing to increase the force resolution in this regime. For topics that would go beyond the scope of this chapter - as, for example, the details about different cantilever calibration methods - references are provided. PMID:21909892

Bornschlögl, Thomas; Rief, Matthias

2011-01-01

322

Protein folding and unfolding studied at atomic resolution by fast two-dimensional NMR spectroscopy.  

PubMed

Atom-resolved real-time studies of kinetic processes in proteins have been hampered in the past by the lack of experimental techniques that yield sufficient temporal and atomic resolution. Here we present band-selective optimized flip-angle short transient (SOFAST) real-time 2D NMR spectroscopy, a method that allows simultaneous observation of reaction kinetics for a large number of nuclear sites along the polypeptide chain of a protein with an unprecedented time resolution of a few seconds. SOFAST real-time 2D NMR spectroscopy combines fast NMR data acquisition techniques with rapid sample mixing inside the NMR magnet to initiate the kinetic event. We demonstrate the use of SOFAST real-time 2D NMR to monitor the conformational transition of alpha-lactalbumin from a molten globular to the native state for a large number of amide sites along the polypeptide chain. The kinetic behavior observed for the disappearance of the molten globule and the appearance of the native state is monoexponential and uniform along the polypeptide chain. This observation confirms previous findings that a single transition state ensemble controls folding of alpha-lactalbumin from the molten globule to the native state. In a second application, the spontaneous unfolding of native ubiquitin under nondenaturing conditions is characterized by amide hydrogen exchange rate constants measured at high pH by using SOFAST real-time 2D NMR. Our data reveal that ubiquitin unfolds in a gradual manner with distinct unfolding regimes. PMID:17592113

Schanda, Paul; Forge, Vincent; Brutscher, Bernhard

2007-06-25

323

Protein folding and unfolding studied at atomic resolution by fast two-dimensional NMR spectroscopy  

PubMed Central

Atom-resolved real-time studies of kinetic processes in proteins have been hampered in the past by the lack of experimental techniques that yield sufficient temporal and atomic resolution. Here we present band-selective optimized flip-angle short transient (SOFAST) real-time 2D NMR spectroscopy, a method that allows simultaneous observation of reaction kinetics for a large number of nuclear sites along the polypeptide chain of a protein with an unprecedented time resolution of a few seconds. SOFAST real-time 2D NMR spectroscopy combines fast NMR data acquisition techniques with rapid sample mixing inside the NMR magnet to initiate the kinetic event. We demonstrate the use of SOFAST real-time 2D NMR to monitor the conformational transition of ?-lactalbumin from a molten globular to the native state for a large number of amide sites along the polypeptide chain. The kinetic behavior observed for the disappearance of the molten globule and the appearance of the native state is monoexponential and uniform along the polypeptide chain. This observation confirms previous findings that a single transition state ensemble controls folding of ?-lactalbumin from the molten globule to the native state. In a second application, the spontaneous unfolding of native ubiquitin under nondenaturing conditions is characterized by amide hydrogen exchange rate constants measured at high pH by using SOFAST real-time 2D NMR. Our data reveal that ubiquitin unfolds in a gradual manner with distinct unfolding regimes.

Schanda, Paul; Forge, Vincent; Brutscher, Bernhard

2007-01-01

324

About the structural role of disulfide bridges in serum albumins: evidence from protein simulated unfolding.  

PubMed

The role of the 17 disulfide (S-S) bridges in preserving the native conformation of human serum albumin (HSA) is investigated by performing classical molecular dynamics (MD) simulations on protein structures with intact and, respectively, reduced S-S bridges. The thermal unfolding simulations predict a clear destabilization of the protein secondary structure upon reduction of the S-S bridges as well as a significant distortion of the tertiary structure that is revealed by the changes in the protein native contacts fraction. The effect of the S-S bridges reduction on the protein compactness was tested by calculating Gibbs free energy profiles with respect to the protein gyration radius. The theoretical results obtained using the OPLS-AA and the AMBER ff03 force fields are in agreement with the available experimental data. Beyond the validation of the simulation method, the results here reported provide new insights into the mechanism of the protein reductive/oxidative unfolding/folding processes. It is predicted that in the native conformation of the protein, the thiol (-SH) groups belonging to the same reduced S-S bridge are located in potential wells that maintain them in contact. The -SH pairs can be dispatched by specific conformational transitions of the peptide chain located in the neighborhood of the cysteine residues. PMID:22899364

Paris, Guillaume; Kraszewski, Sebastian; Ramseyer, Christophe; Enescu, Mironel

2012-11-01

325

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

326

Water-protein interaction in native and partially unfolded equine cytochrome c  

NASA Astrophysics Data System (ADS)

The problem of the interaction of water solvent with proteins has been addressed by investigating the water 1H nuclear magnetic relaxation dispersion (NMRD) profiles of cytochrome c solutions. It is shown that the 1H NMRD profiles are accounted for by 1, a sizeable contribution from exchangeable protein protons (mostly from lysine side chains) and 2, a modest contribution from long-lived water. It is also shown that the number of exchangeable protons is sizeably increased in the oxidized but not in the reduced protein in the presence of the unfolding agent guanidinium chloride at a 3M concentration. This additional contribution arises mostly from backbone protons, as evidenced by high resolution NMR data which provide significant and independent data on the structure and the dynamic behaviour of the partly unfolded oxidized protein. Higher accessibility to short lived water molecules is proposed also. For the analysis of the 1H NMRD data a complete relaxation matrix approach is presented that is analogous, but not identical, to one recently described. This approach permits the simultaneous incorporation of exchangeable protein protons and an unlimited number of water molecules in pre-defined protein binding sites.

Banci, Lucia

1998-12-01

327

Fibronectin in aging extracellular matrix fibrils is progressively unfolded by cells and elicits an enhanced rigidity response  

PubMed Central

While the mechanical properties of a substrate or engineered scaffold can govern numerous aspects of cell behavior, cells quickly start to assemble their own matrix and will ultimately respond to their self-made extracellular matrix (ECM) microenvironments. Using fluorescence resonance energy transfer (FRET), we detected major changes in the conformation of a constituent ECM protein, fibronectin (Fn), as cells fabricated a thick three-dimensional (3D) matrix over the course of three days. These data provide the first evidence that matrix maturation occurs and that aging is associated with increased stretching of fibronectin fibrils, which leads to at least partial unfolding of the secondary structure of individual protein modules. A comparison of the conformations of Fn in these 3D matrices with those constructed by cells on rigid and flexible polyacrylamide surfaces suggests that cells in maturing matrices experience a microenviroment of gradually increasing rigidity. In addition, further matrix stiffening is caused by active Fn fiber alignment parallel to the contractile axis of the elongated fibroblasts, a cell-driven effect previously described for other fibrillar matrices. The fibroblasts, therefore, not only cause matrix unfolding, but reciprocally respond to the altered Fn matrix properties by up-regulating their own rigidity response. Consequently, our data demonstrate for the first time that a matured and aged matrix has distinctly different physical and biochemical properties compared to a newly assembled matrix. This might allow cells to specifically recognise the age of a matrix.

Antia, Meher; Baneyx, Gretchen; Kubow, Kristopher E.; Vogel, Viola

2008-01-01

328

Unfolding of quadruplex structure in the G-rich strand of the minisatellite repeat by the binding protein UP1.  

PubMed

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. PMID:12235355

Fukuda, Hirokazu; Katahira, Masato; Tsuchiya, Naoto; Enokizono, Yoshiaki; Sugimura, Takashi; Nagao, Minako; Nakagama, Hitoshi

2002-09-16

329

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

330

Probing Single-Molecule Protein Conformational Folding-Unfolding Dynamics: The multiple-State and Multiple-Channel Energy Landscape  

NASA Astrophysics Data System (ADS)

The folding-unfolding dynamics of protein provides an important understanding of the protein conformational dynamics and functions. We have used single-molecule fluorescence resonance energy transfer combined with statistical data analysis to characterize enzyme and signaling protein fundamental conformational dynamics of Calmodulin (CaM) and kinase (6-Hydroxymethyl-7,8-dihydropterin pyrophosphokinase, HPPK). The concentration dependence of FRET efficiency of GdmCl indicates the unfolding conformational transition of the proteins. At 2M of denaturant solvent, the majority of the HPPK and CaM protein molecules are under fluctuating folding-unfolding conformational changes, spending about half time in their native state and half time in their unfolded state. We obtained the fluctuation rates from the autocorrelation function analyses of the protein conformational fluctuation trajectories, and we have identified multiple intermediate states involving in bunched time dynamics and the related energy landscape. We had also analyzed the protein folding-unfolding pathways using detailed balance theoretical model analysis in order to understand the complex multiple-state and multiple-channel protein dynamics.

Lu, H. Peter; Wang, Zhijiang; He, Yufan

2013-03-01

331

Inhibition of Unfolding and Aggregation of Lens Protein Human Gamma D Crystallin by Sodium Citrate  

PubMed Central

Cataract affects 1 in 6 Americans over the age of 40, and is considered a global health problem. Cataract is caused by the aggregation of unfolded or damaged proteins in the lens, which accumulate as an individual ages. Currently, surgery is the only available treatment for cataract, however, small molecules have been suggested as potential preventative therapies. In this work, we study the effect of sodium citrate on the stability of Human ?D Crystallin (H?D-Crys), a structural protein of the eye lens, and two cataract-related mutants, L5S H?D-Crys and I90F H?D-Crys. In equilibrium unfolding-refolding studies, the presence of 250 mM sodium citrate increased the transition midpoint of the N-td of WT H?D-Crys and L5S H?D-Crys by 0.3 M GuHCl, the C-td by 0.6M GuHCl, and the single transition of I90F H?D-Crys by 0.4M GuHCl. In kinetic unfolding reactions, sodium citrate demonstrates a measurable stabilization effect only for the mutant I90F H?D-Crys. In the presence of citrate, a kinetic unfolding intermediate of I90F H?D-Crys can be observed, which was not observed in the absence of citrate. Rate of aggregation was measured using solution turbidity, and sodium citrate demonstrates negligible effect on rate of aggregation of WT H?D-Crys, but considerably slows the rate of aggregation of both L5S H?D-Crys and I90F H?D-Crys. The presence of sodium citrate dramatically slows refolding of WT H?D-Crys and I90F H?D-Crys, but has a significantly smaller effect on the refolding of L5S H?D-Crys. The differential stabilizing effect of sodium citrate suggests that the ion is binding to a partially unfolded conformation of the C-td, but a solution-based Hofmeister effect cannot be eliminated as a possible explanation for the effects observed. These results suggest that sodium citrate may be a potential preventative agent for cataract.

Goulet, Daniel R.; Knee, Kelly M.; King, Jonathan A.

2012-01-01

332

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

333

NMR unfolding studies on a liver bile acid binding protein reveal a global two-state unfolding and localized singular behaviors.  

PubMed

The folding properties of a bile acid binding protein, belonging to a subfamily of the fatty acid binding proteins, have been here investigated both by hydrogen exchange measurements, using the SOFAST NMR approach, and urea denaturation experiments. The urea unfolding profiles of individual residues, acting as single probes, were simultaneously analyzed through a global fit, according to a two-state unfolding model. The resulting conformational stability DeltaG(U)(H(2)O)=7.2+/-0.25kcal mol(-1) is in good agreement with hydrogen exchange stability DeltaG(op). While the majority of protein residues satisfy this model, few amino-acids display a singular behavior, not directly amenable to the presence of a folding intermediate, as reported for other fatty acid binding proteins. These residues are part of a protein patch characterized by enhanced plasticity. To explain this singular behavior a tentative model has been proposed which takes into account the interplay between the dynamic features and the formation of transient aggregates. A functional role for this plasticity, related to translocation across the nuclear membrane, is discussed. PMID:18977333

D'Onofrio, Mariapina; Ragona, Laura; Fessas, Dimitrios; Signorelli, Marco; Ugolini, Raffaella; Pedò, Massimo; Assfalg, Michael; Molinari, Henriette

2008-10-21

334

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

PubMed

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. PMID:24121352

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

2013-10-02

335

The liquid amide transfer model and the unfolding thermodynamics of small globular proteins.  

PubMed

In this paper, the solid cyclic dipeptide model developed by Murphy and Gill is analysed in order to point out that, apart from general thermodynamic features shown by well-characterized small globular proteins, only the polar and apolar contributions to the net denaturation heat capacity change are necessary to calculate the so-called protein stability curve, delta dGzero versus temperature. We propose that these specific heat capacity contributions can be determined in a reliable manner by a group additivity analysis of the transfer process of liquid amides from pure liquid phase into water. This suggests that the unfolding process, thought of as the transfer of amino acid residues from the protein 'core' to contact with water molecules, can be modelled based on the transfer process of organic amides. The reliability of the model is tested in comparison with literature data. PMID:8580089

Barone, G; del Vecchio, P; Giancola, C; Graziano, G

1995-10-01

336

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

337

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

338

Temperature Dependent Equilibrium Native to Unfolded Protein Dynamics and Properties Observed with IR Absorption and 2D IR Vibrational Echo Experiments  

PubMed Central

Dynamic and structural properties of carbonmonoxy (CO)-coordinated cytochrome c552 from Hydrogenobacter thermophilus (Ht-M61A) at different temperatures under thermal equilibrium conditions were studied with infrared absorption spectroscopy and ultrafast two dimensional infrared (2D IR) vibrational echo experiments using the heme-bound CO as the vibrational probe. Depending on the temperature, the stretching mode of CO shows two distinct bands corresponding to the native and unfolded proteins. As the temperature is increased from low temperature, a new absorption band for the unfolded protein grows in and the native band decreases in amplitude. Both the temperature dependent circular dichroism and the IR absorption area ratio RA(T), defined as the ratio of the area under the unfolded band to the sum of the areas of the native and unfolded bands, suggest a two-state transition from the native to the unfolded protein. However, it is found that the absorption spectrum of the unfolded protein increases its inhomogeneous linewidth and the center frequency shifts as the temperature is increased. The changes in linewidth and center frequency demonstrate that the unfolding does not follow simple two-state behavior. The temperature dependent 2D IR vibrational echo experiments show that the fast dynamics of the native protein are virtually temperature independent. In contrast, the fast dynamics of the unfolded protein are slower than those of the native protein, and the unfolded protein fast dynamics and at least a portion of the slower dynamics of the unfolded protein change significantly, becoming faster as the temperature is raised. The temperature dependence of the absorption spectrum and the changes in dynamics measured with the 2D IR experiments confirm that the unfolded ensemble of conformers continuously changes its nature as unfolding proceeds, in contrast to the native state, which displays a temperature independent distribution of structures.

Chung, Jean K.; Thielges, Megan C.; Bowman, Sarah E. J.; Bren, Kara L.; Fayer, M. D.

2011-01-01

339

The Stability and Formation of Native Proteins from Unfolded Monomers Is Increased through Interactions with Unrelated Proteins  

PubMed Central

The intracellular concentration of protein may be as high as 400 mg per ml; thus it seems inevitable that within the cell, numerous protein-protein contacts are constantly occurring. A basic biochemical principle states that the equilibrium of an association reaction can be shifted by ligand binding. This indicates that if within the cell many protein-protein interactions are indeed taking place, some fundamental characteristics of proteins would necessarily differ from those observed in traditional biochemical systems. Accordingly, we measured the effect of eight different proteins on the formation of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM) from guanidinium chloride unfolded monomers. The eight proteins at concentrations of micrograms per ml induced an important increase on active dimer formation. Studies on the mechanism of this phenomenon showed that the proteins stabilize the dimeric structure of TbTIM, and that this is the driving force that promotes the formation of active dimers. Similar data were obtained with TIM from three other species. The heat changes that occur when TbTIM is mixed with lysozyme were determined by isothermal titration calorimetry; the results provided direct evidence of the weak interaction between apparently unrelated proteins. The data, therefore, are strongly suggestive that the numerous protein-protein interactions that occur in the intracellular space are an additional control factor in the formation and stability of proteins.

Rodriguez-Almazan, Claudia; Torner, Francisco J.; Costas, Miguel; Perez-Montfort, Ruy; de Gomez-Puyou, Marieta Tuena; Puyou, Armando Gomez

2007-01-01

340

Engineered oligomerization state of OmpF protein through computational design decouples oligomer dissociation from unfolding  

PubMed Central

Biogenesis of ?-barrel membrane proteins is a complex, multi-step, and as yet incompletely characterized process. The bacterial porin family is perhaps the best studied protein family among the ?-barrel membrane proteins that allows diffusion of small solutes across the bacterial outer membrane. In this study, we have identified residues that contribute significantly to the protein-protein interaction (PPI) interface between the chains of Outer Membrane Protein F (OmpF), a trimeric porin, using an empirical energy function in conjunction with an evolutionary analysis. By replacing these residues through site-directed mutagenesis, either with energetically favorable residues or substitutions that do not occur in natural bacterial outer membrane proteins, we succeeded in engineering OmpF mutants with dimeric and monomeric instead of trimeric oligomerization state. Moreover, our results suggest that the oligomerization of OmpF proceeds through a series of interactions involving two distinct regions of the extensive PPI interface: Two monomers interact to form a dimer through the PPI interface near G19. This dimer than interacts with another monomer through the PPI interface near G135 to form a trimer. We have found that perturbing the PPI interface near G19 results in the formation of the monomeric OmpF only. Thermal de-naturation of the designed dimeric OmpF mutant suggests that the oligomer dissociation can be separated from the process of protein unfolding. Furthermore, the conserved site near G57, G59 is important for the PPI interface and might provide the essential scaffold for protein-protein interactions.

Naveed, Hammad; Jimenez-Morales, David; Tian, Jun; Pasupuleti, Volga; Kenney, Linda J.; Liang, Jie

2013-01-01

341

Alternative Computational Protocols for Supercharging Protein Surfaces for Reversible Unfolding and Retention of Stability  

PubMed Central

Reengineering protein surfaces to exhibit high net charge, referred to as “supercharging”, can improve reversibility of unfolding by preventing aggregation of partially unfolded states. Incorporation of charged side chains should be optimized while considering structural and energetic consequences, as numerous mutations and accumulation of like-charges can also destabilize the native state. A previously demonstrated approach deterministically mutates flexible polar residues (amino acids DERKNQ) with the fewest average neighboring atoms per side chain atom (AvNAPSA). Our approach uses Rosetta-based energy calculations to choose the surface mutations. Both protocols are available for use through the ROSIE web server. The automated Rosetta and AvNAPSA approaches for supercharging choose dissimilar mutations, raising an interesting division in surface charging strategy. Rosetta-supercharged variants of GFP (RscG) ranging from ?11 to ?61 and +7 to +58 were experimentally tested, and for comparison, we re-tested the previously developed AvNAPSA-supercharged variants of GFP (AscG) with +36 and ?30 net charge. Mid-charge variants demonstrated ?3-fold improvement in refolding with retention of stability. However, as we pushed to higher net charges, expression and soluble yield decreased, indicating that net charge or mutational load may be limiting factors. Interestingly, the two different approaches resulted in GFP variants with similar refolding properties. Our results show that there are multiple sets of residues that can be mutated to successfully supercharge a protein, and combining alternative supercharge protocols with experimental testing can be an effective approach for charge-based improvement to refolding.

Der, Bryan S.; Kluwe, Christien; Miklos, Aleksandr E.; Jacak, Ron; Lyskov, Sergey; Gray, Jeffrey J.; Georgiou, George; Ellington, Andrew D.; Kuhlman, Brian

2013-01-01

342

Alternative computational protocols for supercharging protein surfaces for reversible unfolding and retention of stability.  

PubMed

Reengineering protein surfaces to exhibit high net charge, referred to as "supercharging", can improve reversibility of unfolding by preventing aggregation of partially unfolded states. Incorporation of charged side chains should be optimized while considering structural and energetic consequences, as numerous mutations and accumulation of like-charges can also destabilize the native state. A previously demonstrated approach deterministically mutates flexible polar residues (amino acids DERKNQ) with the fewest average neighboring atoms per side chain atom (AvNAPSA). Our approach uses Rosetta-based energy calculations to choose the surface mutations. Both protocols are available for use through the ROSIE web server. The automated Rosetta and AvNAPSA approaches for supercharging choose dissimilar mutations, raising an interesting division in surface charging strategy. Rosetta-supercharged variants of GFP (RscG) ranging from -11 to -61 and +7 to +58 were experimentally tested, and for comparison, we re-tested the previously developed AvNAPSA-supercharged variants of GFP (AscG) with +36 and -30 net charge. Mid-charge variants demonstrated ?3-fold improvement in refolding with retention of stability. However, as we pushed to higher net charges, expression and soluble yield decreased, indicating that net charge or mutational load may be limiting factors. Interestingly, the two different approaches resulted in GFP variants with similar refolding properties. Our results show that there are multiple sets of residues that can be mutated to successfully supercharge a protein, and combining alternative supercharge protocols with experimental testing can be an effective approach for charge-based improvement to refolding. PMID:23741319

Der, Bryan S; Kluwe, Christien; Miklos, Aleksandr E; Jacak, Ron; Lyskov, Sergey; Gray, Jeffrey J; Georgiou, George; Ellington, Andrew D; Kuhlman, Brian

2013-05-31

343

Two-dimensional infrared spectroscopy of the thermal unfolding of proteins  

NASA Astrophysics Data System (ADS)

Steady-state and transient conformational changes upon the thermal unfolding of ubiquitin were investigated with femtosecond infrared spectroscopy of the amide I vibrations. Equilibrium temperature-dependent 2D IR spectroscopy reveals the unfolding of the ?-sheet of ubiquitin through the loss of cross peaks formed between transitions arising from vibrations of the ?-sheet. Transient unfolding following a nanosecond temperature jump is monitored with vibrational echo spectroscopy, a projection of the 2D IR spectrum. While the equilibrium study follows a simple two-state unfolding, the transient experiments observe complex relaxation behavior that differs for various spectral components and spans time scales from nanoseconds to milliseconds. By modeling the amide I vibrations of ubiquitin, this observation is explained as unfolding of the less stable strands III-V of the ?-sheet prior to unfolding of the hairpin that forms part of the hydrophobic core.

Tokmakoff, Andrei

2005-03-01

344

Quantification of free cysteines in membrane and soluble proteins using a fluorescent dye and thermal unfolding.  

PubMed

Cysteine is an extremely useful site for selective attachment of labels to proteins for many applications, including the study of protein structure in solution by electron paramagnetic resonance (EPR), fluorescence spectroscopy and medical imaging. The demand for quantitative data for these applications means that it is important to determine the extent of the cysteine labeling. The efficiency of labeling is sensitive to the 3D context of cysteine within the protein. Where the label or modification is not directly measurable by optical or magnetic spectroscopy, for example, in cysteine modification to dehydroalanine, assessing labeling efficiency is difficult. We describe a simple assay for determining the efficiency of modification of cysteine residues, which is based on an approach previously used to determine membrane protein stability. The assay involves a reaction between the thermally unfolded protein and a thiol-specific coumarin fluorophore that is only fluorescent upon conjugation with thiols. Monitoring fluorescence during thermal denaturation of the protein in the presence of the dye identifies the temperature at which the maximum fluorescence occurs; this temperature differs among proteins. Comparison of the fluorescence intensity at the identified temperature between modified, unmodified (positive control) and cysteine-less protein (negative control) allows for the quantification of free cysteine. We have quantified both site-directed spin labeling and dehydroalanine formation. The method relies on a commonly available fluorescence 96-well plate reader, which rapidly screens numerous samples within 1.5 h and uses <100 ?g of material. The approach is robust for both soluble and detergent-solubilized membrane proteins. PMID:24091556

Branigan, Emma; Pliotas, Christos; Hagelueken, Gregor; Naismith, James H

2013-10-03

345

Contrasting the individual reactive pathways in protein unfolding and disulfide bond reduction observed within a single protein.  

PubMed

Identifying the dynamics of individual molecules along their reactive pathways remains a major goal of modern chemistry. For simple chemical reactions, the transition state position is thought to be highly localized. Conversely, in the case of more complex reactions involving proteins, the potential energy surfaces become rougher, resulting in heterogeneous reaction pathways with multiple transition state structures. Force-clamp spectroscopy experimentally probes the individual reaction pathways sampled by a single protein under the effect of a constant stretching force. Herein, we examine the distribution of conformations that populate the transition state of two different reactions; the unfolding of a single protein and the reduction of a single disulfide bond, both occurring within the same single protein. By applying the recently developed static disorder theory, we quantify the variance of the barrier heights, ?(2), governing each distinct reaction. We demonstrate that the unfolding of the I27 protein follows a nonexponential kinetics, consistent with a high value of ?(2) ? 18 (pN nm)(2). Interestingly, shortening of the protein upon introduction of a rigid disulfide bond significantly modulates the disorder degree, spanning from ?(2) ? 8 to ?21 (pN nm)(2). These results are in sharp contrast with the exponential distribution of times measured for an S(N)2 chemical reaction, implying the absence of static disorder ?(2) ? 0 (pN nm)(2). Our results demonstrate the high sensitivity of the force-clamp technique to capture the signatures of disorder in the individual pathways that define two distinct force-induced reactions, occurring within the core of a single protein. PMID:21309561

Garcia-Manyes, Sergi; Kuo, Tzu-Ling; Fernández, Julio M

2011-02-10

346

The unfolded state of the murine prion protein and properties of single-point mutants related to human prion diseases.  

PubMed

The prion protein can exist both in a normal cellular isoform and in a pathogenic conformational isoform. The latter is responsible for the development of different neurodegenerative diseases, for example Creutzfeldt-Jakob disease or fatal familial insomnia. To convert the native benign state of the protein into a highly ordered fibrillar aggregate, large-scale rearrangements of the tertiary structure are necessary during the conversion process and intermediates that are at least partially unfolded are present during fibril formation. In addition to the sporadic conversion into the pathogenic isoform, more than 20 familial diseases are known that are caused by single point mutations increasing the probability of aggregation and neurodegeneration. Here, we demonstrate that the chemically denatured states of the mouse and human prion proteins have very similar structural and dynamic characteristics. Initial studies on the single point mutants E196K, F198S, V203I and R208H of the oxidized mouse construct, which are related to human prion diseases, reveal significant differences in the rate of aggregation. Aggregation for mutants V203I and R208H is slower than it is for the wild type, and the constructs E196K and F198S show accelerated aggregation. These differences in aggregation behaviour are not correlated with the thermal stability of the mutants, indicating different mechanisms promoting the conformational conversion process. PMID:20541558

Gerum, Christian; Schlepckow, Kai; Schwalbe, Harald

2010-06-10

347

Measurement of protein unfolding/refolding kinetics and structural characterization of hidden intermediates by NMR relaxation dispersion  

PubMed Central

Detailed understanding of protein function and malfunction hinges on the ability to characterize transiently populated states and the transitions between them. Here, we use 15N, , and 13CO NMR R2 relaxation dispersion to investigate spontaneous unfolding and refolding events of native apomyoglobin. Above pH 5.0, dispersion is dominated by processes involving fluctuations of the F-helix region, which is invisible in NMR spectra. Measurements of R2 dispersion for residues contacted by the F-helix region in the native (N) structure reveal a transient state formed by local unfolding of helix F and undocking from the protein core. A similar state was detected at pH 4.75–4.95 and determined to be an on-pathway intermediate (I1) in a linear three-state unfolding scheme (N?I1?MG) leading to a transiently populated molten globule (MG) state. The slowest steps in unfolding and refolding are N ? I1 (36 s-1) and MG ? I1 (26 s-1), respectively. Differences in chemical shift between N and I1 are very small, except in regions adjacent to helix F, showing that their core structures are similar. Chemical shift changes between the N and MG states, obtained from R2 dispersion, reveal that the transient MG state is structurally similar to the equilibrium MG observed previously at high temperature and low pH. Analysis of MG state chemical shifts shows the location of residual helical structure in the transient intermediate and identifies regions that unfold or rearrange into nonnative structure during the N ? MG transition. The experiments also identify regions of energetic frustration that “crack” during unfolding and impede the refolding process.

Meinhold, Derrick W.; Wright, Peter E.

2011-01-01

348

A similarity measure for partially folded proteins: application to unfolded and native-like conformational fluctuations  

NASA Astrophysics Data System (ADS)

Computing the root-mean-square deviation (RMSD) of a partially folded protein structure from the folded state requires the two structures to be translationally and rotationally aligned. We examine the constraint matrix L that preserves orthogonality of the rotation matrix during minimization of the RMSD. L is proportional to the sensitivity of the RMSD to the rotational alignment matrix. Its trace yields an isotropic reaction coordinate, while its off-diagonal matrix elements are related to the moment of inertia derivative tensor that encodes anisotropic information about the structure. We use L to compare ?-repressor fragment 6-85 (? 6-85) to several partially folded structures obtained from molecular dynamics simulation (MD), and find that L as a reaction coordinate indeed encodes some information about protein topology. We also apply C ? RMSD, L and tryptophan sidechain mobility as criteria for native state structural fluctuations of several ? 6-85 mutants. The mutants' denaturation curves and fluorescence quenching are measured experimentally for comparison. The results are in accord with a recent proposal that structural fluctuations near the chromophore can induce increased native state fluorescence or hyperfluorescence during unfolding of proteins.

Larios, Edgar; Yang, Wei Y.; Schulten, K.; Gruebele, M.

2004-12-01

349

Slow unfolded-state structuring in Acyl-CoA binding protein folding revealed by simulation and experiment.  

PubMed

Protein folding is a fundamental process in biology, key to understanding many human diseases. Experimentally, proteins often appear to fold via simple two- or three-state mechanisms involving mainly native-state interactions, yet recent network models built from atomistic simulations of small proteins suggest the existence of many possible metastable states and folding pathways. We reconcile these two pictures in a combined experimental and simulation study of acyl-coenzyme A binding protein (ACBP), a two-state folder (folding time ~10 ms) exhibiting residual unfolded-state structure, and a putative early folding intermediate. Using single-molecule FRET in conjunction with side-chain mutagenesis, we first demonstrate that the denatured state of ACBP at near-zero denaturant is unusually compact and enriched in long-range structure that can be perturbed by discrete hydrophobic core mutations. We then employ ultrafast laminar-flow mixing experiments to study the folding kinetics of ACBP on the microsecond time scale. These studies, along with Trp-Cys quenching measurements of unfolded-state dynamics, suggest that unfolded-state structure forms on a surprisingly slow (~100 ?s) time scale, and that sequence mutations strikingly perturb both time-resolved and equilibrium smFRET measurements in a similar way. A Markov state model (MSM) of the ACBP folding reaction, constructed from over 30 ms of molecular dynamics trajectory data, predicts a complex network of metastable stables, residual unfolded-state structure, and kinetics consistent with experiment but no well-defined intermediate preceding the main folding barrier. Taken together, these experimental and simulation results suggest that the previously characterized fast kinetic phase is not due to formation of a barrier-limited intermediate but rather to a more heterogeneous and slow acquisition of unfolded-state structure. PMID:22747188

Voelz, Vincent A; Jäger, Marcus; Yao, Shuhuai; Chen, Yujie; Zhu, Li; Waldauer, Steven A; Bowman, Gregory R; Friedrichs, Mark; Bakajin, Olgica; Lapidus, Lisa J; Weiss, Shimon; Pande, Vijay S

2012-07-19

350

Reduction potentials of blue and purple copper proteins in their unfolded states: a closer look at rack-induced coordination  

Microsoft Academic Search

Cyclic voltammetry has been used to determine the reduction potentials of blue (Pseudomonas aeruginosa azurin) and purple (Thermus thermophilus CuA domain) copper proteins unfolded by guanidine hydrochloride. These Cu(II\\/I) potentials [456 (azurin); 453 (CuA) mV vs., NHE] are higher than those of the folded proteins. The downshift of the potential in the folded state can be accounted\\u000a for by assuming

Pernilla Wittung-Stafshede; Michael G. Hill; Ester Gomez; Angel J. Di Bilio; B. Göran Karlsson; Johan Leckner; Jay R. Winkler; Harry B. Gray; Bo G. Malmström

1998-01-01

351

Interaction of oxidized chaperonin GroEL with an unfolded protein at low temperatures.  

PubMed

The chaperonin GroEL binds to non-native substrate proteins via hydrophobic interactions, preventing their aggregation, which is minimized at low temperatures. In the present study, we investigated the refolding of urea-denatured rhodanese at low temperatures, in the presence of ox-GroEL (oxidized GroEL), which contains increased exposed hydrophobic surfaces and retains its ability to hydrolyse ATP. We found that ox-GroEL could efficiently bind the urea-unfolded rhodanese at 4°C, without requiring excess amount of chaperonin relative to normal GroEL (i.e. non-oxidized). The release/reactivation of rhodanese from GroEL was minimal at 4°C, but was found to be optimal between 22 and 37°C. It was found that the loss of the ATPase activity of ox-GroEL at 4°C prevented the release of rhodanese from the GroEL-rhodanese complex. Thus ox-GroEL has the potential to efficiently trap recombinant or non-native proteins at 4°C and release them at higher temperatures under appropriate conditions. PMID:22273181

Melkani, Girish C; Sielaff, Robin; Zardeneta, Gustavo; Mendoza, Jose A

2012-06-01

352

The role of hydration in protein stability: comparison of the cold and heat unfolded states of Yfh1.  

PubMed

Protein unfolding occurs at both low and high temperatures, although in most cases, only the high-temperature transition can be experimentally studied. A pressing question is how much the low- and high-temperature denatured states, although thermodynamically equivalent, are structurally and kinetically similar. We have combined experimental and computational approaches to compare the high- and low-temperature unfolded states of Yfh1, a natural protein that, at physiologic pH, undergoes cold and heat denaturation around 0 °C and 40 °C without the help of ad hoc destabilization. We observe that the two denatured states have similar but not identical residual secondary structures, different kinetics and compactness and a remarkably different degree of hydration. We use molecular dynamics simulations to rationalize the role of solvation and its effect on protein stability. PMID:22342930

Adrover, Miquel; Martorell, Gabriel; Martin, Stephen R; Urosev, Dunja; Konarev, Petr V; Svergun, Dmitri I; Daura, Xavier; Temussi, Pierandrea; Pastore, Annalisa

2012-02-14

353

Irradiation of the Porphyrin Causes Unfolding of the Protein in the Protoporphyrin IX/?-Lactoglobulin Noncovalent Complex  

PubMed Central

Porphyrins such as protoporphyrin IX (PPIX) are known to occasionally cause conformational changes in proteins for which they are specific ligands. It has also been established that irradiation of porphyrins noncovalently intercalated between bases or bound to one of the grooves can cause conformational effects on DNA. Conversely, there is no evidence reported in the literature of conformational changes caused by noncovalently bound PPIX to globular proteins for which the porphyrin is not a specific ligand. This study shows that the irradiation of the porphyrin in the PPIX/lactoglobulin noncovalent complex indeed causes a local and limited (~7%) unfolding of the protein near the location of Trp19. This event causes the intrinsic fluorescence spectrum of the protein to shift to the red by 2 nm and the average decay lifetime to lengthen by approximately 0.5 ns. The unfolding of lactoglobulin occurs only at pH > 7 because of the increased instability of the protein at alkaline pH. The photoinduced unfolding does not depend on the presence of O2 in solution; therefore, it is not mediated by formation of singlet oxygen and is likely the result of electron transfer between the porphyrin and amino acid residues.

Fernandez, Nicholas F.; Sansone, Samuel; Mazzini, Alberto; Brancaleon, Lorenzo

2012-01-01

354

Loss of dispersion energy changes the stability and folding/unfolding equilibrium of the Trp-cage protein.  

PubMed

The structure of proteins as well as their folding/unfolding equilibrium are commonly attributed to H-bonding and hydrophobic interactions. We have used the molecular dynamic simulations in an explicit water environment based on the standard empirical potential as well as more accurately (and thus also more reliably) on the QM/MM potential. The simulations where the dispersion term was suppressed have led to a substantial change of the tryptophan-cage protein structure (unfolded structure). This structure cannot fold without the dispersion energy term, whereas, if it is covered fully, the system finds its native structure relatively quickly. This implies that after such physical factors as temperature and pH, the dispersion energy is an important factor in protein structure determination as well as in the protein folding/unfolding equilibrium. The loss of dispersion also affected the R-helical structure. On the other hand, weakening the electrostatic interactions (and thus H-bonding) affected the R-helical structure only to a minor extent. PMID:19444987

Cerný, Jirí; Vondrásek, Jirí; Hobza, Pavel

2009-04-23

355

The search for local native-like nucleation centers in the unfolded state of beta -sheet proteins.  

PubMed

An approach involving the systematic computational conformational analysis of all overlapping hexapeptide segments in the protein sequence has found fragments with the higher than average propensity to adopt the native-like three-dimensional structure and other regular nonrandom structures in the unfolded states of four beta-sheet proteins, namely IFABP (intestinal fatty acid-binding protein), ILBP (ileal fatty acid-binding protein), CRABP I (cellular retinoic acid-binding protein), and CRBP II (cellular retinal binding protein). The native three-dimensional structures of these four proteins are very similar even though they possess as little as approximately 30% sequence similarity. The computational results were validated by comparison with the experimental data of the heteronuclear sequential quantum correlation NMR spectroscopy obtained earlier for IFABP at high urea concentrations. On this basis, a molecular model of the unfolded state of IFABP has been developed. The model presumes a dynamic equilibrium between various nonrandom structures (including the native-like structure) and random coil in the local segments of the protein sequence. The model explains experimental observations obtained earlier for folding of several mutants of IFABP, as well as the observed differences in molecular mechanisms of folding for the four beta-sheet proteins. Because the computational approach itself does not employ any experimentally derived information in advance, it is not necessarily limited to the beta-sheet proteins. PMID:12140369

Nikiforovich, Gregory V; Frieden, Carl

2002-07-24

356

FoldIndex copyright: a simple tool to predict whether a given protein sequence is intrinsically unfolded  

Microsoft Academic Search

Summary: 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 hydrophobi- city and net charge of the sequence. FoldIndex© has an error rate comparable to that of more sophisticated fold prediction methods. Sliding

Jaime Prilusky; Clifford E. Felder; Tzviya Zeev-ben-mordehai; Edwin H. Rydberg; Orna Man; Jacques S. Beckmann; Israel Silman; Joel L. Sussman

2005-01-01

357

Interesting Features of Liquid-Vapor Interfaces Observed by X-ray Reflection: Protein Unfolding at Interfaces  

NASA Astrophysics Data System (ADS)

X-ray reflectivity profiles of human lactoferrin absorbed at water interfaces have calculated using the structure determined by neutron reflection measurements [Lu et al., Langmuir, 21, 3354 (2005)]. A significant difference in the expected profiles was observed between the unfolding lactoferrin at the interfaces and aqueous subphase itself. Time-resolved X-ray reflectivity measurements are proposed to probe the protein adsorbing process at interfaces.

Yano, Yohko F.

2007-03-01

358

Cyclosporin A induces the unfolded protein response in keratinocytes  

Microsoft Academic Search

Psoriasis vulgaris is a chronic inflammatory disorder of the skin, in which activation of keratinocytes and crosstalk between\\u000a keratinocytes and T cells or dendritic cells are considered to be involved in the pathogenesis of psoriasis vulgaris. Cyclosporin\\u000a (Cy) A, an immunomodulator, has been used for the treatment of psoriasis vulgaris, but the mechanism of its action on keratinocytes\\u000a has not

Michiko Hibino; Kazumitsu Sugiura; Yoshinao Muro; Yoshie Shimoyama; Yasushi Tomita