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Sample records for unfolded protein response

  1. Translation Attenuation Mechanism in Unfolded Protein Response

    NASA Astrophysics Data System (ADS)

    Trusina, Ala; Papa, Feroz; Tang, Chao

    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 β-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.

  2. Melanoma and the Unfolded Protein Response

    PubMed Central

    Sykes, Erin K.; Mactier, Swetlana; Christopherson, Richard I.

    2016-01-01

    The UPR (unfolded protein response) has been identified as a key factor in the progression and metastasis of cancers, notably melanoma. Several mediators of the UPR are upregulated in cancers, e.g., high levels of GRP78 (glucose-regulator protein 78 kDa) correlate with progression and poor outcome in melanoma patients. The proliferative burden of cancer induces stress and activates several cellular stress responses. The UPR is a tightly orchestrated stress response that is activated upon the accumulation of unfolded proteins within the ER (endoplasmic reticulum). The UPR is designed to mediate two conflicting outcomtes, recovery and apoptosis. As a result, the UPR initiates a widespread signaling cascade to return the cell to homeostasis and failing to achieve cellular recovery, initiates UPR-induced apoptosis. There is evidence that ER stress and subsequently the UPR promote tumourigenesis and metastasis. The complete role of the UPR has yet to be defined. Understanding how the UPR allows for adaption to stress and thereby assists in cancer progression is important in defining an archetype of melanoma pathology. In addition, elucidation of the mechanisms of the UPR may lead to development of effective treatments of metastatic melanoma. PMID:26927180

  3. The mitochondrial unfolded protein response - synchronizing genomes

    PubMed Central

    Jovaisaite, Virginija; Auwerx, Johan

    2014-01-01

    Maintenance of the mitochondrial proteome is performed primarily by chaperones, which fold and assemble proteins, and by proteases, which degrade excess damaged proteins. Upon various types of mitochondrial stress, triggered genetically or pharmacologically, dysfunction of the proteome is sensed and communicated to the nucleus, where an extensive transcriptional program, aimed to repair the damage, is activated. This feedback loop, termed the mitochondrial unfolded protein response (UPRmt), synchronizes the activity of the mitochondrial and nuclear genomes and as such ensures the quality of the mitochondrial proteome. Here we review the recent advances in the UPRmt field and discuss its induction, signaling, communication with the other mitochondrial and major cellular regulatory pathways and its potential implications on health and lifespan. PMID:25543897

  4. The Unfolded Protein Response and Gastrointestinal Disease

    PubMed Central

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

    2013-01-01

    As the inner lining of the gastrointestinal tract, the intestinal epithelium serves an essential role in innate immune function at the interface between the host and microbiota. Given the unique environmental challenges and thus physiologic secretory functions of this surface, it is exquisitely sensitive to perturbations that affect its capacity to resolve endoplasmic reticulum (ER) stress. Genetic deletion of factors involved in the unfolded protein response (UPR), which functions in the resolution of ER stress that arises from misfolded proteins, result in spontaneous intestinal inflammation closely mimicking human inflammatory bowel disease (IBD). This is demonstrated by observations wherein deletion of genes such as Xbp1 and Agr2 profoundly affects the intestinal epithelium and results in spontaneous intestinal inflammation. Moreover, both genes, along with others (e.g. ORDML3) represent genetic risk factors for human IBD, both Crohn’s disease and ulcerative colitis. Here we review the current mechanistic understanding for how unresolved ER stress can lead to intestinal inflammation, and highlight the findings that implicate ER stress as a genetically affected biological pathway in IBD. We further discuss environmental and microbial factors that might impact on the epithelium’s capacity to resolve ER stress, and which may constitute exogenous factors that may precipitate disease in genetically susceptible individuals. PMID:23588234

  5. Endoplasmic Reticulum Stress Sensing in the Unfolded Protein Response

    PubMed Central

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

    2013-01-01

    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

  6. The unfolded protein response: mechanisms and therapy of neurodegeneration

    PubMed Central

    Smith, Heather L.

    2016-01-01

    Activation of the unfolded protein response is emerging as a common theme in protein-misfolding neurodegenerative diseases, with relevant markers observed in patient tissue and mouse models. Genetic and pharmacological manipulation of the pathway in several mouse models has shown that this is not a passive consequence of the neurodegeneration process. Rather, overactivation of the protein kinase RNA-like ER kinase (PERK, encoded by EIF2AK3) branch of the unfolded protein response directly contributes to disease pathogenesis through the critical reduction in neuronal protein synthesis rates, essential for learning and memory and for neuronal survival. The pharmacological inhibition of this process in these models is strikingly neuroprotective, resulting in the discovery of the first small molecule preventing neurodegeneration and clinical disease in vivo. This now represents a potential generic approach for boosting memory and preventing neurodegeneration across the spectrum of these disorders, albeit with some exceptions, independent of disease-specific proteins. Targeting the unfolded protein response, and particularly PERK-branch mediated translational failure is thus an increasingly compelling strategy for new treatments for dementia and neurodegenerative disease. PMID:27190028

  7. The unfolded protein response: mechanisms and therapy of neurodegeneration.

    PubMed

    Smith, Heather L; Mallucci, Giovanna R

    2016-08-01

    Activation of the unfolded protein response is emerging as a common theme in protein-misfolding neurodegenerative diseases, with relevant markers observed in patient tissue and mouse models. Genetic and pharmacological manipulation of the pathway in several mouse models has shown that this is not a passive consequence of the neurodegeneration process. Rather, overactivation of the protein kinase RNA-like ER kinase (PERK, encoded by EIF2AK3) branch of the unfolded protein response directly contributes to disease pathogenesis through the critical reduction in neuronal protein synthesis rates, essential for learning and memory and for neuronal survival. The pharmacological inhibition of this process in these models is strikingly neuroprotective, resulting in the discovery of the first small molecule preventing neurodegeneration and clinical disease in vivo This now represents a potential generic approach for boosting memory and preventing neurodegeneration across the spectrum of these disorders, albeit with some exceptions, independent of disease-specific proteins. Targeting the unfolded protein response, and particularly PERK-branch mediated translational failure is thus an increasingly compelling strategy for new treatments for dementia and neurodegenerative disease. PMID:27190028

  8. Unfolded protein response in cancer: the Physician's perspective

    PubMed Central

    2011-01-01

    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. PMID:21345215

  9. The unfolded protein response in immunity and inflammation.

    PubMed

    Grootjans, Joep; Kaser, Arthur; Kaufman, Randal J; Blumberg, Richard S

    2016-08-01

    The unfolded protein response (UPR) is a highly conserved pathway that allows the cell to manage endoplasmic reticulum (ER) stress that is imposed by the secretory demands associated with environmental forces. In this role, the UPR has increasingly been shown to have crucial functions in immunity and inflammation. In this Review, we discuss the importance of the UPR in the development, differentiation, function and survival of immune cells in meeting the needs of an immune response. In addition, we review current insights into how the UPR is involved in complex chronic inflammatory diseases and, through its role in immune regulation, antitumour responses. PMID:27346803

  10. Cellular unfolded protein response against viruses used in gene therapy

    PubMed Central

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

    2014-01-01

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

  11. Targeting the unfolded protein response in heart diseases

    PubMed Central

    Liu, Man; Dudley, Samuel C

    2016-01-01

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

  12. The unfolded protein response in skeletal development and homeostasis.

    PubMed

    Horiuchi, Keisuke; Tohmonda, Takahide; Morioka, Hideo

    2016-08-01

    Osteoblasts and chondrocytes produce a large number of extracellular matrix proteins to generate and maintain the skeletal system. To cope with their functions as secretory cells, these cells must acquire a considerable capacity for protein synthesis and also the machinery for the quality-control and transport of newly synthesized secreted proteins. The unfolded protein response (UPR) plays a crucial role during the differentiation of these cells to achieve this goal. Unexpectedly, however, studies in the past several years have revealed that the UPR has more extensive functions in skeletal development than was initially assumed, and the UPR critically orchestrates many facets of skeletal development and homeostasis. This review focuses on recent findings on the functions of the UPR in the differentiation of osteoblasts, chondrocytes, and osteoclasts. These findings may have a substantial impact on our understanding of bone metabolism and also on establishing treatments for congenital and acquired skeletal disorders. PMID:27002737

  13. Inhibition of the Unfolded Protein Response Mechanism Prevents Cardiac Fibrosis

    PubMed Central

    Jung, Joanna; Dyck, Jason R. B.; Lopaschuk, Gary D.; Agellon, Luis B.; Michalak, Marek

    2016-01-01

    Background Cardiac fibrosis attributed to excessive deposition of extracellular matrix proteins is a major cause of heart failure and death. Cardiac fibrosis is extremely difficult and challenging to treat in a clinical setting due to lack of understanding of molecular mechanisms leading to cardiac fibrosis and effective anti-fibrotic therapies. The objective in this study was to examine whether unfolded protein response (UPR) pathway mediates cardiac fibrosis and whether a pharmacological intervention to modulate UPR can prevent cardiac fibrosis and preserve heart function. Methodology/Principal Findings We demonstrate here that the mechanism leading to development of fibrosis in a mouse with increased expression of calreticulin, a model of heart failure, stems from impairment of endoplasmic reticulum (ER) homeostasis, transient activation of the unfolded protein response (UPR) pathway and stimulation of the TGFβ1/Smad2/3 signaling pathway. Remarkably, sustained pharmacologic inhibition of the UPR pathway by tauroursodeoxycholic acid (TUDCA) is sufficient to prevent cardiac fibrosis, and improved exercise tolerance. Conclusions We show that the mechanism leading to development of fibrosis in a mouse model of heart failure stems from transient activation of UPR pathway leading to persistent remodelling of cardiac tissue. Blocking the activation of the transiently activated UPR pathway by TUDCA prevented cardiac fibrosis, and improved prognosis. These findings offer a window for additional interventions that can preserve heart function. PMID:27441395

  14. Dynamics of unfolded protein response in recombinant CHO cells.

    PubMed

    Prashad, Kamal; Mehra, Sarika

    2015-03-01

    Genes in the protein secretion pathway have been targeted to increase productivity of monoclonal antibodies in Chinese hamster ovary cells. The results have been highly variable depending on the cell type and the relative amount of recombinant and target proteins. This paper presents a comprehensive study encompassing major components of the protein processing pathway in the endoplasmic reticulum (ER) to elucidate its role in recombinant cells. mRNA profiles of all major ER chaperones and unfolded protein response (UPR) pathway genes are measured at a series of time points in a high-producing cell line under the dynamic environment of a batch culture. An initial increase in IgG heavy chain mRNA levels correlates with an increase in productivity. We observe a parallel increase in the expression levels of majority of chaperones. The chaperone levels continue to increase until the end of the batch culture. In contrast, calreticulin and ERO1-L alpha, two of the lowest expressed genes exhibit transient time profiles, with peak induction on day 3. In response to increased ER stress, both the GCN2/PKR-like ER kinase and inositol-requiring enzyme-1alpha (Ire1α) signalling branch of the UPR are upregulated. Interestingly, spliced X-Box binding protein 1 (XBP1s) transcription factor from Ire1α pathway is detected from the beginning of the batch culture. Comparison with the expression levels in a low producer, show much lower induction at the end of the exponential growth phase. Thus, the unfolded protein response strongly correlates with the magnitude and timing of stress in the course of the batch culture. PMID:24504562

  15. Unfolded protein response in hepatitis C virus infection

    PubMed Central

    Chan, Shiu-Wan

    2014-01-01

    Hepatitis C virus (HCV) is a single-stranded, positive-sense RNA virus of clinical importance. The virus establishes a chronic infection and can progress from chronic hepatitis, steatosis to fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). The mechanisms of viral persistence and pathogenesis are poorly understood. Recently the unfolded protein response (UPR), a cellular homeostatic response to endoplasmic reticulum (ER) stress, has emerged to be a major contributing factor in many human diseases. It is also evident that viruses interact with the host UPR in many different ways and the outcome could be pro-viral, anti-viral or pathogenic, depending on the particular type of infection. Here we present evidence for the elicitation of chronic ER stress in HCV infection. We analyze the UPR signaling pathways involved in HCV infection, the various levels of UPR regulation by different viral proteins and finally, we propose several mechanisms by which the virus provokes the UPR. PMID:24904547

  16. Unfolded Protein Response Pathways in Bloodstream-Form Trypanosoma brucei?

    PubMed

    Tiengwe, Calvin; Brown, Abigail E N A; Bangs, James D

    2015-11-01

    The unfolded protein response (UPR) is a stress mechanism to cope with misfolded proteins in the early secretory pathway, the hallmark being transcriptional upregulation of endoplasmic reticulum (ER) molecular chaperones such as BiP and protein disulfide isomerase. Despite the lack of transcriptional regulation and the absence of the classical UPR machinery, African trypanosomes apparently respond to persistent ER stress by a UPR-like response, including upregulation of BiP, and a related spliced leader silencing (SLS) response whereby SL RNA transcription is shut down. Initially observed by knockdown of the secretory protein translocation machinery, both responses are also induced by chemical agents known to elicit UPR in mammalian cells (H. Goldshmidt, D. Matas, A. Kabi, A. Carmi, R. Hope, S. Michaeli, PLoS Pathog 6:e1000731, 2010, http://dx.doi.org/10.1371/journal.ppat.1000731). As these findings were generated primarily in procyclic-stage trypanosomes, we have investigated both responses in pathogenic bloodstream-stage parasites. RNA interference (RNAi) silencing of the core translocon subunit Trypanosoma brucei Sec61α (TbSec61α) failed to induce either response. Interestingly, cell growth halted within 16 h of silencing, but sufficient TbSec61α remained to allow full competence for translocation of nascent secretory proteins for up to 24 h, indicating that replication is finely coupled with the capacity to synthesize and transport secretory cargo. Tunicamycin and thapsigargin at concentrations compatible with short-term (4 h) and long-term (24 h) viability also failed to induce any of the indicators of UPR-like or SLS responses. Dithiothreitol (DTT) was lethal at all concentrations tested. These results indicate that UPR-like and SLS responses to persistent ER stress do not occur in bloodstream-stage trypanosomes. PMID:26318397

  17. Unfolded Protein Response Pathways in Bloodstream-Form Trypanosoma brucei?

    PubMed Central

    Tiengwe, Calvin; Brown, Abigail E. N. A.

    2015-01-01

    The unfolded protein response (UPR) is a stress mechanism to cope with misfolded proteins in the early secretory pathway, the hallmark being transcriptional upregulation of endoplasmic reticulum (ER) molecular chaperones such as BiP and protein disulfide isomerase. Despite the lack of transcriptional regulation and the absence of the classical UPR machinery, African trypanosomes apparently respond to persistent ER stress by a UPR-like response, including upregulation of BiP, and a related spliced leader silencing (SLS) response whereby SL RNA transcription is shut down. Initially observed by knockdown of the secretory protein translocation machinery, both responses are also induced by chemical agents known to elicit UPR in mammalian cells (H. Goldshmidt, D. Matas, A. Kabi, A. Carmi, R. Hope, S. Michaeli, PLoS Pathog 6:e1000731, 2010, http://dx.doi.org/10.1371/journal.ppat.1000731). As these findings were generated primarily in procyclic-stage trypanosomes, we have investigated both responses in pathogenic bloodstream-stage parasites. RNA interference (RNAi) silencing of the core translocon subunit Trypanosoma brucei Sec61α (TbSec61α) failed to induce either response. Interestingly, cell growth halted within 16 h of silencing, but sufficient TbSec61α remained to allow full competence for translocation of nascent secretory proteins for up to 24 h, indicating that replication is finely coupled with the capacity to synthesize and transport secretory cargo. Tunicamycin and thapsigargin at concentrations compatible with short-term (4 h) and long-term (24 h) viability also failed to induce any of the indicators of UPR-like or SLS responses. Dithiothreitol (DTT) was lethal at all concentrations tested. These results indicate that UPR-like and SLS responses to persistent ER stress do not occur in bloodstream-stage trypanosomes. PMID:26318397

  18. Oxidative Stress, Unfolded Protein Response, and Apoptosis in Developmental Toxicity

    PubMed Central

    Kupsco, Allison; Schlenk, Daniel

    2016-01-01

    Physiological development requires precise spatiotemporal regulation of cellular and molecular processes. Disruption of these key events can generate developmental toxicity in the form of teratogenesis or mortality. The mechanism behind many developmental toxicants remains unknown. While recent work has focused on the unfolded protein response (UPR), oxidative stress, and apoptosis in the pathogenesis of disease, few studies have addressed their relationship in developmental toxicity. Redox regulation, UPR, and apoptosis are essential for physiological development and can be disturbed by a variety of endogenous and exogenous toxicants to generate lethality and diverse malformations. This review examines the current knowledge of the role of oxidative stress, UPR, and apoptosis in physiological development as well as in developmental toxicity, focusing on studies and advances in vertebrates model systems. PMID:26008783

  19. The unfolded protein response affects readthrough of premature termination codons

    PubMed Central

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

    2014-01-01

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

  20. The mitochondrial unfolded protein response in mammalian physiology

    PubMed Central

    Mottis, Adrienne; Jovaisaite, Virginija; Auwerx, Johan

    2014-01-01

    Mitochondria, the main site of cellular energy harvesting, are derived from proteobacteria that evolved within our cells in endosymbiosis. Mitochondria retained vestiges of their proteobacterial genome, the circular mitochondrial DNA (mtDNA), which encodes 13 subunits of the oxidative phosphorylation (OXPHOS) multiprotein complexes in the electron transport chain (ETC), while the remaining ~80 ETC components are encoded in the nuclear DNA (nDNA). A further ~1,400 proteins, which are essential for mitochondrial function are also encoded in nDNA. Thus the majority of mitochondrial proteins are translated in the cytoplasm, then imported, processed, and assembled in the mitochondria. An intricate protein quality control (PQC) network, constituted of chaperones and proteases that refold or degrade defective proteins, maintains mitochondrial proteostasis and ensures the cell and organism health. The mitochondrial unfolded protein response (UPRmt) is a relatively recently discovered PQC pathway, which senses the proteostatic disturbances specifically in the mitochondria and resolves the stress by retrograde signaling to the nucleus and consequent transcriptional activation of protective genes. This PQC system does not only transiently resolves the local stress, but can have long lasting effects on whole body metabolism, fitness and longevity. A delicate tuning of its activation levels might constitute a treatment of various diseases, such as metabolic diseases, cancer and neurodegenerative disorders. PMID:24898297

  1. Legionella suppresses the host unfolded protein response via multiple mechanisms

    PubMed Central

    Treacy-Abarca, Sean; Mukherjee, Shaeri

    2015-01-01

    The intracellular pathogen, Legionella pneumophila, secretes ∼300 effector proteins to modulate the host environment. Given the intimate interaction between L. pneumophila and the endoplasmic reticulum, we investigated the role of the host unfolded protein response (UPR) during L. pneumophila infection. Interestingly, we show that the host identifies L. pneumophila infection as a form of endoplasmic reticulum stress and the sensor pATF6 is processed to generate pATF6(N), a transcriptional activator of downstream UPR genes. However, L. pneumophila is able to suppress the UPR and block the translation of prototypical UPR genes, BiP and CHOP. Furthermore, biochemical studies reveal that L. pneumophila uses two effectors (Lgt1 and Lgt2) to inhibit the splicing of XBP1u mRNA to spliced XBP1 (XBP1s), an UPR response regulator. Thus, we demonstrate that L. pneumophila is able to inhibit the UPR by multiple mechanisms including blocking XBP1u splicing and causing translational repression. This observation highlights the utility of L. pneumophila as a powerful tool for studying a critical protein homeostasis regulator. PMID:26219498

  2. Chloroplast unfolded protein response, a new plastid stress signaling pathway?

    PubMed

    Ramundo, Silvia; Rochaix, Jean-David

    2014-01-01

    A unique feature of the ATP-dependent ClpP protease of eukaryotic photosynthetic organisms is that its catalytic subunit ClpP1 is encoded by the chloroplast genome. Attempts to inactivate this subunit through chloroplast transformation have failed because it is essential for cell survival. To study the function of ClpP we have developed a repressible chloroplast gene expression system in Chlamydomonas reinhardtii. This system is based on the use of a chimeric nuclear gene in which the vitamin-repressible MetE promoter and Thi4 riboswitch have been fused to the coding sequence of Nac2. Upon entry into the chloroplast the Nac2 protein specifically interacts with the psbD 5'UTR and is required for the proper processing/translation of the psbD mRNA. This property can be conveyed to any chloroplast mRNA by replacing its 5'UTR with that of psbD. In this study we have chosen clpP1 as plastid target gene and examined the cellular events induced upon depletion of ClpP through transcriptomic, proteomic, biochemical and electron microscope analysis. Among the most striking features, a massive increase in protein abundance occurs for plastid chaperones, proteases and proteins involved in membrane assembly/disassembly strongly suggesting the existence of a chloroplast unfolded protein response. PMID:25482768

  3. The unfolded protein response is required for dendrite morphogenesis

    PubMed Central

    Wei, Xing; Howell, Audrey S; Dong, Xintong; Taylor, Caitlin A; Cooper, Roshni C; Zhang, Jianqi; Zou, Wei; Sherwood, David R; Shen, Kang

    2015-01-01

    Precise patterning of dendritic fields is essential for the formation and function of neuronal circuits. During development, dendrites acquire their morphology by exuberant branching. How neurons cope with the increased load of protein production required for this rapid growth is poorly understood. Here we show that the physiological unfolded protein response (UPR) is induced in the highly branched Caenorhabditis elegans sensory neuron PVD during dendrite morphogenesis. Perturbation of the IRE1 arm of the UPR pathway causes loss of dendritic branches, a phenotype that can be rescued by overexpression of the ER chaperone HSP-4 (a homolog of mammalian BiP/ grp78). Surprisingly, a single transmembrane leucine-rich repeat protein, DMA-1, plays a major role in the induction of the UPR and the dendritic phenotype in the UPR mutants. These findings reveal a significant role for the physiological UPR in the maintenance of ER homeostasis during morphogenesis of large dendritic arbors. DOI: http://dx.doi.org/10.7554/eLife.06963.001 PMID:26052671

  4. The unfolded protein response: the dawn of a new field

    PubMed Central

    MORI, Kazutoshi

    2015-01-01

    Originating from cancer research in mammalian cultured cells, the entirely new field of the unfolded protein response (UPR) was born in 1988. The UPR is a transcriptional induction program coupled with intracellular signaling from the endoplasmic reticulum (ER) to the nucleus to maintain the homeostasis of the ER, an organelle which controls the quality of proteins destined for the secretory pathway. Extremely competitive analyses using the budding yeast Saccharomyces cerevisiae revealed that although signaling from both the ER and cell surface is initiated by activation of a transmembrane protein kinase, the mechanism downstream of ER-resident Ire1p, a sensor molecule of the UPR, is unique. Thus, unconventional spliceosome-independent mRNA splicing is utilized to produce the highly active transcription factor Hac1p. This is the autobiographical story of how a young and not yet independent scientist competed with a very famous full professor in the early days of UPR research, which ultimately lead to their sharing Lasker Basic Medical Research Award in 2014. PMID:26560836

  5. Drosophila as a model for unfolded protein response research

    PubMed Central

    Ryoo, Hyung Don

    2015-01-01

    Endoplasmic Reticulum (ER) is an organelle where most secretory and membrane proteins are synthesized, folded, and undergo further maturation. As numerous conditions can perturb such ER function, eukaryotic cells are equipped with responsive signaling pathways, widely referred to as the Unfolded Protein Response (UPR). Chronic conditions of ER stress that cannot be fully resolved by UPR, or conditions that impair UPR signaling itself, are associated with many metabolic and degenerative diseases. In recent years, Drosophila has been actively employed to study such connections between UPR and disease. Notably, the UPR pathways are largely conserved between Drosophila and humans, and the mediating genes are essential for development in both organisms, indicating their requirement to resolve inherent stress. By now, many Drosophila mutations are known to impose stress in the ER, and a number of these appear similar to those that underlie human diseases. In addition, studies have employed the strategy of overexpressing human mutations in Drosophila tissues to perform genetic modifier screens. The fact that the basic UPR pathways are conserved, together with the availability of many human disease models in this organism, makes Drosophila a powerful tool for studying human disease mechanisms. [BMB Reports 2015; 48(8): 445-453] PMID:25999177

  6. Signaling the Unfolded Protein Response in primary brain cancers.

    PubMed

    Le Reste, Pierre-Jean; Avril, Tony; Quillien, Véronique; Morandi, Xavier; Chevet, Eric

    2016-07-01

    The Unfolded Protein Response (UPR) is an adaptive cellular program used by eukaryotic cells to cope with protein misfolding stress in the Endoplasmic Reticulum (ER). During tumor development, cancer cells are facing intrinsic (oncogene activation) and extrinsic (limiting nutrient or oxygen supply; exposure to chemotherapies) challenges, with which they must cope to survive. Primary brain tumors are relatively rare but deadly and present a significant challenge in the determination of risk factors in the population. These tumors are inherently difficult to cure because of their protected location in the brain. As such surgery, radiation and chemotherapy options carry potentially lasting patient morbidity and incomplete tumor cure. Some of these tumors, such as glioblastoma, were reported to present features of ER stress and to depend on UPR activation to sustain growth, but to date there is no clear general representation of the ER stress status in primary brain tumors. In this review, we describe the key molecular mechanisms controlling the UPR and their implication in cancers. Then we extensively review the literature reporting the status of ER stress in various primary brain tumors and discuss the potential impact of such observation on patient stratification and on the possibility of developing appropriate targeted therapies using the UPR as therapeutic target. PMID:27016056

  7. Unfolded protein response in Gaucher disease: from human to Drosophila

    PubMed Central

    2013-01-01

    Background In Gaucher disease (GD), resulting from mutations in the GBA gene, mutant β-glucocerebrosidase (GCase) molecules are recognized as misfolded in the endoplasmic reticulum (ER). They are retrotranslocated to the cytoplasm, where they are ubiquitinated and undergo proteasomal degradation in a process known as the ER Associated Degradation (ERAD). We have shown in the past that the degree of ERAD of mutant GCase correlates with GD severity. Persistent presence of mutant, misfolded protein molecules in the ER leads to ER stress and evokes the unfolded protein response (UPR). Methods We investigated the presence of UPR in several GD models, using molecular and behavioral assays. Results Our results show the existence of UPR in skin fibroblasts from GD patients and carriers of GD mutations. We could recapitulate UPR in two different Drosophila models for carriers of GD mutations: flies heterozygous for the endogenous mutant GBA orthologs and flies expressing the human N370S or L444P mutant GCase variants. We encountered early death in both fly models, indicating the deleterious effect of mutant GCase during development. The double heterozygous flies, and the transgenic flies, expressing mutant GCase in dopaminergic/serotonergic cells developed locomotion deficit. Conclusion Our results strongly suggest that mutant GCase induces the UPR in GD patients as well as in carriers of GD mutations and leads to development of locomotion deficit in flies heterozygous for GD mutations. PMID:24020503

  8. Ethanol Cellular Defense Induce Unfolded Protein Response in Yeast.

    PubMed

    Navarro-Tapia, Elisabet; Nana, Rebeca K; Querol, Amparo; Pérez-Torrado, Roberto

    2016-01-01

    Ethanol is a valuable industrial product and a common metabolite used by many cell types. However, this molecule produces high levels of cytotoxicity affecting cellular performance at several levels. In the presence of ethanol, cells must adjust some of their components, such as the membrane lipids to maintain homeostasis. In the case of microorganism as Saccharomyces cerevisiae, ethanol is one of the principal products of their metabolism and is the main stress factor during fermentation. Although, many efforts have been made, mechanisms of ethanol tolerance are not fully understood and very little evidence is available to date for specific signaling by ethanol in the cell. This work studied two S. cerevisiae strains, CECT10094, and Temohaya-MI26, isolated from flor wine and agave fermentation (a traditional fermentation from Mexico) respectively, which differ in ethanol tolerance, in order to understand the molecular mechanisms underlying the ethanol stress response and the reasons for different ethanol tolerance. The transcriptome was analyzed after ethanol stress and, among others, an increased activation of genes related with the unfolded protein response (UPR) and its transcription factor, Hac1p, was observed in the tolerant strain CECT10094. We observed that this strain also resist more UPR agents than Temohaya-MI26 and the UPR-ethanol stress correlation was corroborated observing growth of 15 more strains and discarding UPR correlation with other stresses as thermal or oxidative stress. Furthermore, higher activation of UPR pathway in the tolerant strain CECT10094 was observed using a UPR mCherry reporter. Finally, we observed UPR activation in response to ethanol stress in other S. cerevisiae ethanol tolerant strains as the wine strains T73 and EC1118. This work demonstrates that the UPR pathway is activated under ethanol stress occurring in a standard fermentation and links this response to an enhanced ethanol tolerance. Thus, our data suggest that there

  9. Ethanol Cellular Defense Induce Unfolded Protein Response in Yeast

    PubMed Central

    Pérez-Torrado, Roberto

    2016-01-01

    Ethanol is a valuable industrial product and a common metabolite used by many cell types. However, this molecule produces high levels of cytotoxicity affecting cellular performance at several levels. In the presence of ethanol, cells must adjust some of their components, such as the membrane lipids to maintain homeostasis. In the case of microorganism as Saccharomyces cerevisiae, ethanol is one of the principal products of their metabolism and is the main stress factor during fermentation. Although, many efforts have been made, mechanisms of ethanol tolerance are not fully understood and very little evidence is available to date for specific signaling by ethanol in the cell. This work studied two S. cerevisiae strains, CECT10094, and Temohaya-MI26, isolated from flor wine and agave fermentation (a traditional fermentation from Mexico) respectively, which differ in ethanol tolerance, in order to understand the molecular mechanisms underlying the ethanol stress response and the reasons for different ethanol tolerance. The transcriptome was analyzed after ethanol stress and, among others, an increased activation of genes related with the unfolded protein response (UPR) and its transcription factor, Hac1p, was observed in the tolerant strain CECT10094. We observed that this strain also resist more UPR agents than Temohaya-MI26 and the UPR-ethanol stress correlation was corroborated observing growth of 15 more strains and discarding UPR correlation with other stresses as thermal or oxidative stress. Furthermore, higher activation of UPR pathway in the tolerant strain CECT10094 was observed using a UPR mCherry reporter. Finally, we observed UPR activation in response to ethanol stress in other S. cerevisiae ethanol tolerant strains as the wine strains T73 and EC1118. This work demonstrates that the UPR pathway is activated under ethanol stress occurring in a standard fermentation and links this response to an enhanced ethanol tolerance. Thus, our data suggest that there

  10. The unfolded protein response (UPR) pathway in Cryptococcus

    PubMed Central

    Cheon, Seon Ah; Jung, Kwang-Woo; Bahn, Yong-Sun; Kang, Hyun Ah

    2014-01-01

    Unique and evolutionarily conserved signaling pathways allow an organism to sense, respond to, and adapt to internal and external environmental cues at its biological niche. In eukaryotic cells, the unfolded protein response (UPR) pathway regulates endoplasmic reticulum (ER) homeostasis upon exposure to environmental changes causing ER stress. The UPR pathway of Cryptococcus neoformans, an opportunistic fungal pathogen, which causes life-threatening meningoencephalitis in immunocompromised individuals, consists of the evolutionarily conserved Ire1 kinase, a unique bZIP transcription factor, Hxl1, and the ER-resident molecular chaperone Kar2/BiP. Although the Cryptococcus UPR pathway regulates ER stress, antifungal drug resistance, and virulence in an Ire1/Hxl1-dependent manner, Ire1 has Hxl1-independent roles in capsule biosynthesis and thermotolerance. In this review, we highlight the conserved and unique features of the Cryptococcus UPR pathway compared with other fungal UPR systems and its importance in the pathogenesis of cryptococcosis and discuss future challenges in this field. PMID:24504058

  11. The role of the unfolded protein response in axial spondyloarthritis.

    PubMed

    Smith, Judith A

    2016-06-01

    Susceptibility to ankylosing spondylitis is highly genetic, with a heritability greater than 90 %. Presence of the HLA-B27 MHC class I allele remains the greatest genetic risk factor identified to date. Beyond its nominal role in antigen presentation, HLA-B27 displays interesting and possibly unique biochemical characteristics which may contribute to disease pathogenesis. During its biosynthesis in the endoplasmic reticulum (ER), HLA-B27 folds very slowly and misfolds, inducing ER stress. Herein, we describe a major outcome of ER stress, the unfolded protein response (UPR), as well as consequences of the UPR for inflammation and autophagy. The ability of the UPR to augment inflammatory cytokine production is particularly intriguing given the centrality of cytokines in spondyloarthritis. Evidence for the relevance of an HLA-B27-related UPR to spondyloarthritis pathogenesis in animal models and human subjects will be reviewed. As greater pharmacologic capacity to modulate ER stress becomes available, improved understanding of the role of the UPR in spondyloarthritis may yield new therapeutic targets. PMID:26567900

  12. The unfolded protein response selectively targets active smoothened mutants.

    PubMed

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

    2013-06-01

    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

  13. Induction of the Unfolded Protein Response by Constitutive G-protein Signaling in Rod Photoreceptor Cells*

    PubMed Central

    Wang, Tian; Chen, Jeannie

    2014-01-01

    Phototransduction is a G-protein signal transduction cascade that converts photon absorption to a change in current at the plasma membrane. Certain genetic mutations affecting the proteins in the phototransduction cascade cause blinding disorders in humans. Some of these mutations serve as a genetic source of “equivalent light” that activates the cascade, whereas other mutations lead to amplification of the light response. How constitutive phototransduction causes photoreceptor cell death is poorly understood. We showed that persistent G-protein signaling, which occurs in rod arrestin and rhodopsin kinase knock-out mice, caused a rapid and specific induction of the PERK pathway of the unfolded protein response. These changes were not observed in the cGMP-gated channel knock-out rods, an equivalent light condition that mimics light-stimulated channel closure. Thus transducin signaling, but not channel closure, triggers rapid cell death in light damage caused by constitutive phototransduction. Additionally, we show that in the albino light damage model cell death was not associated with increase in global protein ubiquitination or unfolded protein response induction. Taken together, these observations provide novel mechanistic insights into the cell death pathway caused by constitutive phototransduction and identify the unfolded protein response as a potential target for therapeutic intervention. PMID:25183010

  14. Distinct transcriptional responses elicited by unfolded nuclear or cytoplasmic protein in mammalian cells

    PubMed Central

    Miyazaki, Yusuke; Chen, Ling-chun; Chu, Bernard W; Swigut, Tomek; Wandless, Thomas J

    2015-01-01

    Eukaryotic cells possess a variety of signaling pathways that prevent accumulation of unfolded and misfolded proteins. Chief among these is the heat shock response (HSR), which is assumed to respond to unfolded proteins in the cytosol and nucleus alike. In this study, we probe this axiom further using engineered proteins called ‘destabilizing domains’, whose folding state we control with a small molecule. The sudden appearance of unfolded protein in mammalian cells elicits a robust transcriptional response, which is distinct from the HSR and other known pathways that respond to unfolded proteins. The cellular response to unfolded protein is strikingly different in the nucleus and the cytosol, although unfolded protein in either compartment engages the p53 network. This response provides cross-protection during subsequent proteotoxic stress, suggesting that it is a central component of protein quality control networks, and like the HSR, is likely to influence the initiation and progression of human pathologies. DOI: http://dx.doi.org/10.7554/eLife.07687.001 PMID:26314864

  15. Thermal unfolding of proteins

    NASA Astrophysics Data System (ADS)

    Cieplak, Marek; Sułkowska, Joanna I.

    2005-11-01

    Thermal unfolding of proteins is compared to folding and mechanical stretching in a simple topology-based dynamical model. We define the unfolding time and demonstrate its low-temperature divergence. Below a characteristic temperature, contacts break at separate time scales and unfolding proceeds approximately in a way reverse to folding. Features in these scenarios agree with experiments and atomic simulations on titin.

  16. The mitochondrial unfolded protein response, a conserved stress response pathway with implications in health and disease

    PubMed Central

    Jovaisaite, Virginija; Mouchiroud, Laurent; Auwerx, Johan

    2014-01-01

    The ability to respond to various intracellular and/or extracellular stresses allows the organism to adapt to changing environmental conditions and drives evolution. It is now well accepted that a progressive decline of the efficiency of stress response pathways occurs with aging. In this context, a correct proteostasis is essential for the functionality of the cell, and its dysfunction has been associated with protein aggregation and age-related degenerative diseases. Complex response mechanisms have evolved to deal with unfolded protein stress in different subcellular compartments and their moderate activation translates into positive effects on health. In this review, we focus on the mitochondrial unfolded protein response (UPRmt), a response to proteotoxic stress specifically in mitochondria, an organelle with a wide array of fundamental functions, most notably the harvesting of energy from food and the control of cell death. We compare UPRmt with the extensively characterized cytosolic heat shock response (HSR) and the unfolded protein response in endoplasmic reticulum (UPRER), and discuss the current knowledge about UPRmt signaling pathways as well as their potential involvement in physiology. PMID:24353213

  17. The unfolded protein response triggers site-specific regulatory ubiquitylation of 40S ribosomal proteins

    PubMed Central

    Rising, Lisa; Mak, Raymond; Webb, Kristofor; Kaiser, Stephen E.; Zuzow, Nathan; Riviere, Paul; Yang, Bing; Fenech, Emma; Tang, Xin; Lindsay, Scott A.; Christianson, John C.; Hampton, Randolph Y.; Wasserman, Steven A.; Bennett, Eric J.

    2015-01-01

    Summary Insults to endoplasmic reticulum (ER) homeostasis activate the unfolded protein response (UPR), which elevates protein folding and degradation capacity and attenuates protein synthesis. While a role for ubiquitin in regulating the degradation of misfolded ER-resident proteins is well described, ubiquitin-dependent regulation of translational reprogramming during the UPR remains uncharacterized. Using global quantitative ubiquitin proteomics, we identify evolutionarily conserved, site-specific regulatory ubiquitylation of 40S ribosomal proteins. We demonstrate that these events occur on assembled cytoplasmic ribosomes and are stimulated by both UPR activation and translation inhibition. We further show that ER stress-stimulated regulatory 40S ribosomal ubiquitylation occurs on a timescale similar to eIF2α phosphorylation, is dependent upon PERK signaling, and is required for optimal cell survival during chronic UPR activation. In total, these results reveal regulatory 40S ribosomal ubiquitylation as a previously uncharacterized and important facet of eukaryotic translational control. PMID:26051182

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

    PubMed Central

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

    2011-01-01

    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. PMID:21474436

  19. Yeast flavin-containing monooxygenase is induced by the unfolded protein response

    PubMed Central

    Suh, Jung-Keun; Robertus, Jon D.

    2000-01-01

    Flavin-containing monooxygenase from yeast (yFMO) carries out the O2- and NADPH-dependent oxidation of biological thiols, including oxidizing glutathione to glutathione disulfide. FMO provides a large fraction of the oxidizing necessary for proper folding of disulfide bond-containing proteins; deletion of the enzyme reduces proper folding of endogenous carboxypeptidase Y by about 40%. The enzyme is not essential to cell viability because other enzymes can generate a significant fraction of the oxidizing equivalents required by the cell. However, yFMO is vital to the yeast response to reductive stress. FMO1 deletion mutants grow poorly under reductive stress, and carboxypeptidase Y activity is less than 10% of that in a stressed wild type. The FMO1 gene appears to be under control of an unfolded protein response element and is inducible by factors, such as reductive stress, that elicit the unfolded protein response. Reductive stress can increase yFMO activity at least 6-fold. This increased activity allows the cell to process endogenous disulfide bond-containing proteins and also to allow correct folding of disulfide-bonded proteins expressed from multicopy plasmids. The unfolded protein response is mediated by the Hac1p transcription factor that mediates virtually all of the induction of yFMO triggered by exogenous reducing agents. PMID:10618381

  20. Implication of Unfolded Protein Response and Autophagy in the Treatment of BRAF Inhibitor Resistant Melanoma.

    PubMed

    Meng, Xiao-Xiao; Xu, Hong-Xi; Yao, Mu; Dong, Qihan; Zhang, Xu Dong

    2016-01-01

    The continuous activation of the mitogen-activated protein kinase signaling cascade, typified by the BRAFV600E mutation, is one of the key alterations in melanoma. Accordingly, two BRAF inhibitors (BRAFi), vemurafenib and dabrafenib are utilized to treat melanoma and resulted in an excellent clinical outcome. However, the clinical success is not long-lasting, and the BRAFi resistance and disease progression inevitably occurs in nearly all patients. Endoplasmic reticulum stress-induced unfolded protein response and autophagy have emerged as potential pro-survival mechanisms adopted by melanoma cells in response to BRAFi. In this review, we discuss the role of unfolded protein response and autophagy that are implicated in the development of BRAFi-resistant melanoma and the corresponding strategy aiming at overcoming the intractable clinical problem. PMID:26419469

  1. Divergent roles of IRE1alpha and PERK in the unfolded protein response.

    PubMed

    Schröder, Martin; Kaufman, Randal J

    2006-02-01

    The endoplasmic reticulum (ER) provides unique machinery for the folding and posttranslational modification of many secretory and transmembrane proteins in eukaryotic cells. The unfolded protein response (UPR) is a signal transduction network from the ER to the nucleus activated when the folding demand imposed by nascent, unfolded polypeptide chains exceeds the capacity of the ER protein folding machinery. In all eukaryotes the UPR maintains the physiological balance between folding demand and capacity of the ER by regulating adaptive responses to this stress situation. These include an increase in the folding capacity of the ER through induction of ER resident molecular chaperones and protein foldases, and a decrease in the folding demand on the ER by upregulation of ER associated degradation (ERAD), attenuation of general translation in metazoans, and stimulation of ER synthesis to dilute the unfolded protein load. In higher eukaryotes the UPR gained control over inflammatory and immune responses by controlling the activity of the transcription factor NF-kappaB to combat viral infections associated with an increased synthesis of viral glycoproteins. Similarly, in multicellular organisms apoptotic programs are controlled by the UPR to eliminate cells whose folding problems in the ER cannot be resolved by coordinated regulation of adaptive, inflammatory, and immune responses. In this review we will summarize our current understanding of signal transduction mechanisms involved in the mammalian UPR, and discuss examples to highlight the regulation of adaptive, inflammatory, immune, and apoptotic responses by the UPR. PMID:16472110

  2. The Unfolded Protein Response and the Role of Protein Disulfide Isomerase in Neurodegeneration

    PubMed Central

    Perri, Emma R.; Thomas, Colleen J.; Parakh, Sonam; Spencer, Damian M.; Atkin, Julie D.

    2016-01-01

    The maintenance and regulation of proteostasis is a critical function for post-mitotic neurons and its dysregulation is increasingly implicated in neurodegenerative diseases. Despite having different clinical manifestations, these disorders share similar pathology; an accumulation of misfolded proteins in neurons and subsequent disruption to cellular proteostasis. The endoplasmic reticulum (ER) is an important component of proteostasis, and when the accumulation of misfolded proteins occurs within the ER, this disturbs ER homeostasis, giving rise to ER stress. This triggers the unfolded protein response (UPR), distinct signaling pathways that whilst initially protective, are pro-apoptotic if ER stress is prolonged. ER stress is increasingly implicated in neurodegenerative diseases, and emerging evidence highlights the complexity of the UPR in these disorders, with both protective and detrimental components being described. Protein Disulfide Isomerase (PDI) is an ER chaperone induced during ER stress that is responsible for the formation of disulfide bonds in proteins. Whilst initially considered to be protective, recent studies have revealed unconventional roles for PDI in neurodegenerative diseases, distinct from its normal function in the UPR and the ER, although these mechanisms remain poorly defined. However, specific aspects of PDI function may offer the potential to be exploited therapeutically in the future. This review will focus on the evidence linking ER stress and the UPR to neurodegenerative diseases, with particular emphasis on the emerging functions ascribed to PDI in these conditions. PMID:26779479

  3. Pancreatic adaptive responses in alcohol abuse: Role of the unfolded protein response.

    PubMed

    Lugea, Aurelia; Waldron, Richard T; Pandol, Stephen J

    2015-07-01

    The majority of those who drink excessive amounts of alcohol do not develop pancreatic disease. One overarching hypothesis is that alcohol abuse requires additional risk factors, either environmental or genetic, for disease to occur. However, another reason be a result of alcohol-induced activation of adaptive systems that protect the pancreas from the toxic effects of alcohol. We show that mechanisms within the unfolded protein response (UPR) of the endoplasmic reticulum (ER) that can lead to protection of the pancreas from pancreatic diseases with alcohol abuse. The remarkable ability of the pancreas to adapt its machinery to alcohol abuse using UPR systems and continue functioning is the likely reason that pancreatitis from alcohol abuse does not occur in the majority of heavy drinkers. These findings indicate that methods to enhance the protective responses of the UPR can provide opportunities for prevention and treatment of pancreatic diseases. PMID:25736240

  4. Unfolding of Proteins: Thermal and Mechanical Unfolding

    NASA Technical Reports Server (NTRS)

    Hur, Joe S.; Darve, Eric

    2004-01-01

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

  5. Zebrafish Fukutin family proteins link the unfolded protein response with dystroglycanopathies

    PubMed Central

    Lin, Yung-Yao; White, Richard J.; Torelli, Silvia; Cirak, Sebahattin; Muntoni, Francesco; Stemple, Derek L.

    2011-01-01

    Allelic mutations in putative glycosyltransferase genes, fukutin and fukutin-related protein (fkrp), lead to a wide range of muscular dystrophies associated with hypoglycosylation of α-dystroglycan, commonly referred to as dystroglycanopathies. Defective glycosylation affecting dystroglycan–ligand interactions is considered to underlie the disease pathogenesis. We have modelled dystroglycanopathies in zebrafish using a novel loss-of-function dystroglycan allele and by inhibition of Fukutin family protein activities. We show that muscle pathology in embryos lacking Fukutin or FKRP is different from loss of dystroglycan. In addition to hypoglycosylated α-dystroglycan, knockdown of Fukutin or FKRP leads to a notochord defect and a perturbation of laminin expression before muscle degeneration. These are a consequence of endoplasmic reticulum stress and activation of the unfolded protein response (UPR), preceding loss of dystroglycan–ligand interactions. Together, our results suggest that Fukutin family proteins may play important roles in protein secretion and that the UPR may contribute to the phenotypic spectrum of some dystroglycanopathies in humans. PMID:21317159

  6. The unfolded protein response triggers selective mRNA release from the endoplasmic reticulum.

    PubMed

    Reid, David W; Chen, Qiang; Tay, Angeline S-L; Shenolikar, Shirish; Nicchitta, Christopher V

    2014-09-11

    The unfolded protein response (UPR) is a stress response program that reprograms cellular translation and gene expression in response to proteotoxic stress in the endoplasmic reticulum (ER). One of the primary means by which the UPR alleviates this stress is by reducing protein flux into the ER via a general suppression of protein synthesis and ER-specific mRNA degradation. We report here an additional UPR-induced mechanism for the reduction of protein flux into the ER, where mRNAs that encode signal sequences are released from the ER to the cytosol. By removing mRNAs from the site of translocation, this mechanism may serve as a potent means to transiently reduce ER protein folding load and restore proteostasis. These findings identify the dynamic subcellular localization of mRNAs and translation as a selective and rapid regulatory feature of the cellular response to protein folding stress. PMID:25215492

  7. Bacteria, the ER and the Unfolded Protein Response: Friends or Foes?

    PubMed Central

    Celli, Jean; Tsolis, Renée M.

    2015-01-01

    The Unfolded Protein Response (UPR) is a cytoprotective response aimed at restoring cellular homeostasis following physiological stress exerted on the endoplasmic reticulum (ER) that also invokes innate immune signaling in response to invading microorganisms. While the UPR is modulated by various viruses, recent evidence indicates that it also plays multiple roles during bacterial infections. In this Review, we describe how bacteria adapt to live in the ER and discuss the intricacies of bacterial interactions with the UPR, including how UPR subversion promotes the proliferation of intracellular bacterial pathogens and how the UPR contributes to innate immune responses against invading bacteria. PMID:25534809

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

    PubMed Central

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

    2012-01-01

    Objective We determined whether postburn hyperglycemia and insulin resistance are associated with endoplasmic reticulum (ER) stress/unfolded protein response (UPR) activation leading to impaired insulin receptor signaling. Background Inflammation and cellular stress, hallmarks of severely burned and critically ill patients, have been causally linked to insulin resistance in type 2 diabetes via induction of ER stress and the UPR. Methods Twenty severely burned pediatric patients were compared with 36 nonburned children. Clinical markers, protein, and GeneChip analysis were used to identify transcriptional changes in ER stress and UPR and insulin resistance–related signaling cascades in peripheral blood leukocytes, fat, and muscle at admission and up to 466 days postburn. Results Burn-induced inflammatory and stress responses are accompanied by profound insulin resistance and hyperglycemia. Genomic and protein analysis revealed that burn injury was associated with alterations in the signaling pathways that affect insulin resistance, ER/sarcoplasmic reticulum stress, inflammation, and cell growth/apoptosis up to 466 days postburn. Conclusion Burn-induced insulin resistance is associated with persistent ER/sarcoplasmic reticulum stress/UPR and subsequent suppressed insulin receptor signaling over a prolonged period of time. PMID:22241293

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

    PubMed

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

    2014-03-13

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

  10. Spliced X-box Binding Protein 1 Couples the Unfolded Protein Response to Hexosamine Biosynthetic Pathway

    PubMed Central

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

    2014-01-01

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

  11. Advances and New Concepts in Alcohol-Induced Organelle Stress, Unfolded Protein Responses and Organ Damage

    PubMed Central

    Ji, Cheng

    2015-01-01

    Alcohol is a simple and consumable biomolecule yet its excessive consumption disturbs numerous biological pathways damaging nearly all organs of the human body. One of the essential biological processes affected by the harmful effects of alcohol is proteostasis, which regulates the balance between biogenesis and turnover of proteins within and outside the cell. A significant amount of published evidence indicates that alcohol and its metabolites directly or indirectly interfere with protein homeostasis in the endoplasmic reticulum (ER) causing an accumulation of unfolded or misfolded proteins, which triggers the unfolded protein response (UPR) leading to either restoration of homeostasis or cell death, inflammation and other pathologies under severe and chronic alcohol conditions. The UPR senses the abnormal protein accumulation and activates transcription factors that regulate nuclear transcription of genes related to ER function. Similarly, this kind of protein stress response can occur in other cellular organelles, which is an evolving field of interest. Here, I review recent advances in the alcohol-induced ER stress response as well as discuss new concepts on alcohol-induced mitochondrial, Golgi and lysosomal stress responses and injuries. PMID:26047032

  12. Unfolded Protein Response and Activated Degradative Pathways Regulation in GNE Myopathy

    PubMed Central

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

    2013-01-01

    Although intracellular beta amyloid (Aβ) accumulation is known as an early upstream event in the degenerative course of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE) myopathy, the process by which Aβdeposits initiate various degradative pathways, and their relationship have not been fully clarified. We studied the possible secondary responses after amyloid beta precursor protein (AβPP) deposition including unfolded protein response (UPR), ubiquitin proteasome system (UPS) activation and its correlation with autophagy system. Eight GNE myopathy patients and five individuals with normal muscle morphology were included in this study. We performed immunofluorescence and immunoblotting to investigate the expression of AβPP, phosphorylated tau (p-tau) and endoplasmic reticulum molecular chaperones. Proteasome activities were measured by cleavage of fluorogenic substrates. The expression of proteasome subunits and linkers between proteasomal and autophagy systems were also evaluated by immunoblotting and relative quantitative real-time RT-PCR. Four molecular chaperones, glucose-regulated protein 94 (GRP94), glucose-regulated protein 78 (GRP78), calreticulin and calnexin and valosin containing protein (VCP) were highly expressed in GNE myopathy. 20S proteasome subunits, three main proteasome proteolytic activities, and the factors linking UPS and autophagy system were also increased. Our study suggests that AβPP deposition results in endoplasmic reticulum stress (ERS) and highly expressed VCP deliver unfolded proteins from endoplasmic reticulum to proteosomal system which is activated in endoplasmic reticulum associated degradation (ERAD) in GNE myopathy. Excessive ubiquitinated unfolded proteins are exported by proteins that connect UPS and autophagy to autophagy system, which is activated as an alternative pathway for degradation. PMID:23472144

  13. Estrogen receptor α inhibitor activates the unfolded protein response, blocks protein synthesis, and induces tumor regression.

    PubMed

    Andruska, Neal D; Zheng, Xiaobin; Yang, Xujuan; Mao, Chengjian; Cherian, Mathew M; Mahapatra, Lily; Helferich, William G; Shapiro, David J

    2015-04-14

    Recurrent estrogen receptor α (ERα)-positive breast and ovarian cancers are often therapy resistant. Using screening and functional validation, we identified BHPI, a potent noncompetitive small molecule ERα biomodulator that selectively blocks proliferation of drug-resistant ERα-positive breast and ovarian cancer cells. In a mouse xenograft model of breast cancer, BHPI induced rapid and substantial tumor regression. Whereas BHPI potently inhibits nuclear estrogen-ERα-regulated gene expression, BHPI is effective because it elicits sustained ERα-dependent activation of the endoplasmic reticulum (EnR) stress sensor, the unfolded protein response (UPR), and persistent inhibition of protein synthesis. BHPI distorts a newly described action of estrogen-ERα: mild and transient UPR activation. In contrast, BHPI elicits massive and sustained UPR activation, converting the UPR from protective to toxic. In ERα(+) cancer cells, BHPI rapidly hyperactivates plasma membrane PLCγ, generating inositol 1,4,5-triphosphate (IP3), which opens EnR IP3R calcium channels, rapidly depleting EnR Ca(2+) stores. This leads to activation of all three arms of the UPR. Activation of the PERK arm stimulates phosphorylation of eukaryotic initiation factor 2α (eIF2α), resulting in rapid inhibition of protein synthesis. The cell attempts to restore EnR Ca(2+) levels, but the open EnR IP3R calcium channel leads to an ATP-depleting futile cycle, resulting in activation of the energy sensor AMP-activated protein kinase and phosphorylation of eukaryotic elongation factor 2 (eEF2). eEF2 phosphorylation inhibits protein synthesis at a second site. BHPI's novel mode of action, high potency, and effectiveness in therapy-resistant tumor cells make it an exceptional candidate for further mechanistic and therapeutic exploration. PMID:25825714

  14. The Unfolded Protein Response in Retinal Vascular Diseases: Implications and Therapeutic Potential Beyond Protein Folding

    PubMed Central

    Zhang, Sarah X.; Ma, Jacey H.; Bhatta, Maulasri; Fliesler, Steven J.; Wang, Joshua J.

    2015-01-01

    Angiogenesis is a complex, step-wise process of new vessel formation that is involved in both normal embryonic development as well as postnatal pathological processes, such as cancer, cardiovascular disease, and diabetes. Aberrant blood vessel growth, also known as neovascularization, in the retina and the choroid is a major cause of vision loss in severe eye diseases, such as diabetic retinopathy, age-related macular degeneration, retinopathy of prematurity, and central and branch retinal vein occlusion. Yet, retinal neovascularization is causally and dynamically associated with vasodegeneration, ischemia, and vascular remodeling in retinal tissues. Understanding the mechanisms of retinal neovascularization is an urgent unmet need for developing new treatments for these devastating diseases. Accumulating evidence suggests a vital role for the unfolded protein response (UPR) in regulation of angiogenesis, in part through coordinating the secretion of pro-angiogenic growth factors, such as VEGF, and modulating endothelial cell survival and activity. Herein, we summarize current research in the context of endoplasmic reticulum (ER) stress and UPR signaling in retinal angiogenesis and vascular remodeling, highlighting potential implications of targeting these stress response pathways in the prevention and treatment of retinal vascular diseases that result in visual deficits and blindness. PMID:25529848

  15. Discovery of Sulfonamidebenzamides as Selective Apoptotic CHOP Pathway Activators of the Unfolded Protein Response

    PubMed Central

    2015-01-01

    Cellular proteins that fail to fold properly result in inactive or disfunctional proteins that can have toxic functions. The unfolded protein response (UPR) is a two-tiered cellular mechanism initiated by eukaryotic cells that have accumulated misfolded proteins within the endoplasmic reticulum (ER). An adaptive pathway facilitates the clearance of the undesired proteins; however, if overwhelmed, cells trigger apoptosis by upregulating transcription factors such as C/EBP-homologous protein (CHOP). A high throughput screen was performed directed at identifying compounds that selectively upregulate the apoptotic CHOP pathway while avoiding adaptive signaling cascades, resulting in a sulfonamidebenzamide chemotype that was optimized. These efforts produced a potent and selective CHOP inducer (AC50 = 0.8 μM; XBP1 > 80 μM), which was efficacious in both mouse embryonic fibroblast cells and a human oral squamous cell cancer cell line, and demonstrated antiproliferative effects for multiple cancer cell lines in the NCI-60 panel. PMID:25530830

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

    PubMed Central

    Blázquez, Ana-Belén; Escribano-Romero, Estela; Merino-Ramos, Teresa; Saiz, Juan-Carlos; Martín-Acebes, Miguel A.

    2014-01-01

    The Flavivirus is a genus of RNA viruses that includes multiple long known human, animal, and zoonotic pathogens such as Dengue virus, yellow fever virus, West Nile virus, or Japanese encephalitis virus, as well as other less known viruses that represent potential threats for human and animal health such as Usutu or Zika viruses. Flavivirus replication is based on endoplasmic reticulum-derived structures. Membrane remodeling and accumulation of viral factors induce endoplasmic reticulum stress that results in activation of a cellular signaling response termed unfolded protein response (UPR), which can be modulated by the viruses for their own benefit. Concomitant with the activation of the UPR, an upregulation of the autophagic pathway in cells infected with different flaviviruses has also been described. This review addresses the current knowledge of the relationship between endoplasmic reticulum stress, UPR, and autophagy in flavivirus-infected cells and the growing evidences for an involvement of these cellular pathways in the replication and pathogenesis of these viruses. PMID:24917859

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

    PubMed Central

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

    2013-01-01

    Disturbance of cellular functions results in the activation of stress-signaling pathways that aim at restoring homeostasis. We performed a genome-wide screen to identify components of the signal transduction of the mitochondrial unfolded protein response (UPRmt) to a nuclear chaperone promoter. We used the ROS generating complex I inhibitor paraquat to induce the UPRmt, and we employed RNAi exposure post-embryonically to allow testing genes whose knockdown results in embryonic lethality. We identified 54 novel regulators of the ROS–induced UPRmt. Activation of the UPRmt, but not of other stress-signaling pathways, failed when homeostasis of basic cellular mechanisms such as translation and protein transport were impaired. These mechanisms are monitored by a recently discovered surveillance system that interprets interruption of these processes as pathogen attack and depends on signaling through the JNK-like MAP-kinase KGB-1. Mutation of kgb-1 abrogated the inhibition of ROS–induced UPRmt, suggesting that surveillance-activated defenses specifically inhibit the UPRmt but do not compromise activation of the heat shock response, the UPR of the endoplasmic reticulum, or the SKN-1/Nrf2 mediated response to cytosolic stress. In addition, we identified PIFK-1, the orthologue of the Drosophila PI 4-kinase four wheel drive (FWD), and found that it is the only known factor so far that is essential for the unfolded protein responses of both mitochondria and endoplasmic reticulum. This suggests that both UPRs may share a common membrane associated mechanism. PMID:23516373

  18. Review: Modulating the unfolded protein response to prevent neurodegeneration and enhance memory.

    PubMed

    Halliday, Mark; Mallucci, Giovanna R

    2015-06-01

    Recent evidence has placed the unfolded protein response (UPR) at the centre of pathological processes leading to neurodegenerative disease. The translational repression caused by UPR activation starves neurons of the essential proteins they need to function and survive. Restoration of protein synthesis, via genetic or pharmacological means, is neuroprotective in animal models, prolonging survival. This is of great interest due to the observation of UPR activation in the post mortem brains of patients with Alzheimer's, Parkinson's, tauopathies and prion diseases. Protein synthesis is also an essential step in the formation of new memories. Restoring translation in disease or increasing protein synthesis from basal levels has been shown to improve memory in numerous models. As neurodegenerative diseases often present with memory impairments, targeting the UPR to both provide neuroprotection and enhance memory provides an extremely exciting novel therapeutic target. PMID:25556298

  19. Role for the Unfolded Protein Response in Heart Disease and Cardiac Arrhythmias

    PubMed Central

    Liu, Man; Dudley, Samuel C.

    2015-01-01

    The unfolded protein response (UPR) has been extensively investigated in neurological diseases and diabetes, while its function in heart disease is less well understood. Activated UPR participates in multiple cardiac conditions and can either protect or impair heart function. Recently, the UPR has been found to play a role in arrhythmogenesis during human heart failure by affecting cardiac ion channels expression, and blocking UPR has an antiarrhythmic effect. This review will discuss the rationale for and challenges to targeting UPR in heart disease for treatment of arrhythmias. PMID:26729106

  20. Emerging Roles for the Unfolded Protein Response in the Developing Nervous System.

    PubMed

    Godin, Juliette D; Creppe, Catherine; Laguesse, Sophie; Nguyen, Laurent

    2016-06-01

    The unfolded protein response (UPR) is a homeostatic signaling pathway triggered by protein misfolding in the endoplasmic reticulum (ER). Beyond its protective role, it plays important functions during normal development in response to elevated demand for protein folding. Several UPR effectors show dynamic temporal and spatial expression patterns that correlate with milestones of the central nervous system (CNS) development. Here, we discuss recent studies suggesting that a dynamic regulation of UPR supports generation, maturation, and maintenance of differentiated neurons in the CNS. We further highlight studies supporting a developmental vulnerability of CNS to UPR dysregulation, which underlies neurodevelopmental disorders. We believe that a better understanding of UPR functions may provide novel opportunities for therapeutic strategies to fight ER/UPR-associated human neurological disorders. PMID:27130659

  1. Mitochondrial unfolded protein response controls matrix pre-RNA processing and translation.

    PubMed

    Münch, Christian; Harper, J Wade

    2016-06-30

    The mitochondrial matrix is unique in that it must integrate the folding and assembly of proteins derived from the nuclear and mitochondrial genomes. In Caenorhabditis elegans, the mitochondrial unfolded protein response (UPRmt) senses matrix protein misfolding and induces a program of nuclear gene expression, including mitochondrial chaperonins, to promote mitochondrial proteostasis. While misfolded mitochondrial-matrix-localized ornithine transcarbamylase induces chaperonin expression, our understanding of mammalian UPRmt is rudimentary, reflecting a lack of acute triggers for UPRmt activation. This limitation has prevented analysis of the cellular responses to matrix protein misfolding and the effects of UPRmt on mitochondrial translation to control protein folding loads. Here we combine pharmacological inhibitors of matrix-localized HSP90/TRAP1 (ref. 8) or LON protease, which promote chaperonin expression, with global transcriptional and proteomic analysis to reveal an extensive and acute response of human cells to UPRmt. This response encompasses widespread induction of nuclear genes, including matrix-localized proteins involved in folding, pre-RNA processing and translation. Functional studies revealed rapid but reversible translation inhibition in mitochondria occurring concurrently with defects in pre-RNA processing caused by transcriptional repression and LON-dependent turnover of the mitochondrial pre-RNA processing nuclease MRPP3 (ref. 10). This study reveals that acute mitochondrial protein folding stress activates both increased chaperone availability within the matrix and reduced matrix-localized protein synthesis through translational inhibition, and provides a framework for further dissection of mammalian UPRmt. PMID:27350246

  2. Regulation of the unfolded protein response via S-nitrosylation of sensors of endoplasmic reticulum stress

    PubMed Central

    Nakato, Ryosuke; Ohkubo, Yu; Konishi, Akari; Shibata, Mari; Kaneko, Yuki; Iwawaki, Takao; Nakamura, Tomohiro; Lipton, Stuart A.; Uehara, Takashi

    2015-01-01

    Protein S-nitrosylation modulates important cellular processes, including neurotransmission, vasodilation, proliferation, and apoptosis in various cell types. We have previously reported that protein disulfide isomerase (PDI) is S-nitrosylated in brains of patients with sporadic neurodegenerative diseases. This modification inhibits PDI enzymatic activity and consequently leads to the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) lumen. Here, we describe S-nitrosylation of additional ER pathways that affect the unfolded protein response (UPR) in cell-based models of Parkinson’s disease (PD). We demonstrate that nitric oxide (NO) can S-nitrosylate the ER stress sensors IRE1α and PERK. While S-nitrosylation of IRE1α inhibited its ribonuclease activity, S-nitrosylation of PERK activated its kinase activity and downstream phosphorylation/inactivation or eIF2α. Site-directed mutagenesis of IRE1α(Cys931) prevented S-nitrosylation and inhibition of its ribonuclease activity, indicating that Cys931 is the predominant site of S-nitrosylation. Importantly, cells overexpressing mutant IRE1α(C931S) were resistant to NO-induced damage. Our findings show that nitrosative stress leads to dysfunctional ER stress signaling, thus contributing to neuronal cell death. PMID:26446798

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

    PubMed

    Smith, Judith A

    2014-01-01

    THE IMMUNE SYSTEM DEPENDS UPON COMBINATIONS OF SIGNALS TO MOUNT APPROPRIATE RESPONSES: pathogen specific signals in the context of co-stimulatory "danger" signals drive immune strength and accuracy. Viral infections trigger anti-viral type I interferon (IFN) responses by stimulating endosomal and cytosolic pattern recognition receptors (PRRs). However, viruses have also evolved many strategies to counteract IFN responses. Are there intracellular danger signals that enhance immune responses to viruses? During infection, viruses place a heavy demand on the protein folding machinery of the host endoplasmic reticulum (ER). To survive ER stress, host cells mount an unfolded protein response (UPR) to decrease ER protein load and enhance protein-folding capacity. Viruses also directly elicit the UPR to enhance their replication. Increasing evidence supports an intersection between the host UPR and inflammation, in particular the production of pro-inflammatory cytokines and type I IFN. The UPR directly activates pro-inflammatory cytokine transcription factors and dramatically enhances cytokine production in response to viral PRR engagement. Additionally, viral PRR engagement may stimulate specific pathways within the UPR to enhance cytokine production. Through these mechanisms, viral detection via the UPR and inflammatory cytokine production are intertwined. Consequently, the UPR response is perfectly poised to act as an infection-triggered "danger" signal. The UPR may serve as an internal "co-stimulatory" signal that (1) provides specificity and (2) critically augments responses to overcome viral subterfuge. Further work is needed to test this hypothesis during viral infections. PMID:24904537

  4. Co-opting the unfolded protein response to elicit olfactory receptor feedback

    PubMed Central

    Dalton, Ryan P.; Lyons, David B.

    2013-01-01

    Summary Olfactory receptor (OR) expression requires the transcriptional activation of one out of thousands of OR alleles and a feedback signal that preserves this transcriptional choice. The mechanism by which olfactory sensory neurons (OSNs) detect ORs to signal to the nucleus remains elusive. Here, we show that OR proteins generate this feedback by activating the unfolded protein response (UPR). OR expression induces Perk-mediated phosphorylation of the translation initiation factor eif2α causing selective translation of Activating Transcription Factor 5 (ATF5). ATF5 induces the transcription of Adenylyl Cyclase 3 (Adcy3), which relieves the UPR. Our data provide a novel role for the UPR in defining neuronal identity and cell fate commitment and support a two-step model for the feedback signal: first OR protein, as a stress stimulus, alters the translational landscape of the OSN and induces Adcy3 expression; then, Adcy3 relieves that stress, restores global translation and makes OR choice permanent. PMID:24120133

  5. Selective inhibition of unfolded protein response induces apoptosis in pancreatic cancer cells

    PubMed Central

    Sun, Qiao-Yang; Torres-Fernandez, Lucia A; Tan, Siew Zhuan; Xiao, Jinfen; Lim, Su Lin; Garg, Manoj; Lee, Kian Leong; Kitajima, Shojiro; Takao, Sumiko; Leong, Wei Zhong; Sun, Haibo; Tokatly, Itay; Poellinger, Lorenz; Gery, Sigal; Koeffler, Phillip H

    2014-01-01

    Endoplasmic reticulum stress from unfolded proteins is associated with the proliferation of pancreatic tumor cells, making the many regulatory molecules of this pathway appealing targets for therapy. The objective of our study was to assess potential therapeutic efficacy of inhibitors of unfolded protein response (UPR) in pancreatic cancers focusing on IRE1α inhibitors. IRE1α-mediated XBP-1 mRNA splicing encodes a transcription factor that enhances transcription of chaperone proteins in order to reverse UPR. Proliferation assays using a panel of 14 pancreatic cancer cell lines showed a dose- and time-dependent growth inhibition by IRE1α-specific inhibitors (STF-083010, 2-Hydroxy-1-naphthaldehyde, 3-Ethoxy-5,6-dibromosalicylaldehyde, toyocamycin). Growth inhibition was also noted using a clonogenic growth assay in soft agar, as well as a xenograft in vivo model of pancreatic cancer. Cell cycle analysis showed that these IRE1α inhibitors caused growth arrest at either the G1 or G2/M phases (SU8686, MiaPaCa2) and induced apoptosis (Panc0327, Panc0403). Western blot analysis showed cleavage of caspase 3 and PARP, and prominent induction of the apoptotic molecule BIM. In addition, synergistic effects were found between either STF-083010, 2-Hydroxy-1-naphthaldehyde, 3-Ethoxy-5,6-dibromosalicylaldehyde, or toyocamycin and either gemcitabine or bortezomib. Our data suggest that use of an IRE1α inhibitor is a novel therapeutic approach for treatment of pancreatic cancers. PMID:24952679

  6. Selective inhibition of unfolded protein response induces apoptosis in pancreatic cancer cells.

    PubMed

    Chien, Wenwen; Ding, Ling-Wen; Sun, Qiao-Yang; Torres-Fernandez, Lucia A; Tan, Siew Zhuan; Xiao, Jinfen; Lim, Su Lin; Garg, Manoj; Lee, Kian Leong; Kitajima, Shojiro; Takao, Sumiko; Leong, Wei Zhong; Sun, Haibo; Tokatly, Itay; Poellinger, Lorenz; Gery, Sigal; Koeffler, Phillip H

    2014-07-15

    Endoplasmic reticulum stress from unfolded proteins is associated with the proliferation of pancreatic tumor cells, making the many regulatory molecules of this pathway appealing targets for therapy. The objective of our study was to assess potential therapeutic efficacy of inhibitors of unfolded protein response (UPR) in pancreatic cancers focusing on IRE1α inhibitors. IRE1α-mediated XBP-1 mRNA splicing encodes a transcription factor that enhances transcription of chaperone proteins in order to reverse UPR. Proliferation assays using a panel of 14 pancreatic cancer cell lines showed a dose- and time-dependent growth inhibition by IRE1α-specific inhibitors (STF-083010, 2-Hydroxy-1-naphthaldehyde, 3-Ethoxy-5,6-dibromosalicylaldehyde, toyocamycin). Growth inhibition was also noted using a clonogenic growth assay in soft agar, as well as a xenograft in vivo model of pancreatic cancer. Cell cycle analysis showed that these IRE1α inhibitors caused growth arrest at either the G1 or G2/M phases (SU8686, MiaPaCa2) and induced apoptosis (Panc0327, Panc0403). Western blot analysis showed cleavage of caspase 3 and PARP, and prominent induction of the apoptotic molecule BIM. In addition, synergistic effects were found between either STF-083010, 2-Hydroxy-1-naphthaldehyde, 3-Ethoxy-5,6-dibromosalicylaldehyde, or toyocamycin and either gemcitabine or bortezomib. Our data suggest that use of an IRE1α inhibitor is a novel therapeutic approach for treatment of pancreatic cancers. PMID:24952679

  7. Unfolded protein response is required for Aspergillus oryzae growth under conditions inducing secretory hydrolytic enzyme production.

    PubMed

    Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya

    2015-12-01

    Unfolded protein response (UPR) is an intracellular signaling pathway for adaptation to endoplasmic reticulum (ER) stress. In yeast UPR, Ire1 cleaves the unconventional intron of HAC1 mRNA, and the functional Hac1 protein translated from the spliced HAC1 mRNA induces the expression of ER chaperone genes and ER-associated degradation genes for the refolding or degradation of unfolded proteins. In this study, we constructed an ireA (IRE1 ortholog) conditionally expressing strain of Aspergillus oryzae, a filamentous fungus producing a large amount of amylolytic enzymes, and examined the contribution of UPR to ER stress adaptation under physiological conditions. Repression of ireA completely blocked A. oryzae growth under conditions inducing the production of hydrolytic enzymes, such as amylases and proteases. This growth defect was restored by the introduction of unconventional intronless hacA (hacA-i). Furthermore, UPR was observed to be induced by amylolytic gene expression, and the disruption of the transcriptional activator for amylolytic genes resulted in partial growth restoration of the ireA-repressing strain. In addition, a homokaryotic ireA disruption mutant was successfully generated using the strain harboring hacA-i as a parental host. These results indicated that UPR is required for A. oryzae growth to alleviate ER stress induced by excessive production of hydrolytic enzymes. PMID:26496881

  8. Role of the Unfolded Protein Response in β Cell Compensation and Failure during Diabetes

    PubMed Central

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

    2014-01-01

    Pancreatic β cell failure leads to diabetes development. During disease progression, β cells adapt their secretory capacity to compensate the elevated glycaemia and the peripheral insulin resistance. This compensatory mechanism involves a fine-tuned regulation to modulate the endoplasmic reticulum (ER) capacity and quality control to prevent unfolded proinsulin accumulation, a major protein synthetized within the β cell. These signalling pathways are collectively termed unfolded protein response (UPR). The UPR machinery is required to preserve ER homeostasis and β cell integrity. Moreover, UPR actors play a key role by regulating ER folding capacity, increasing the degradation of misfolded proteins, and limiting the mRNA translation rate. Recent genetic and biochemical studies on mouse models and human UPR sensor mutations demonstrate a clear requirement of the UPR machinery to prevent β cell failure and increase β cell mass and adaptation throughout the progression of diabetes. In this review we will highlight the specific role of UPR actors in β cell compensation and failure during diabetes. PMID:24812634

  9. Role of the unfolded protein response in β cell compensation and failure during diabetes.

    PubMed

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

    2014-01-01

    Pancreatic β cell failure leads to diabetes development. During disease progression, β cells adapt their secretory capacity to compensate the elevated glycaemia and the peripheral insulin resistance. This compensatory mechanism involves a fine-tuned regulation to modulate the endoplasmic reticulum (ER) capacity and quality control to prevent unfolded proinsulin accumulation, a major protein synthetized within the β cell. These signalling pathways are collectively termed unfolded protein response (UPR). The UPR machinery is required to preserve ER homeostasis and β cell integrity. Moreover, UPR actors play a key role by regulating ER folding capacity, increasing the degradation of misfolded proteins, and limiting the mRNA translation rate. Recent genetic and biochemical studies on mouse models and human UPR sensor mutations demonstrate a clear requirement of the UPR machinery to prevent β cell failure and increase β cell mass and adaptation throughout the progression of diabetes. In this review we will highlight the specific role of UPR actors in β cell compensation and failure during diabetes. PMID:24812634

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

    SciTech Connect

    Honma, Yuichi; Harada, Masaru

    2013-08-15

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

  11. TULP1 Missense Mutations Induces the Endoplasmic Reticulum Unfolded Protein Response Stress Complex (ER-UPR).

    PubMed

    Lobo, Glenn P; Ebke, Lindsey A; Au, Adrian; Hagstrom, Stephanie A

    2016-01-01

    Mutations in the TULP1 gene are associated with early-onset retinitis pigmentosa (RP); however, the molecular mechanisms related to the deleterious effects of TULP1 mutations remains unknown. Several studies have shown that misfolded proteins secondary to genetic mutations can accumulate within the endoplasmic reticulum (ER), causing activation of the unfolded protein response (UPR) complex followed by cellular apoptosis. We hypothesize that TULP1 mutations produce misfolded protein products that accumulate in the ER and induce cellular apoptosis via the UPR. To test our hypothesis, we first performed three in-silico analyses of TULP1 missense mutations (I459K, R420P and F491L), which predicted misfolded protein products. Subsequently, the three mutant TULP1-GFP constructs and wild-type (wt) TULP1-GFP were transiently transfected into hTERT-RPE-1 cells. Staining of cells using ER tracker followed by confocal microscopy showed wt-TULP1 localized predominantly to the cytoplasm and plasma membrane. In contrast, all three mutant TULP1 proteins revealed cytoplasmic punctate staining which co-localized with the ER. Furthermore, western blot analysis of cells expressing mutant TULP1 proteins revealed induction of downstream targets of the ER-UPR complex, including BiP/GPR-78, phosphorylated-PERK (Thr980) and CHOP. Our in-vitro analyses suggest that mutant TULP1 proteins are misfolded and accumulate within the ER leading to induction of the UPR stress response complex. PMID:26427415

  12. A chemical screen to identify inducers of the mitochondrial unfolded protein response in C. elegans

    PubMed Central

    Rauthan, Manish; Pilon, Marc

    2015-01-01

    We previously showed that inhibition of the mevalonate pathway in C. elegans causes inhibition of protein prenylation, developmental arrest and lethality. We also showed that constitutive activation of the mitochondrial unfolded protein response, UPRmt, is an effective way for C. elegans to become resistant to the negative effects of mevalonate pathway inhibition. This was an important finding since statins, a drug class prescribed to lower cholesterol levels in patients, act by inhibiting the mevalonate pathway, and it is therefore possible that some of their undesirable side effects could be alleviated by activating the UPRmt. Here, we screened a chemical library and identified 4 compounds that specifically activated the UPRmt. One of these compounds, methacycline hydrochloride (a tetracycline antibiotic) also protected C. elegans and mammalian cells from statin toxicity. Methacycline hydrochloride and ethidium bromide, a known UPRmt activator, were also tested in mice: only ethidium bromide significantly activate the UPRmt in skeletal muscles. PMID:27123370

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

    PubMed Central

    Holstein, Sarah A.; Hohl, Raymond J.

    2010-01-01

    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. PMID:20828814

  14. Neuroserpin Polymers Activate NF-κB by a Calcium Signaling Pathway That Is Independent of the Unfolded Protein Response*

    PubMed Central

    Davies, Mark J.; Miranda, Elena; Roussel, Benoit D.; Kaufman, Randal J.; Marciniak, Stefan J.; Lomas, David A.

    2009-01-01

    The autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies is characterized by the accumulation of ordered polymers of mutant neuroserpin within the endoplasmic reticulum of neurones. We show here that intracellular neuroserpin polymers activate NF-κB by a pathway that is independent of the IRE1, ATF6, and PERK limbs of the canonical unfolded protein response but is dependent on intracellular calcium. This pathway provides a mechanism for cells to sense and react to the accumulation of folded structures of mutant serpins within the endoplasmic reticulum. Our results provide strong support for the endoplasmic reticulum overload response being independent of the unfolded protein response. PMID:19423713

  15. Amyloidogenesis of Natively Unfolded Proteins

    PubMed Central

    Uversky, Vladimir N.

    2009-01-01

    Aggregation and subsequent development of protein deposition diseases originate from conformational changes in corresponding amyloidogenic proteins. The accumulated data support the model where protein fibrillogenesis proceeds via the formation of a relatively unfolded amyloidogenic conformation, which shares many structural properties with the pre-molten globule state, a partially folded intermediate first found during the equilibrium and kinetic (un)folding studies of several globular proteins and later described as one of the structural forms of natively unfolded proteins. The flexibility of this structural form is essential for the conformational rearrangements driving the formation of the core cross-beta structure of the amyloid fibril. Obviously, molecular mechanisms describing amyloidogenesis of ordered and natively unfolded proteins are different. For ordered protein to fibrillate, its unique and rigid structure has to be destabilized and partially unfolded. On the other hand, fibrillogenesis of a natively unfolded protein involves the formation of partially folded conformation; i.e., partial folding rather than unfolding. In this review recent findings are surveyed to illustrate some unique features of the natively unfolded proteins amyloidogenesis. PMID:18537543

  16. Unfolded protein response-induced ERdj3 secretion links ER stress to extracellular proteostasis

    PubMed Central

    Genereux, Joseph C; Qu, Song; Zhou, Minghai; Ryno, Lisa M; Wang, Shiyu; Shoulders, Matthew D; Kaufman, Randal J; Lasmézas, Corinne I; Kelly, Jeffery W; Wiseman, R Luke

    2015-01-01

    The Unfolded Protein Response (UPR) indirectly regulates extracellular proteostasis through transcriptional remodeling of endoplasmic reticulum (ER) proteostasis pathways. This remodeling attenuates secretion of misfolded, aggregation-prone proteins during ER stress. Through these activities, the UPR has a critical role in preventing the extracellular protein aggregation associated with numerous human diseases. Here, we demonstrate that UPR activation also directly influences extracellular proteostasis through the upregulation and secretion of the ER HSP40 ERdj3/DNAJB11. Secreted ERdj3 binds misfolded proteins in the extracellular space, substoichiometrically inhibits protein aggregation, and attenuates proteotoxicity of disease-associated toxic prion protein. Moreover, ERdj3 can co-secrete with destabilized, aggregation-prone proteins in a stable complex under conditions where ER chaperoning capacity is overwhelmed, preemptively providing extracellular chaperoning of proteotoxic misfolded proteins that evade ER quality control. This regulated co-secretion of ERdj3 with misfolded clients directly links ER and extracellular proteostasis during conditions of ER stress. ERdj3 is, to our knowledge, the first metazoan chaperone whose secretion into the extracellular space is regulated by the UPR, revealing a new mechanism by which UPR activation regulates extracellular proteostasis. PMID:25361606

  17. Endoplasmic Reticulum Stress and the Unfolded Protein Responses in Retinal Degeneration

    PubMed Central

    Zhang, Sarah X.; Sanders, Emily; Fliesler, Steven J.; Wang, Joshua J.

    2014-01-01

    The endoplasmic reticulum (ER) is the primary intracellular organelle responsible for protein and lipid biosynthesis, protein folding and trafficking, calcium homeostasis, and several other vital processes in cell physiology. Disturbance in ER function results in ER stress and subsequent activation of the unfolded protein response (UPR). The UPR up-regulates ER chaperones, reduces protein translation, and promotes clearance of cytotoxic misfolded proteins to restore ER homeostasis. If this vital process fails, the cell will be signaled to enter apoptosis, resulting in cell death. Sustained ER stress also can trigger an inflammatory response and exacerbate oxidative stress, both of which contribute synergistically to tissue damage. Studies performed over the past decade have implicated ER stress in a broad range of human diseases, including neurodegenerative diseases, cancer, diabetes, and vascular disorders. Several of these diseases also entail retinal dysfunction and degeneration caused by injury to retinal neurons and/or to the blood vessels that supply retinal cells with nutrients, trophic and homeostatic factors, oxygen, and other essential molecules, as well as serving as a conduit for removal of waste products and potentially toxic substances from the retina. Collectively, such injuries represent the leading cause of blindness world-wide in all age groups. Herein, we summarize recent progress on the study of ER stress and UPR signaling in retinal biology and discuss the molecular mechanisms and the potential clinical applications of targeting ER stress as a new therapeutic approach to prevent and treat neuronal degeneration in the retina. PMID:24792589

  18. Role of the unfolded protein response, GRP78 and GRP94 in organ homeostasis.

    PubMed

    Zhu, Genyuan; Lee, Amy S

    2015-07-01

    The endoplasmic reticulum (ER) is a cellular organelle where secretory and membrane proteins, as well as lipids, are synthesized and modified. When cells are subjected to ER stress, an adaptive mechanism referred to as the Unfolded Protein Response (UPR) is triggered to allow the cells to restore homeostasis. Evidence has accumulated that the UPR pathways provide specialized and unique roles in diverse development and metabolic processes. The glucose regulated proteins (GRPs) are traditionally regarded as ER proteins with chaperone and calcium binding properties. The GRPs are constitutively expressed at basal levels in all organs, and as stress-inducible ER chaperones, they are major players in protein folding, assembly and degradation. This conventional concept is augmented by recent discoveries that GRPs can be actively translocated to other cellular locations such as the cell surface, where they assume novel functions that regulate signaling, proliferation, apoptosis and immunity. Recent construction and characterization of mouse models where the gene encoding for the UPR components and the GRPs is genetically altered provide new insights on the physiological contribution of these proteins in vivo. This review highlights recent progress towards the understanding of the role of the UPR and two major GRPs (GRP78 and GRP94) in regulating homeostasis of organs arising from the endoderm, mesoderm and ectoderm. GRP78 and GRP94 exhibit shared and unique functions, and in specific organs their depletion elicits adaptive responses with physiological consequences. PMID:25546813

  19. Apoptosis, autophagy and unfolded protein response pathways in Arbovirus replication and pathogenesis.

    PubMed

    Iranpour, Mahmoud; Moghadam, Adel Rezaei; Yazdi, Mina; Ande, Sudharsana R; Alizadeh, Javad; Wiechec, Emilia; Lindsay, Robbin; Drebot, Michael; Coombs, Kevin M; Ghavami, Saeid

    2016-01-01

    Arboviruses are pathogens that widely affect the health of people in different communities around the world. Recently, a few successful approaches toward production of effective vaccines against some of these pathogens have been developed, but treatment and prevention of the resulting diseases remain a major health and research concern. The arbovirus infection and replication processes are complex, and many factors are involved in their regulation. Apoptosis, autophagy and the unfolded protein response (UPR) are three mechanisms that are involved in pathogenesis of many viruses. In this review, we focus on the importance of these pathways in the arbovirus replication and infection processes. We provide a brief introduction on how apoptosis, autophagy and the UPR are initiated and regulated, and then discuss the involvement of these pathways in regulation of arbovirus pathogenesis. PMID:26781343

  20. Gestational stress induces the unfolded protein response, resulting in heart defects.

    PubMed

    Shi, Hongjun; O'Reilly, Victoria C; Moreau, Julie L M; Bewes, Therese R; Yam, Michelle X; Chapman, Bogdan E; Grieve, Stuart M; Stocker, Roland; Graham, Robert M; Chapman, Gavin; Sparrow, Duncan B; Dunwoodie, Sally L

    2016-07-15

    Congenital heart disease (CHD) is an enigma. It is the most common human birth defect and yet, even with the application of modern genetic and genomic technologies, only a minority of cases can be explained genetically. This is because environmental stressors also cause CHD. Here we propose a plausible non-genetic mechanism for induction of CHD by environmental stressors. We show that exposure of mouse embryos to short-term gestational hypoxia induces the most common types of heart defect. This is mediated by the rapid induction of the unfolded protein response (UPR), which profoundly reduces FGF signaling in cardiac progenitor cells of the second heart field. Thus, UPR activation during human pregnancy might be a common cause of CHD. Our findings have far-reaching consequences because the UPR is activated by a myriad of environmental or pathophysiological conditions. Ultimately, our discovery could lead to preventative strategies to reduce the incidence of human CHD. PMID:27436040

  1. Low intensity focused ultrasound (LOFU) modulates unfolded protein response and sensitizes prostate cancer to 17AAG

    PubMed Central

    Saha, Subhrajit; Bhanja, Payel; Partanen, Ari; Zhang, Wei; Liu, Laibin; Tomé, Wolfgang; Guha, Chandan

    2014-01-01

    The hypoxic tumor microenvironment generates oxidative Endoplasmic Reticulum (ER) stress, resulting in protein misfolding and unfolded protein response (UPR). UPR induces several molecular chaperones including heat-shock protein 90 (HSP90), which corrects protein misfolding and improves survival of cancer cells and resistance to tumoricidal therapy although prolonged activation of UPR induces cell death. The HSP90 inhibitor, 17AAG, has shown promise against various solid tumors, including prostate cancer (PC). However, therapeutic doses of 17AAG elicit systemic toxicity. In this manuscript, we describe a new paradigm where the combination therapy of a non-ablative and non-invasive low energy focused ultrasound (LOFU) and a non-toxic, low dose 17AAG causes synthetic lethality and significant tumoricidal effects in mouse and human PC xenografts. LOFU induces ER stress and UPR in tumor cells without inducing cell death. Treatment with a non-toxic dose of 17AAG further increased ER stress in LOFU treated PC and switch UPR from a cytoprotective to an apoptotic response in tumors resulting significant induction of apoptosis and tumor growth retardation. These observations suggest that LOFU-induced ER stress makes the ultrasound-treated tumors more susceptible to chemotherapeutic agents, such as 17AAG. Thus, a novel therapy of LOFU-induced chemosensitization may be designed for locally advanced and recurrent tumors. PMID:25594042

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

    PubMed Central

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

    2014-01-01

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

  3. Venezuelan Equine Encephalitis Virus Induces Apoptosis through the Unfolded Protein Response Activation of EGR1

    PubMed Central

    Baer, Alan; Lundberg, Lindsay; Swales, Danielle; Waybright, Nicole; Pinkham, Chelsea; Dinman, Jonathan D.

    2016-01-01

    ABSTRACT Venezuelan equine encephalitis virus (VEEV) is a previously weaponized arthropod-borne virus responsible for causing acute and fatal encephalitis in animal and human hosts. The increased circulation and spread in the Americas of VEEV and other encephalitic arboviruses, such as eastern equine encephalitis virus and West Nile virus, underscore the need for research aimed at characterizing the pathogenesis of viral encephalomyelitis for the development of novel medical countermeasures. The host-pathogen dynamics of VEEV Trinidad donkey-infected human astrocytoma U87MG cells were determined by carrying out RNA sequencing (RNA-Seq) of poly(A) and mRNAs. To identify the critical alterations that take place in the host transcriptome following VEEV infection, samples were collected at 4, 8, and 16 h postinfection and RNA-Seq data were acquired using an Ion Torrent PGM platform. Differential expression of interferon response, stress response factors, and components of the unfolded protein response (UPR) was observed. The protein kinase RNA-like endoplasmic reticulum kinase (PERK) arm of the UPR was activated, as the expression of both activating transcription factor 4 (ATF4) and CHOP (DDIT3), critical regulators of the pathway, was altered after infection. Expression of the transcription factor early growth response 1 (EGR1) was induced in a PERK-dependent manner. EGR1−/− mouse embryonic fibroblasts (MEFs) demonstrated lower susceptibility to VEEV-induced cell death than isogenic wild-type MEFs, indicating that EGR1 modulates proapoptotic pathways following VEEV infection. The influence of EGR1 is of great importance, as neuronal damage can lead to long-term sequelae in individuals who have survived VEEV infection. IMPORTANCE Alphaviruses represent a group of clinically relevant viruses transmitted by mosquitoes to humans. In severe cases, viral spread targets neuronal tissue, resulting in significant and life-threatening inflammation dependent on a combination

  4. Coronavirus Infection Modulates the Unfolded Protein Response and Mediates Sustained Translational Repression▿

    PubMed Central

    Bechill, John; Chen, Zhongbin; Brewer, Joseph W.; Baker, Susan C.

    2008-01-01

    During coronavirus replication, viral proteins induce the formation of endoplasmic reticulum (ER)-derived double-membrane vesicles for RNA synthesis, and viral structural proteins assemble virions at the ER-Golgi intermediate compartment. We hypothesized that the association and intense utilization of the ER during viral replication would induce the cellular unfolded protein response (UPR), a signal transduction cascade that acts to modulate translation, membrane biosynthesis, and the levels of ER chaperones. Here, we report that infection by the murine coronavirus mouse hepatitis virus (MHV) triggers the proximal UPR transducers, as revealed by monitoring the IRE1-mediated splicing of XBP-1 mRNA and the cleavage of ATF6α. However, we detected minimal downstream induction of UPR target genes, including ERdj4, ER degradation-enhancing α-mannosidase-like protein, and p58IPK, or expression of UPR reporter constructs. Translation initiation factor eIF2α is highly phosphorylated during MHV infection, and translation of cellular mRNAs is attenuated. Furthermore, we found that the critical homeostasis regulator GADD34, which recruits protein phosphatase 1 to dephosphorylate eIF2α during the recovery phase of the UPR, is not expressed during MHV infection. These results suggest that MHV modifies the UPR by impeding the induction of UPR-responsive genes, thereby favoring a sustained shutdown of the synthesis of host cell proteins while the translation of viral proteins escalates. The role of this modified response and its potential relevance to viral mechanisms for the evasion of innate defense signaling pathways during coronavirus replication are discussed. PMID:18305036

  5. The critical roles of endoplasmic reticulum chaperones and unfolded protein response in tumorigenesis and anticancer therapies.

    PubMed

    Luo, B; Lee, A S

    2013-02-14

    Cancer progression is characterized by rapidly proliferating cancer cells that are in need of increased protein synthesis. Therefore, enhanced endoplasmic reticulum (ER) activity is required to facilitate the folding, assembly and transportation of membrane and secretory proteins. These functions are carried out by ER chaperones. It is now becoming clear that the ER chaperones have critical functions outside of simply facilitating protein folding. For example, cancer progression requires glucose regulated protein (GRP) 78 for cancer cell survival and proliferation, as well as angiogenesis in the microenvironment. GRP78 can translocate to the cell surface acting as a receptor regulating oncogenic signaling and cell viability. Calreticulin, another ER chaperone, can translocate to the cell surface of apoptotic cancer cells and induce immunogenic cancer cell death and antitumor responses in vivo. Tumor-secreted GRP94 has been shown to elicit antitumor immune responses when used as antitumor vaccines. Protein disulfide isomerase is another ER chaperone that demonstrates pro-oncogenic and pro-survival functions. Because of intrinsic alterations of cellular metabolism and extrinsic factors in the tumor microenvironment, cancer cells are under ER stress, and they respond to this stress by activating the unfolded protein response (UPR). Depending on the severity and duration of ER stress, the signaling branches of the UPR can activate adaptive and pro-survival signals, or induce apoptotic cell death. The protein kinase RNA-like ER kinase signaling branch of the UPR has a dual role in cancer proliferation and survival, and is also required for ER stress-induced autophagy. The activation of the inositol-requiring kinase 1α branch promotes tumorigenesis, cancer cell survival and regulates tumor invasion. In summary, perturbance of ER homeostasis has critical roles in tumorigenesis, and therapeutic modulation of ER chaperones and/or UPR components presents potential antitumor

  6. Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in plants.

    PubMed

    Wan, Shucen; Jiang, Liwen

    2016-05-01

    Being a major factory for protein synthesis, assembly, and export, the endoplasmic reticulum (ER) has a precise and robust ER quality control (ERQC) system monitoring its product line. However, when organisms are subjected to environmental stress, whether biotic or abiotic, the levels of misfolded proteins may overwhelm the ERQC system, tilting the balance between the capacity of and demand for ER quality control and resulting in a scenario termed ER stress. Intense or prolonged ER stress may cause damage to the ER as well as to other organelles, or even lead to cell death in extreme cases. To avoid such serious consequences, cells activate self-rescue programs to restore protein homeostasis in the ER, either through the enhancement of protein-folding and degradation competence or by alleviating the demands for such reactions. These are collectively called the unfolded protein response (UPR). Long investigated in mammalian cells and yeasts, the UPR is also of great interest to plant scientists. Among the three branches of UPR discovered in mammals, two have been studied in plants with plant homologs existing of the ER-membrane-associated activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1 (IRE1). This review discusses the molecular mechanisms of these two types of UPR in plants, as well as the consequences of insufficient UPR, with a focus on experiments using model plants. PMID:26060134

  7. Killing Me Softly: Connotations to Unfolded Protein Response and Oxidative Stress in Alzheimer's Disease

    PubMed Central

    Pająk, Beata; Kania, Elżbieta; Orzechowski, Arkadiusz

    2016-01-01

    This review is focused on the possible causes of mitochondrial dysfunction in AD, underlying molecular mechanisms of this malfunction, possible causes and known consequences of APP, Aβ, and hyperphosphorylated tau presence in mitochondria, and the contribution of altered lipid metabolism (nonsterol isoprenoids) to pathological processes leading to increased formation and accumulation of the aforementioned hallmarks of AD. Abnormal protein folding and unfolded protein response seem to be the outcomes of impaired glycosylation due to metabolic disturbances in geranylgeraniol intermediary metabolism. The origin and consecutive fate of APP, Aβ, and tau are emphasized on intracellular trafficking apparently influenced by inaccurate posttranslational modifications. We hypothesize that incorrect intracellular processing of APP determines protein translocation to mitochondria in AD. Similarly, without obvious reasons, the passage of Aβ and tau to mitochondria is observed. APP targeted to mitochondria blocks the activity of protein translocase complex resulting in poor import of proteins central to oxidative phosphorylation. Besides, APP, Aβ, and neurofibrillary tangles of tau directly or indirectly impair mitochondrial biochemistry and bioenergetics, with concomitant generation of oxidative/nitrosative stress. Limited protective mechanisms are inadequate to prevent the free radical-mediated lesions. Finally, neuronal loss is observed in AD-affected brains typically by pathologic apoptosis. PMID:26881014

  8. ER stress and unfolded protein response in amyotrophic lateral sclerosis-a controversial role of protein disulphide isomerase.

    PubMed

    Jaronen, Merja; Goldsteins, Gundars; Koistinaho, Jari

    2014-01-01

    Accumulation of proteins in aberrant conformation occurs in many neurodegenerative diseases. Furthermore, dysfunctions in protein handling in endoplasmic reticulum (ER) and the following ER stress have been implicated in a vast number of diseases, such as amyotrophic lateral sclerosis (ALS). During excessive ER stress unfolded protein response (UPR) is activated to return ER to its normal physiological balance. The exact mechanisms of protein misfolding, accumulation and the following ER stress, which could lead to neurodegeneration, and the question whether UPR is a beneficial compensatory mechanism slowing down the neurodegenerative processes, are of interest. Protein disulphide isomerase (PDI) is a disulphide bond-modulating ER chaperone, which can also facilitate the ER-associated degradation (ERAD) of misfolded proteins. In this review we discuss the recent findings of ER stress, UPR and especially the role of PDI in ALS. PMID:25520620

  9. Identification of Novel Components of the Unfolded Protein Response in Arabidopsis

    PubMed Central

    Hossain, Md. Amir; Henríquez-Valencia, Carlos; Gómez-Páez, Marcela; Medina, Joaquín; Orellana, Ariel; Vicente-Carbajosa, Jesús; Zouhar, Jan

    2016-01-01

    Unfavorable environmental and developmental conditions may cause disturbances in protein folding in the endoplasmic reticulum (ER) that are recognized and counteracted by components of the Unfolded Protein Response (UPR) signaling pathways. The early cellular responses include transcriptional changes to increase the folding and processing capacity of the ER. In this study, we systematically screened a collection of inducible transgenic Arabidopsis plants expressing a library of transcription factors for resistance toward UPR-inducing chemicals. We identified 23 candidate genes that may function as novel regulators of the UPR and of which only three genes (bZIP10, TBF1, and NF-YB3) were previously associated with the UPR. The putative role of identified candidate genes in the UPR signaling is supported by favorable expression patterns in both developmental and stress transcriptional analyses. We demonstrated that WRKY75 is a genuine regulator of the ER-stress cellular responses as its expression was found to be directly responding to ER stress-inducing chemicals. In addition, transgenic Arabidopsis plants expressing WRKY75 showed resistance toward salt stress, connecting abiotic and ER-stress responses. PMID:27242851

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

    PubMed Central

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

    2014-01-01

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

  11. Tunicamycin-induced unfolded protein response in the developing mouse brain

    SciTech Connect

    Wang, Haiping; Wang, Xin; Ke, Zun-Ji; Comer, Ashley L.; Xu, Mei; Frank, Jacqueline A.; Zhang, Zhuo; Shi, Xianglin; Luo, Jia

    2015-03-15

    Accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) causes ER stress, resulting in the activation of the unfolded protein response (UPR). ER stress and UPR are associated with many neurodevelopmental and neurodegenerative disorders. The developing brain is particularly susceptible to environmental insults which may cause ER stress. We evaluated the UPR in the brain of postnatal mice. Tunicamycin, a commonly used ER stress inducer, was administered subcutaneously to mice of postnatal days (PDs) 4, 12 and 25. Tunicamycin caused UPR in the cerebral cortex, hippocampus and cerebellum of mice of PD4 and PD12, which was evident by the upregulation of ATF6, XBP1s, p-eIF2α, GRP78, GRP94 and MANF, but failed to induce UPR in the brain of PD25 mice. Tunicamycin-induced UPR in the liver was observed at all stages. In PD4 mice, tunicamycin-induced caspase-3 activation was observed in layer II of the parietal and optical cortex, CA1–CA3 and the subiculum of the hippocampus, the cerebellar external germinal layer and the superior/inferior colliculus. Tunicamycin-induced caspase-3 activation was also shown on PD12 but to a much lesser degree and mainly located in the dentate gyrus of the hippocampus, deep cerebellar nuclei and pons. Tunicamycin did not activate caspase-3 in the brain of PD25 mice and the liver of all stages. Similarly, immature cerebellar neurons were sensitive to tunicamycin-induced cell death in culture, but became resistant as they matured in vitro. These results suggest that the UPR is developmentally regulated and the immature brain is more susceptible to ER stress. - Highlights: • Tunicamycin caused a development-dependent UPR in the mouse brain. • Immature brain was more susceptible to tunicamycin-induced endoplasmic reticulum stress. • Tunicamycin caused more neuronal death in immature brain than mature brain. • Tunicamycin-induced neuronal death is region-specific.

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

    PubMed Central

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

    2013-01-01

    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 β-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

  13. Drop in endo/sarcoplasmic calcium precedes the unfolded protein response in Brefeldin A-treated vascular smooth muscle cells.

    PubMed

    Ziomek, Gabriela; van Breemen, Cornelis; Esfandiarei, Mitra

    2015-10-01

    The present study addresses the causal relationship between induction of endo/sarcoplasmic reticulum stress and dysregulation of calcium transport, while examining whether the most widely-used experimental endo/sarcoplasmic reticulum stressors can be considered appropriate for elucidating underlying cellular mechanisms involved during the progression of the unfolded protein response in vascular smooth muscle cells. Brefeldin A is most commonly cited as inducing the stress response through an accumulation of unfolded proteins in the lumen as a result of a blockage of protein transport from the endo/sarcoplasmic reticulum to the Golgi apparatus. We investigated the effects of Brefeldin A on cellular calcium regulation during the the unfolded protein response in cultured rat vascular smooth muscle cells. Acute exposure of cells to Brefeldin A caused a small transient increase in cytoplasmic calcium, which did not cause a significant decrease in endo/sarcoplasmic reticulum calcium content. However, over the time course of 0-12 h post-treatment with Brefeldin A, we observed that the endo/sarcoplasmic reticulum of vascular smooth muscle cells exhibited an approximate fifty percent decrease in calcium concentration after the first hour of exposure, which is maintained over the next eleven hours, whereas concentrations of unfolded protein response markers only began to increase significantly around nine to twelve hours post-treatment. We have concluded that the endo/sarcoplasmic reticulum calcium drop, which up to now, has been considered as a characteristic of the late onset of cellular stress response, occurs prior to the initiation of the unfolded protein response, rather than as a result of its many corrective pathways. PMID:26172080

  14. Inhibition of host cell translation elongation by Legionella pneumophila blocks the host cell unfolded protein response

    PubMed Central

    Hempstead, Andrew D.; Isberg, Ralph R.

    2015-01-01

    Cells of the innate immune system recognize bacterial pathogens by detecting common microbial patterns as well as pathogen-specific activities. One system that responds to these stimuli is the IRE1 branch of the unfolded protein response (UPR), a sensor of endoplasmic reticulum (ER) stress. Activation of IRE1, in the context of Toll-like receptor (TLR) signaling, induces strong proinflammatory cytokine induction. We show here that Legionella pneumophila, an intravacuolar pathogen that replicates in an ER-associated compartment, blocks activation of the IRE1 pathway despite presenting pathogen products that stimulate this response. L. pneumophila TLR ligands induced the splicing of mRNA encoding XBP1s, the main target of IRE1 activity. L. pneumophila was able to inhibit both chemical and bacterial induction of XBP1 splicing via bacterial translocated proteins that interfere with host protein translation. A strain lacking five translocated translation elongation inhibitors was unable to block XBP1 splicing, but this could be rescued by expression of a single such inhibitor, consistent with limitation of the response by translation elongation inhibitors. Chemical inhibition of translation elongation blocked pattern recognition receptor-mediated XBP1 splicing, mimicking the effects of the bacterial translation inhibitors. In contrast, host cell-promoted inhibition of translation initiation in response to the pathogen was ineffective in blocking XBP1 splicing, demonstrating the need for the elongation inhibitors for protection from the UPR. The inhibition of host translation elongation may be a common strategy used by pathogens to limit the innate immune response by interfering with signaling via the UPR. PMID:26598709

  15. The loss of LRPPRC function induces the mitochondrial unfolded protein response.

    PubMed

    Köhler, Fabian; Müller-Rischart, Anne Kathrin; Conradt, Barbara; Rolland, Stéphane Guy

    2015-09-01

    The inactivation of the LRPPRC gene, which has previously been associated with the neurodegenerative French Canadian Leigh Syndrome, results in a decrease in the production of mitochondria-encoded subunits of complex IV, thereby causing a reduction in complex IV activity. Previously we have shown that reducing complex IV activity triggers a compensatory and conserved mitochondrial hyperfusion response. We now demonstrate that LRPPRC knock-down in mammalian cells leads to an imbalance between mitochondria-encoded and nuclear-encoded subunits of complex IV and that this imbalance triggers the mitochondrial unfolded protein response (UPR(mt)). The inactivation of the LRPPRC-like gene mma-1 in C. elegans also induces UPR(mt), which demonstrates that this response is conserved. Furthermore, we provide evidence that mitochondrial hyperfusion and UPR(mt) are coordinated but mediated by genetically distinct pathways. We propose that in the context of LRPPRC mma-1 knock-down, mitochondrial hyperfusion helps to transiently maintain mitochondrial ATP production while UPR(mt) participates in the restoration of mitochondrial proteostasis. Mitochondrial proteostasis is not only critical in pathophysiology but also during aging, as proteotoxic stress has been shown to increase with age. Therefore, we speculate that the coordination of these two mitochondrial stress responses plays a more global role in mitochondrial proteostasis. PMID:26412102

  16. The loss of LRPPRC function induces the mitochondrial unfolded protein response

    PubMed Central

    Conradt, Barbara; Rolland, Stéphane Guy

    2015-01-01

    The inactivation of the LRPPRC gene, which has previously been associated with the neurodegenerative French Canadian Leigh Syndrome, results in a decrease in the production of mitochondria-encoded subunits of complex IV, thereby causing a reduction in complex IV activity. Previously we have shown that reducing complex IV activity triggers a compensatory and conserved mitochondrial hyperfusion response. We now demonstrate that LRPPRC knock-down in mammalian cells leads to an imbalance between mitochondria-encoded and nuclear-encoded subunits of complex IV and that this imbalance triggers the mitochondrial unfolded protein response (UPRmt). The inactivation of the LRPPRC-like gene mma-1 in C. elegans also induces UPRmt, which demonstrates that this response is conserved. Furthermore, we provide evidence that mitochondrial hyperfusion and UPRmt are coordinated but mediated by genetically distinct pathways. We propose that in the context of LRPPRC mma-1 knock-down, mitochondrial hyperfusion helps to transiently maintain mitochondrial ATP production while UPRmt participates in the restoration of mitochondrial proteostasis. Mitochondrial proteostasis is not only critical in pathophysiology but also during aging, as proteotoxic stress has been shown to increase with age. Therefore, we speculate that the coordination of these two mitochondrial stress responses plays a more global role in mitochondrial proteostasis. PMID:26412102

  17. Structure of the Ire1 autophosphorylation complex and implications for the unfolded protein response

    PubMed Central

    Ali, Maruf M U; Bagratuni, Tina; Davenport, Emma L; Nowak, Piotr R; Silva-Santisteban, M Cris; Hardcastle, Anthea; McAndrews, Craig; Rowlands, Martin G; Morgan, Gareth J; Aherne, Wynne; Collins, Ian; Davies, Faith E; Pearl, Laurence H

    2011-01-01

    Ire1 (Ern1) is an unusual transmembrane protein kinase essential for the endoplasmic reticulum (ER) unfolded protein response (UPR). Activation of Ire1 by association of its N-terminal ER luminal domains promotes autophosphorylation by its cytoplasmic kinase domain, leading to activation of the C-terminal ribonuclease domain, which splices Xbp1 mRNA generating an active Xbp1s transcriptional activator. We have determined the crystal structure of the cytoplasmic portion of dephosphorylated human Ire1α bound to ADP, revealing the ‘phosphoryl-transfer' competent dimeric face-to-face complex, which precedes and is distinct from the back-to-back RNase ‘active' conformation described for yeast Ire1. We show that the Xbp1-specific ribonuclease activity depends on autophosphorylation, and that ATP-competitive inhibitors staurosporin and sunitinib, which inhibit autophosphorylation in vitro, also inhibit Xbp1 splicing in vivo. Furthermore, we demonstrate that activated Ire1α is a competent protein kinase, able to phosphorylate a heterologous peptide substrate. These studies identify human Ire1α as a target for development of ATP-competitive inhibitors that will modulate the UPR in human cells, which has particular relevance for myeloma and other secretory malignancies. PMID:21317875

  18. Calcineurin β protects brain after injury by activating the unfolded protein response.

    PubMed

    Chen, Yanan; Holstein, Deborah M; Aime, Sofia; Bollo, Mariana; Lechleiter, James D

    2016-10-01

    The Ca(2+)-dependent phosphatase, calcineurin (CN) is thought to play a detrimental role in damaged neurons; however, its role in astrocytes is unclear. In cultured astrocytes, CNβ expression increased after treatment with a sarco/endoplasmic reticulum Ca(2+)-ATPase inhibitor, thapsigargin, and with oxygen and glucose deprivation, an in vitro model of ischemia. Similarly, CNβ was induced in astrocytes in vivo in two different mouse models of brain injury - photothrombotic stroke and traumatic brain injury (TBI). Immunoprecipitation and chemical activation dimerization methods pointed to physical interaction of CNβ with the unfolded protein response (UPR) sensor, protein kinase RNA-like endoplasmic reticulum kinase (PERK). In accordance, induction of CNβ resulted in oligomerization and activation of PERK. Strikingly, the presence of a phosphatase inhibitor did not interfere with CNβ-mediated activation of PERK, suggesting a hitherto undiscovered non-enzymatic role for CNβ. Importantly, the cytoprotective function of CNβ was PERK-dependent both in vitro and in vivo. Loss of CNβ in vivo resulted in a significant increase in cerebral damage, and correlated with a decrease in astrocyte size, PERK activity and glial fibrillary acidic protein (GFAP) expression. Taken together, these data reveal a critical role for the CNβ-PERK axis in not only prolonging astrocyte cell survival but also in modulating astrogliosis after brain injury. PMID:27334877

  19. Exploring the Conserved Role of MANF in the Unfolded Protein Response in Drosophila melanogaster

    PubMed Central

    Lindström, Riitta; Lindholm, Päivi; Kallijärvi, Jukka; Palgi, Mari; Saarma, Mart; Heino, Tapio I.

    2016-01-01

    Disturbances in the homeostasis of endoplasmic reticulum (ER) referred to as ER stress is involved in a variety of human diseases. ER stress activates unfolded protein response (UPR), a cellular mechanism the purpose of which is to restore ER homeostasis. Previous studies show that Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) is an important novel component in the regulation of UPR. In vertebrates, MANF is upregulated by ER stress and protects cells against ER stress-induced cell death. Biochemical studies have revealed an interaction between mammalian MANF and GRP78, the major ER chaperone promoting protein folding. In this study we discovered that the upregulation of MANF expression in response to drug-induced ER stress is conserved between Drosophila and mammals. Additionally, by using a genetic in vivo approach we found genetic interactions between Drosophila Manf and genes encoding for Drosophila homologues of GRP78, PERK and XBP1, the key components of UPR. Our data suggest a role for Manf in the regulation of Drosophila UPR. PMID:26975047

  20. Exploring the Conserved Role of MANF in the Unfolded Protein Response in Drosophila melanogaster.

    PubMed

    Lindström, Riitta; Lindholm, Päivi; Kallijärvi, Jukka; Palgi, Mari; Saarma, Mart; Heino, Tapio I

    2016-01-01

    Disturbances in the homeostasis of endoplasmic reticulum (ER) referred to as ER stress is involved in a variety of human diseases. ER stress activates unfolded protein response (UPR), a cellular mechanism the purpose of which is to restore ER homeostasis. Previous studies show that Mesencephalic Astrocyte-derived Neurotrophic Factor (MANF) is an important novel component in the regulation of UPR. In vertebrates, MANF is upregulated by ER stress and protects cells against ER stress-induced cell death. Biochemical studies have revealed an interaction between mammalian MANF and GRP78, the major ER chaperone promoting protein folding. In this study we discovered that the upregulation of MANF expression in response to drug-induced ER stress is conserved between Drosophila and mammals. Additionally, by using a genetic in vivo approach we found genetic interactions between Drosophila Manf and genes encoding for Drosophila homologues of GRP78, PERK and XBP1, the key components of UPR. Our data suggest a role for Manf in the regulation of Drosophila UPR. PMID:26975047

  1. Cantharidins Induce ER Stress and a Terminal Unfolded Protein Response in OSCC

    PubMed Central

    Xi, Y.; Garshott, D.M.; Brownell, A.L.; Yoo, G.H.; Lin, H.-S.; Freeburg, T.L.; Yoo, N.G.; Kaufman, R.J.; Callaghan, M.U.

    2015-01-01

    Mortality and morbidity associated with oral squamous cell carcinoma (OSCC) remain unacceptably high with disfiguring treatment options and a death rate of 1 per hour in the United States. The approval of cituximab for advanced OSCC has been the only new treatment for these patients since the 1970s, although it has not significantly increased overall survival. To address the paucity of effective new therapies, we undertook a high-throughput screen to discover small molecules and natural products that could induce endoplasmic reticulum (ER) stress and enforce a terminal unfolded protein response (UPR) in OSCC. The terpenoid cantharidin (CNT), previously used to treat various malignancies in culture-specific medical practices for over 2,000 y, emerged as a hit. CNT and its analog, cantharidic acid, potently induced protein and gene expression profiles consistent with the activation of ER stress, the UPR, and apoptosis in OSCC cells. Murine embryonic fibroblasts null for the UPR-associated transcription factors Atf4 or Chop were significantly protected from CNT, implicating a key role for the UPR in the death response. These data validate that our high-throughput screen can identify novel modulators of UPR signaling and that such compounds might provide a new therapeutic approach to treating patients with OSCC. PMID:25425581

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

    PubMed Central

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

    2012-01-01

    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

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

    PubMed Central

    2010-01-01

    . 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. PMID:20346137

  4. Insulin demand regulates β cell number via the unfolded protein response.

    PubMed

    Sharma, Rohit B; O'Donnell, Amy C; Stamateris, Rachel E; Ha, Binh; McCloskey, Karen M; Reynolds, Paul R; Arvan, Peter; Alonso, Laura C

    2015-10-01

    Although stem cell populations mediate regeneration of rapid turnover tissues, such as skin, blood, and gut, a stem cell reservoir has not been identified for some slower turnover tissues, such as the pancreatic islet. Despite lacking identifiable stem cells, murine pancreatic β cell number expands in response to an increase in insulin demand. Lineage tracing shows that new β cells are generated from proliferation of mature, differentiated β cells; however, the mechanism by which these mature cells sense systemic insulin demand and initiate a proliferative response remains unknown. Here, we identified the β cell unfolded protein response (UPR), which senses insulin production, as a regulator of β cell proliferation. Using genetic and physiologic models, we determined that among the population of β cells, those with an active UPR are more likely to proliferate. Moreover, subthreshold endoplasmic reticulum stress (ER stress) drove insulin demand-induced β cell proliferation, through activation of ATF6. We also confirmed that the UPR regulates proliferation of human β cells, suggesting that therapeutic UPR modulation has potential to expand β cell mass in people at risk for diabetes. Together, this work defines a stem cell-independent model of tissue homeostasis, in which differentiated secretory cells use the UPR sensor to adapt organ size to meet demand. PMID:26389675

  5. Insulin demand regulates β cell number via the unfolded protein response

    PubMed Central

    Sharma, Rohit B.; O’Donnell, Amy C.; Stamateris, Rachel E.; Ha, Binh; McCloskey, Karen M.; Reynolds, Paul R.; Arvan, Peter; Alonso, Laura C.

    2015-01-01

    Although stem cell populations mediate regeneration of rapid turnover tissues, such as skin, blood, and gut, a stem cell reservoir has not been identified for some slower turnover tissues, such as the pancreatic islet. Despite lacking identifiable stem cells, murine pancreatic β cell number expands in response to an increase in insulin demand. Lineage tracing shows that new β cells are generated from proliferation of mature, differentiated β cells; however, the mechanism by which these mature cells sense systemic insulin demand and initiate a proliferative response remains unknown. Here, we identified the β cell unfolded protein response (UPR), which senses insulin production, as a regulator of β cell proliferation. Using genetic and physiologic models, we determined that among the population of β cells, those with an active UPR are more likely to proliferate. Moreover, subthreshold endoplasmic reticulum stress (ER stress) drove insulin demand–induced β cell proliferation, through activation of ATF6. We also confirmed that the UPR regulates proliferation of human β cells, suggesting that therapeutic UPR modulation has potential to expand β cell mass in people at risk for diabetes. Together, this work defines a stem cell–independent model of tissue homeostasis, in which differentiated secretory cells use the UPR sensor to adapt organ size to meet demand. PMID:26389675

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

    PubMed Central

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

    2013-01-01

    The unfolded protein response (UPR) is an important regulatory network that responds to perturbations in protein homeostasis in the endoplasmic reticulum (ER). In mammalian cells, the UPR features translational and transcriptional mechanisms of gene expression aimed at restoring proteostatic control. A central feature of the UPR is phosphorylation of the α subunit of eukaryotic initiation factor-2 (eIF2) by PERK (EIF2AK3/PEK), which reduces the influx of nascent proteins into the ER by lowering global protein synthesis, coincident with preferential translation of key transcription activators of genes that function to expand the processing capacity of this secretory organelle. Upon ER stress, the apicomplexan parasite Toxoplasma gondii is known to induce phosphorylation of Toxoplasma eIF2α and lower translation initiation. To characterize the nature of the ensuing UPR in this parasite, we carried out microarray analyses to measure the changes in the transcriptome and in translational control during ER stress. We determined that a collection of transcripts linked with the secretory process are induced in response to ER stress, supporting the idea that a transcriptional induction phase of the UPR occurs in Toxoplasma. Furthermore, we determined that about 500 gene transcripts showed enhanced association with translating ribosomes during ER stress. Many of these target genes are suggested to be involved in gene expression, including JmjC5, which continues to be actively translated during ER stress. This study indicates that Toxoplasma triggers a UPR during ER stress that features both translational and transcriptional regulatory mechanisms, which is likely to be important for parasite invasion and development. PMID:23666622

  7. Induction of the Unfolded Protein Response Drives Enhanced Metabolism and Chemoresistance in Glioma Cells

    PubMed Central

    Merz, Andrea L.; Dechkovskaia, Anjelika M.; Herring, Matthew; Winston, Benjamin A.; Lencioni, Alex M.; Russell, Rae L.; Madsen, Helen; Nega, Meheret; Dusto, Nathaniel L.; White, Jason; Bigner, Darell D.; Nicchitta, Christopher V.; Serkova, Natalie J.; Graner, Michael W.

    2013-01-01

    The unfolded protein response (UPR) is an endoplasmic reticulum (ER)-based cytoprotective mechanism acting to prevent pathologies accompanying protein aggregation. It is frequently active in tumors, but relatively unstudied in gliomas. We hypothesized that UPR stress effects on glioma cells might protect tumors from additional exogenous stress (ie, chemotherapeutics), postulating that protection was concurrent with altered tumor cell metabolism. Using human brain tumor cell lines, xenograft tumors, human samples and gene expression databases, we determined molecular features of glioma cell UPR induction/activation, and here report a detailed analysis of UPR transcriptional/translational/metabolic responses. Immunohistochemistry, Western and Northern blots identified elevated levels of UPR transcription factors and downstream ER chaperone targets in gliomas. Microarray profiling revealed distinct regulation of stress responses between xenograft tumors and parent cell lines, with gene ontology and network analyses linking gene expression to cell survival and metabolic processes. Human glioma samples were examined for levels of the ER chaperone GRP94 by immunohistochemistry and for other UPR components by Western blotting. Gene and protein expression data from patient gliomas correlated poor patient prognoses with increased expression of ER chaperones, UPR target genes, and metabolic enzymes (glycolysis and lipogenesis). NMR-based metabolomic studies revealed increased metabolic outputs in glucose uptake with elevated glycolytic activity as well as increased phospholipid turnover. Elevated levels of amino acids, antioxidants, and cholesterol were also evident upon UPR stress; in particular, recurrent tumors had overall higher lipid outputs and elevated specific UPR arms. Clonogenicity studies following temozolomide treatment of stressed or unstressed cells demonstrated UPR-induced chemoresistance. Our data characterize the UPR in glioma cells and human tumors, and

  8. Induction of the unfolded protein response drives enhanced metabolism and chemoresistance in glioma cells.

    PubMed

    Epple, Laura M; Dodd, Rebecca D; Merz, Andrea L; Dechkovskaia, Anjelika M; Herring, Matthew; Winston, Benjamin A; Lencioni, Alex M; Russell, Rae L; Madsen, Helen; Nega, Meheret; Dusto, Nathaniel L; White, Jason; Bigner, Darell D; Nicchitta, Christopher V; Serkova, Natalie J; Graner, Michael W

    2013-01-01

    The unfolded protein response (UPR) is an endoplasmic reticulum (ER)-based cytoprotective mechanism acting to prevent pathologies accompanying protein aggregation. It is frequently active in tumors, but relatively unstudied in gliomas. We hypothesized that UPR stress effects on glioma cells might protect tumors from additional exogenous stress (ie, chemotherapeutics), postulating that protection was concurrent with altered tumor cell metabolism. Using human brain tumor cell lines, xenograft tumors, human samples and gene expression databases, we determined molecular features of glioma cell UPR induction/activation, and here report a detailed analysis of UPR transcriptional/translational/metabolic responses. Immunohistochemistry, Western and Northern blots identified elevated levels of UPR transcription factors and downstream ER chaperone targets in gliomas. Microarray profiling revealed distinct regulation of stress responses between xenograft tumors and parent cell lines, with gene ontology and network analyses linking gene expression to cell survival and metabolic processes. Human glioma samples were examined for levels of the ER chaperone GRP94 by immunohistochemistry and for other UPR components by Western blotting. Gene and protein expression data from patient gliomas correlated poor patient prognoses with increased expression of ER chaperones, UPR target genes, and metabolic enzymes (glycolysis and lipogenesis). NMR-based metabolomic studies revealed increased metabolic outputs in glucose uptake with elevated glycolytic activity as well as increased phospholipid turnover. Elevated levels of amino acids, antioxidants, and cholesterol were also evident upon UPR stress; in particular, recurrent tumors had overall higher lipid outputs and elevated specific UPR arms. Clonogenicity studies following temozolomide treatment of stressed or unstressed cells demonstrated UPR-induced chemoresistance. Our data characterize the UPR in glioma cells and human tumors, and

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

    SciTech Connect

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

    2013-11-01

    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-eIF2α, 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. - Highlights: • Inorganic arsenic to humans and experimental animals disrupt innate immune responses. • The mechanism underlying arsenic impaired macrophage functions involves UPR signaling. • Chemical chaperon attenuates arsenic-mediated macrophage function impairment. • Antioxidant, NAC blocks impairment in arsenic-treated macrophage functions.

  10. Activation of the unfolded protein response promotes axonal regeneration after peripheral nerve injury.

    PubMed

    Oñate, Maritza; Catenaccio, Alejandra; Martínez, Gabriela; Armentano, Donna; Parsons, Geoffrey; Kerr, Bredford; Hetz, Claudio; Court, Felipe A

    2016-01-01

    Although protein-folding stress at the endoplasmic reticulum (ER) is emerging as a driver of neuronal dysfunction in models of spinal cord injury and neurodegeneration, the contribution of this pathway to peripheral nerve damage remains poorly explored. Here we targeted the unfolded protein response (UPR), an adaptive reaction against ER stress, in mouse models of sciatic nerve injury and found that ablation of the transcription factor XBP1, but not ATF4, significantly delay locomotor recovery. XBP1 deficiency led to decreased macrophage recruitment, a reduction in myelin removal and axonal regeneration. Conversely, overexpression of XBP1s in the nervous system in transgenic mice enhanced locomotor recovery after sciatic nerve crush, associated to an improvement in key pro-regenerative events. To assess the therapeutic potential of UPR manipulation to axonal regeneration, we locally delivered XBP1s or an shRNA targeting this transcription factor to sensory neurons of the dorsal root ganglia using a gene therapy approach and found an enhancement or reduction of axonal regeneration in vivo, respectively. Our results demonstrate a functional role of specific components of the ER proteostasis network in the cellular changes associated to regeneration and functional recovery after peripheral nerve injury. PMID:26906090

  11. Borrelidin Induces the Unfolded Protein Response in Oral Cancer Cells and Chop-Dependent Apoptosis.

    PubMed

    Sidhu, Alpa; Miller, Justin R; Tripathi, Ashootosh; Garshott, Danielle M; Brownell, Amy L; Chiego, Daniel J; Arevang, Carl; Zeng, Qinghua; Jackson, Leah C; Bechler, Shelby A; Callaghan, Michael U; Yoo, George H; Sethi, Seema; Lin, Ho-Sheng; Callaghan, Joseph H; Tamayo-Castillo, Giselle; Sherman, David H; Kaufman, Randal J; Fribley, Andrew M

    2015-11-12

    Oral squamous cell carcinoma (OSCC) is the most common cancer affecting the oral cavity, and US clinics will register about 30,000 new patients in 2015. Current treatment modalities include chemotherapy, surgery, and radiotherapy, which often result in astonishing disfigurement. Cancers of the head and neck display enhanced levels of glucose-regulated proteins and translation initiation factors associated with endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). Previous work demonstrated that chemically enforced UPR could overwhelm these adaptive features and selectively kill malignant cells. The threonyl-tRNA synthetase (ThRS) inhibitor borrelidin and two congeners were discovered in a cell-based chemical genomic screen. Borrelidin increased XBP1 splicing and led to accumulation of phosphorylated eIF2α and UPR-associated genes, prior to death in panel of OSCC cells. Murine embryonic fibroblasts (MEFs) null for GCN2 and PERK were less able to accumulate UPR markers and were resistant to borrelidin. This study demonstrates that UPR induction is a feature of ThRS inhibition and adds to a growing body of literature suggesting ThRS inhibitors might selectively target cancer cells. PMID:26617965

  12. The unfolded protein response signals through high-order assembly of Ire1

    PubMed Central

    Korennykh, Alexei V.; Egea, Pascal F.; Korostelev, Andrei A.; Finer-Moore, Janet; Zhang, Chao; Shokat, Kevan M.; Stroud, Robert M.; Walter, Peter

    2009-01-01

    Aberrant folding of proteins in the endoplasmic reticulum activates the bifunctional transmembrane kinase/endoribonuclease Ire1. Ire1 excises an intron from HAC1 messenger RNA in yeasts and Xbp1 messenger RNA in metozoans encoding homologous transcription factors. This non-conventional mRNA splicing event initiates the unfolded protein response, a transcriptional program that relieves the endoplasmic reticulum stress. Here we show that oligomerization is central to Ire1 function and is an intrinsic attribute of its cytosolic domains. We obtained the 3.2-Å crystal structure of the oligomer of the Ire1 cytosolic domains in complex with a kinase inhibitor that acts as a potent activator of the Ire1 RNase. The structure reveals a rod-shaped assembly that has no known precedence among kinases. This assembly positions the kinase domain for trans-autophosphorylation, orders the RNase domain, and creates an interaction surface for binding of the mRNA substrate. Activation of Ire1 through oligomerization expands the mechanistic repertoire of kinase-based signalling receptors. PMID:19079236

  13. Activation of the unfolded protein response promotes axonal regeneration after peripheral nerve injury

    PubMed Central

    Oñate, Maritza; Catenaccio, Alejandra; Martínez, Gabriela; Armentano, Donna; Parsons, Geoffrey; Kerr, Bredford; Hetz, Claudio; Court, Felipe A.

    2016-01-01

    Although protein-folding stress at the endoplasmic reticulum (ER) is emerging as a driver of neuronal dysfunction in models of spinal cord injury and neurodegeneration, the contribution of this pathway to peripheral nerve damage remains poorly explored. Here we targeted the unfolded protein response (UPR), an adaptive reaction against ER stress, in mouse models of sciatic nerve injury and found that ablation of the transcription factor XBP1, but not ATF4, significantly delay locomotor recovery. XBP1 deficiency led to decreased macrophage recruitment, a reduction in myelin removal and axonal regeneration. Conversely, overexpression of XBP1s in the nervous system in transgenic mice enhanced locomotor recovery after sciatic nerve crush, associated to an improvement in key pro-regenerative events. To assess the therapeutic potential of UPR manipulation to axonal regeneration, we locally delivered XBP1s or an shRNA targeting this transcription factor to sensory neurons of the dorsal root ganglia using a gene therapy approach and found an enhancement or reduction of axonal regeneration in vivo, respectively. Our results demonstrate a functional role of specific components of the ER proteostasis network in the cellular changes associated to regeneration and functional recovery after peripheral nerve injury. PMID:26906090

  14. Interaction with the unfolded protein response reveals a role for stargazin in biosynthetic AMPA receptor transport.

    PubMed

    Vandenberghe, Wim; Nicoll, Roger A; Bredt, David S

    2005-02-01

    The transmembrane protein stargazin enhances levels of functional AMPA receptors at the neuronal plasma membrane and at synapses. To clarify the mechanism for this effect, we studied trafficking of the AMPA receptor subunit glutamate receptor 1 (GluR1) in transfected COS7 cells. GluR1 expressed poorly on the surface of these cells and was primarily retained in the endoplasmic reticulum (ER). Stargazin expression strongly increased the surface fraction of GluR1. This effect was not reduced by a dominant-negative dynamin mutant, suggesting that stargazin does not inhibit AMPA receptor endocytosis. Interestingly, upregulation of ER chaperones as part of the unfolded protein response (UPR) both mimicked and occluded the effect of stargazin, suggesting a role for stargazin in ER processing of AMPA receptors. Consistent with this idea, we detected UPR induction in cerebellar granule cells lacking stargazin. Finally, residual AMPA receptor currents in stargazin-deficient neurons were suppressed by inhibition of the UPR. These findings uncover a role for stargazin in AMPA receptor trafficking through the early compartments of the biosynthetic pathway. Furthermore, they provide evidence for modulation of AMPA receptor trafficking by the UPR. PMID:15689545

  15. A chemical screen to identify inducers of the mitochondrial unfolded protein response in C. elegans.

    PubMed

    Rauthan, Manish; Pilon, Marc

    2015-01-01

    We previously showed that inhibition of the mevalonate pathway in C. elegans causes inhibition of protein prenylation, developmental arrest and lethality. We also showed that constitutive activation of the mitochondrial unfolded protein response, UPR(mt), is an effective way for C. elegans to become resistant to the negative effects of mevalonate pathway inhibition. This was an important finding since statins, a drug class prescribed to lower cholesterol levels in patients, act by inhibiting the mevalonate pathway, and it is therefore possible that some of their undesirable side effects could be alleviated by activating the UPR(mt). Here, we screened a chemical library and identified 4 compounds that specifically activated the UPR(mt). One of these compounds, methacycline hydrochloride (a tetracycline antibiotic) also protected C. elegans and mammalian cells from statin toxicity. Methacycline hydrochloride and ethidium bromide, a known UPR(mt) activator, were also tested in mice: only ethidium bromide significantly activate the UPR(mt) in skeletal muscles. PMID:27123370

  16. Hepatitis B and C virus-induced hepatitis: Apoptosis, autophagy, and unfolded protein response

    PubMed Central

    Yeganeh, Behzad; Rezaei Moghadam, Adel; Alizadeh, Javad; Wiechec, Emilia; Alavian, Seyed Moayed; Hashemi, Mohammad; Geramizadeh, Bita; Samali, Afshin; Bagheri Lankarani, Kamran; Post, Martin; Peymani, Payam; Coombs, Kevin M; Ghavami, Saeid

    2015-01-01

    AIM: To investigate the co-incidence of apoptosis, autophagy, and unfolded protein response (UPR) in hepatitis B (HBV) and C (HCV) infected hepatocytes. METHODS: We performed immunofluorescence confocal microscopy on 10 liver biopsies from HBV and HCV patients and tissue microarrays of HBV positive liver samples. We used specific antibodies for LC3β, cleaved caspase-3, BIP (GRP78), and XBP1 to detect autophagy, apoptosis and UPR, respectively. Anti-HCV NS3 and anti-HBs antibodies were also used to confirm infection. We performed triple blind counting of events to determine the co-incidence of autophagy (LC3β punctuate), apoptosis (cleaved caspase-3), and unfolded protein response (GRP78) with HBV and HCV infection in hepatocytes. All statistical analyses were performed using SPSS software for Windows (Version 16 SPSS Inc, Chicago, IL, United States). P-values < 0.05 were considered statistically significant. Statistical analyses were performed with Mann-Whitney test to compare incidence rates for autophagy, apoptosis, and UPR in HBV- and HCV-infected cells and adjacent non-infected cells. RESULTS: Our results showed that infection of hepatocytes with either HBV and HCV induces significant increase (P < 0.001) in apoptosis (cleavage of caspase-3), autophagy (LC3β punctate), and UPR (increase in GRP78 expression) in the HCV- and HBV-infected cells, as compared to non-infected cells of the same biopsy sections. Our tissue microarray immunohistochemical expression analysis of LC3β in HBVNeg and HBVPos revealed that majority of HBV-infected hepatocytes display strong positive staining for LC3β. Interestingly, although XBP splicing in HBV-infected cells was significantly higher (P < 0.05), our analyses show a slight increase of XBP splicing was in HCV-infected cells (P > 0.05). Furthermore, our evaluation of patients with HBV and HCV infection based on stage and grade of the liver diseases revealed no correlation between these pathological findings and induction of

  17. The unfolded protein response mediates reversible tau phosphorylation induced by metabolic stress

    PubMed Central

    van der Harg, J M; Nölle, A; Zwart, R; Boerema, A S; van Haastert, E S; Strijkstra, A M; Hoozemans, J JM; Scheper, W

    2014-01-01

    The unfolded protein response (UPR) is activated in neurodegenerative tauopathies such as Alzheimer's disease (AD) in close connection with early stages of tau pathology. Metabolic disturbances are strongly associated with increased risk for AD and are a potent inducer of the UPR. Here, we demonstrate that metabolic stress induces the phosphorylation of endogenous tau via activation of the UPR. Strikingly, upon restoration of the metabolic homeostasis, not only the levels of the UPR markers pPERK, pIRE1α and BiP, but also tau phosphorylation are reversed both in cell models as well as in torpor, a physiological hypometabolic model in vivo. Intervention in the UPR using the global UPR inhibitor TUDCA or a specific small-molecule inhibitor of the PERK signaling pathway, inhibits the metabolic stress-induced phosphorylation of tau. These data support a role for UPR-mediated tau phosphorylation as part of an adaptive response to metabolic stress. Failure to restore the metabolic homeostasis will lead to prolonged UPR activation and tau phosphorylation, and may thus contribute to AD pathogenesis. We demonstrate that the UPR is functionally involved in the early stages of tau pathology. Our data indicate that targeting of the UPR may be employed for early intervention in tau-related neurodegenerative diseases. PMID:25165879

  18. Polyunsaturated Branched-Chain Fatty Acid Geranylgeranoic Acid Induces Unfolded Protein Response in Human Hepatoma Cells

    PubMed Central

    Iwao, Chieko; Shidoji, Yoshihiro

    2015-01-01

    The acyclic diterpenoid acid geranylgeranoic acid (GGA) has been reported to induce autophagic cell death in several human hepatoma-derived cell lines; however, the molecular mechanism for this remains unknown. In the present study, several diterpenoids were examined for ability to induce XBP1 splicing and/or lipotoxicity for human hepatoma cell lines. Here we show that three groups of diterpenoids emerged: 1) GGA, 2,3-dihydro GGA and 9-cis retinoic acid induce cell death and XBP1 splicing; 2) all-trans retinoic acid induces XBP1 splicing but little cell death; and 3) phytanic acid, phytenic acid and geranylgeraniol induce neither cell death nor XBP1 splicing. GGA-induced ER stress/ unfolded protein response (UPR) and its lipotoxicity were both blocked by co-treatment with oleic acid. The blocking activity of oleic acid for GGA-induced XBP1 splicing was not attenuated by methylation of oleic acid. These findings strongly suggest that GGA at micromolar concentrations induces the so-called lipid-induced ER stress response/UPR, which is oleate-suppressive, and shows its lipotoxicity in human hepatoma cells. PMID:26186544

  19. Mutations in the unfolded protein response regulator ATF6 cause the cone dysfunction disorder achromatopsia.

    PubMed

    Kohl, Susanne; Zobor, Ditta; Chiang, Wei-Chieh; Weisschuh, Nicole; Staller, Jennifer; Gonzalez Menendez, Irene; Chang, Stanley; Beck, Susanne C; Garcia Garrido, Marina; Sothilingam, Vithiyanjali; Seeliger, Mathias W; Stanzial, Franco; Benedicenti, Francesco; Inzana, Francesca; Héon, Elise; Vincent, Ajoy; Beis, Jill; Strom, Tim M; Rudolph, Günther; Roosing, Susanne; Hollander, Anneke I den; Cremers, Frans P M; Lopez, Irma; Ren, Huanan; Moore, Anthony T; Webster, Andrew R; Michaelides, Michel; Koenekoop, Robert K; Zrenner, Eberhart; Kaufman, Randal J; Tsang, Stephen H; Wissinger, Bernd; Lin, Jonathan H

    2015-07-01

    Achromatopsia (ACHM) is an autosomal recessive disorder characterized by color blindness, photophobia, nystagmus and severely reduced visual acuity. Using homozygosity mapping and whole-exome and candidate gene sequencing, we identified ten families carrying six homozygous and two compound-heterozygous mutations in the ATF6 gene (encoding activating transcription factor 6A), a key regulator of the unfolded protein response (UPR) and cellular endoplasmic reticulum (ER) homeostasis. Patients had evidence of foveal hypoplasia and disruption of the cone photoreceptor layer. The ACHM-associated ATF6 mutations attenuate ATF6 transcriptional activity in response to ER stress. Atf6(-/-) mice have normal retinal morphology and function at a young age but develop rod and cone dysfunction with increasing age. This new ACHM-related gene suggests a crucial and unexpected role for ATF6A in human foveal development and cone function and adds to the list of genes that, despite ubiquitous expression, when mutated can result in an isolated retinal photoreceptor phenotype. PMID:26029869

  20. Celastrol induces unfolded protein response-dependent cell death in head and neck cancer

    PubMed Central

    Fribley, Andrew M.; Miller, Justin R.; Brownell, Amy L.; Garshott, Danielle M.; Zeng, Qinghua; Reist, Tyler E.; Narula, Neha; Cai, Peter; Xi, Yue; Callaghan, Michael U.; Kodali, Vamsi; Kaufman, Randal J.

    2014-01-01

    The survival rate for patients with oral squamous cell carcinoma (OSCC) has not seen marked improvement in recent decades despite enhanced efforts in prevention and the introduction of novel therapies. We have reported that pharmacological exacerbation of the unfolded protein response (UPR) is an effective approach to killing OSCC cells. The UPR is executed via distinct signaling cascades whereby an initial attempt to restore folding homeostasis in the endoplasmic reticulum during stress is complemented by an apoptotic response if the defect cannot be resolved. To identify novel small molecules able to overwhelm the adaptive capacity of the UPR in OSCC cells, we engineered a complementary cell-based assay to screen a broad spectrum of chemical matter. Stably transfected CHO-K1 cells that individually report (luciferase) on the PERK/eIF2α/ATF4/CHOP (apoptotic) or the IRE1/XBP1 (adaptive) UPR pathways, were engineered [1]. The triterpenoids dihydrocelastrol and celastrol were identified as potent inducers of UPR signaling and cell death in a primary screen and confirmed in a panel of OSCC cells and other cancer cell lines. Biochemical and genetic assays using OSCC cells and modified murine embryonic fibroblasts demonstrated that intact PERK-eIF2–ATF4-CHOP signaling is required for pro-apoptotic UPR and OSCC death following celastrol treatment. PMID:25139619

  1. Mutations in the unfolded protein response regulator ATF6 cause the cone dysfunction disorder achromatopsia

    PubMed Central

    Kohl, Susanne; Zobor, Ditta; Chiang, Wei-Chieh; Weisschuh, Nicole; Staller, Jennifer; Menendez, Irene Gonzalez; Chang, Stanley; Beck, Susanne C; Garrido, Marina Garcia; Sothilingam, Vithiyanjali; Seeliger, Mathias W; Stanzial, Franco; Benedicenti, Francesco; Inzana, Francesca; Héon, Elise; Vincent, Ajoy; Beis, Jill; Strom, Tim M; Rudolph, Günther; Roosing, Susanne; den Hollander, Anneke I; Cremers, Frans P M; Lopez, Irma; Ren, Huanan; Moore, Anthony T; Webster, Andrew R; Michaelides, Michel; Koenekoop, Robert K; Zrenner, Eberhart; Kaufman, Randal J; Tsang, Stephen H; Wissinger, Bernd; Lin, Jonathan H

    2015-01-01

    Achromatopsia (ACHM) is an autosomal recessive disorder characterized by color blindness, photophobia, nystagmus and severely reduced visual acuity. Using homozygosity mapping and whole-exome and candidate gene sequencing, we identified ten families carrying six homozygous and two compound-heterozygous mutations in the ATF6 gene (encoding activating transcription factor 6A), a key regulator of the unfolded protein response (UPR) and cellular endoplasmic reticulum (ER) homeostasis. Patients had evidence of foveal hypoplasia and disruption of the cone photoreceptor layer. The ACHM-associated ATF6 mutations attenuate ATF6 transcriptional activity in response to ER stress. Atf6−/− mice have normal retinal morphology and function at a young age but develop rod and cone dysfunction with increasing age. This new ACHM-related gene suggests a crucial and unexpected role for ATF6A in human foveal development and cone function and adds to the list of genes that, despite ubiquitous expression, when mutated can result in an isolated retinal photoreceptor phenotype. PMID:26029869

  2. Identification and characterization of a mitochondrial unfolded protein response transcription factor ATFS-1 in Litopenaeus vannamei.

    PubMed

    Chen, Yong-Gui; Yue, Hai-Tao; Zhang, Ze-Zhi; Yuan, Feng-Hua; Bi, Hai-Tao; Yuan, Kai; Weng, Shao-Ping; He, Jian-Guo; Chen, Yi-Hong

    2016-07-01

    A mitochondrial specific stress response termed mitochondrial unfolded protein response (UPR(mt)) is activated in responding to disturbance of protein homeostasis in mitochondria. The activating transcription factor associated with stress-1 (designated as ATFS-1) is the key regulator of UPR(mt). To investigating the roles of ATFS-1 (LvATFS-1) in Litopenaeus vannamei mitochondrial stress remission and immunity, it's full length cDNA was cloned. The open reading frame of LvATFS-1 was 1, 557 bp in length, deducing to a 268 amino acids protein. LvATFS-1 was highly expressed in muscle, hemocytes and eyestalk. Subcellular location assays showed that N-terminal of LvATFS-1 contained a mitochondrial targeting sequence, which could directed the fused EGFP located to mitochondria. And the C-terminal of LvATFS-1, which had a nuclear localization signal, expressed in nucleus. The in vitro experiments verified that LvATFS-1 could reduced the level of intracellular reactive oxygen species (ROS). And results of real-time RT-PCR indicated that LvATFS-1 might scavenge excess ROS via ROS-eliminating genes regulation. Reporter gene assays showed that LvATFS-1 could upregulated the expression of the antimicrobial peptide genes in Drosophila Schneider 2 cells. Results of real-time RT-PCR showed that Vibrio alginolyticus or white spot syndrome virus (WSSV) infection induced the expression of LvATFS-1. And knocked-down LvATFS-1 by RNAi resulted in a higher cumulative mortality of L. vannamei upon V. alginolyticus or WSSV infection. These results suggested that LvATFS-1 not only rolled in mitochondrial specific stress responding, but also important for L. vannamei immunologic defence. PMID:26481519

  3. Signal peptide peptidase functions in ERAD to cleave the unfolded protein response regulator XBP1u.

    PubMed

    Chen, Chia-yi; Malchus, Nicole S; Hehn, Beate; Stelzer, Walter; Avci, Dönem; Langosch, Dieter; Lemberg, Marius K

    2014-11-01

    Signal peptide peptidase (SPP) catalyzes intramembrane proteolysis of signal peptides at the endoplasmic reticulum (ER), but has also been suggested to play a role in ER-associated degradation (ERAD). Here, we show that SPP forms a complex with the ERAD factor Derlin1 and the E3 ubiquitin ligase TRC8 to cleave the unfolded protein response (UPR) regulator XBP1u. Cleavage occurs within a so far unrecognized type II transmembrane domain, which renders XBP1u as an SPP substrate through specific sequence features. Additionally, Derlin1 acts in the complex as a substrate receptor by recognizing the luminal tail of XBP1u. Remarkably, this interaction of Derlin1 with XBP1u obviates the need for ectodomain shedding prior to SPP cleavage, commonly required for intramembrane cuts. Furthermore, we show that XBP1u inhibits the UPR transcription factor XBP1s by targeting it toward proteasomal degradation. Thus, we identify an ERAD complex that controls the abundance of XBP1u and thereby tunes signaling through the UPR. PMID:25239945

  4. Signal peptide peptidase functions in ERAD to cleave the unfolded protein response regulator XBP1u

    PubMed Central

    Chen, Chia-yi; Malchus, Nicole S; Hehn, Beate; Stelzer, Walter; Avci, Dönem; Langosch, Dieter; Lemberg, Marius K

    2014-01-01

    Signal peptide peptidase (SPP) catalyzes intramembrane proteolysis of signal peptides at the endoplasmic reticulum (ER), but has also been suggested to play a role in ER-associated degradation (ERAD). Here, we show that SPP forms a complex with the ERAD factor Derlin1 and the E3 ubiquitin ligase TRC8 to cleave the unfolded protein response (UPR) regulator XBP1u. Cleavage occurs within a so far unrecognized type II transmembrane domain, which renders XBP1u as an SPP substrate through specific sequence features. Additionally, Derlin1 acts in the complex as a substrate receptor by recognizing the luminal tail of XBP1u. Remarkably, this interaction of Derlin1 with XBP1u obviates the need for ectodomain shedding prior to SPP cleavage, commonly required for intramembrane cuts. Furthermore, we show that XBP1u inhibits the UPR transcription factor XBP1s by targeting it toward proteasomal degradation. Thus, we identify an ERAD complex that controls the abundance of XBP1u and thereby tunes signaling through the UPR. PMID:25239945

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

    SciTech Connect

    Liu, Bao-Qin; Gao, Yan-Yan; Niu, Xiao-Fang; Xie, Ji-Sheng; Meng, Xin; Guan, Yifu; Wang, Hua-Qin

    2010-01-01

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

  6. The Mitochondrial Unfolded Protein Response Protects against Anoxia in Caenorhabditis elegans

    PubMed Central

    Peña, Salvador; Sherman, Teresa; Brookes, Paul S.; Nehrke, Keith

    2016-01-01

    The mitochondrial unfolded protein response (UPRmt) is a surveillance pathway that defends proteostasis in the “powerhouse” of the cell. Activation of the UPRmt protects against stresses imposed by reactive oxygen species, respiratory chain deficits, and pathologic bacteria. Consistent with the UPRmt’s role in adaption, we found that either its pharmacological or genetic activation by ethidium bromide (EtBr) or RNAi of the mitochondrial AAA-protease spg-7 was sufficient to reduce death in an anoxia-based Caenorhabditis elegans model of ischemia-reperfusion injury. The UPRmt-specific transcription factor atfs-1 was necessary for protection and atfs-1 gain-of-function (gf) mutants were endogenously protected from both death and dysfunction. Neurons exhibited less axonal degeneration following non-lethal anoxia-reperfusion (A-R) when the UPRmt was pre-activated, and consistent with the concept of mitochondrial stress leading to cell non-autonomous (ie. “remote”) effects, we found that restricted activation of the UPRmt in neurons decreased A-R death. However, expression of the atfs-1(gf) mutant in neurons, which resulted in a robust activation of a neuronal UPRmt, did not upregulate the UPRmt in distal tissues, nor did it protect the worms from A-R toxicity. These findings suggest that remote signaling requires additional component(s) acting downstream of de facto mitochondrial stress. PMID:27459203

  7. Unfolded protein response activation reduces secretion and extracellular aggregation of amyloidogenic immunoglobulin light chain

    PubMed Central

    Cooley, Christina B.; Ryno, Lisa M.; Plate, Lars; Morgan, Gareth J.; Hulleman, John D.; Kelly, Jeffery W.; Wiseman, R. Luke

    2014-01-01

    Light-chain amyloidosis (AL) is a degenerative disease characterized by the extracellular aggregation of a destabilized amyloidogenic Ig light chain (LC) secreted from a clonally expanded plasma cell. Current treatments for AL revolve around ablating the cancer plasma cell population using chemotherapy regimens. Unfortunately, this approach is limited to the ∼70% of patients who do not exhibit significant organ proteotoxicity and can tolerate chemotherapy. Thus, identifying new therapeutic strategies to alleviate LC organ proteotoxicity should allow AL patients with significant cardiac and/or renal involvement to subsequently tolerate established chemotherapy treatments. Using a small-molecule screening approach, the unfolded protein response (UPR) was identified as a cellular signaling pathway whose activation selectively attenuates secretion of amyloidogenic LC, while not affecting secretion of a nonamyloidogenic LC. Activation of the UPR-associated transcription factors XBP1s and/or ATF6 in the absence of stress recapitulates the selective decrease in amyloidogenic LC secretion by remodeling the endoplasmic reticulum proteostasis network. Stress-independent activation of XBP1s, or especially ATF6, also attenuates extracellular aggregation of amyloidogenic LC into soluble aggregates. Collectively, our results show that stress-independent activation of these adaptive UPR transcription factors offers a therapeutic strategy to reduce proteotoxicity associated with LC aggregation. PMID:25157167

  8. The Mitochondrial Unfolded Protein Response Protects against Anoxia in Caenorhabditis elegans.

    PubMed

    Peña, Salvador; Sherman, Teresa; Brookes, Paul S; Nehrke, Keith

    2016-01-01

    The mitochondrial unfolded protein response (UPRmt) is a surveillance pathway that defends proteostasis in the "powerhouse" of the cell. Activation of the UPRmt protects against stresses imposed by reactive oxygen species, respiratory chain deficits, and pathologic bacteria. Consistent with the UPRmt's role in adaption, we found that either its pharmacological or genetic activation by ethidium bromide (EtBr) or RNAi of the mitochondrial AAA-protease spg-7 was sufficient to reduce death in an anoxia-based Caenorhabditis elegans model of ischemia-reperfusion injury. The UPRmt-specific transcription factor atfs-1 was necessary for protection and atfs-1 gain-of-function (gf) mutants were endogenously protected from both death and dysfunction. Neurons exhibited less axonal degeneration following non-lethal anoxia-reperfusion (A-R) when the UPRmt was pre-activated, and consistent with the concept of mitochondrial stress leading to cell non-autonomous (ie. "remote") effects, we found that restricted activation of the UPRmt in neurons decreased A-R death. However, expression of the atfs-1(gf) mutant in neurons, which resulted in a robust activation of a neuronal UPRmt, did not upregulate the UPRmt in distal tissues, nor did it protect the worms from A-R toxicity. These findings suggest that remote signaling requires additional component(s) acting downstream of de facto mitochondrial stress. PMID:27459203

  9. Nicotinamide mononucleotide adenylyltransferase promotes hypoxic survival by activating the mitochondrial unfolded protein response

    PubMed Central

    Mao, X R; Kaufman, D M; Crowder, C M

    2016-01-01

    Gain-of-function mutations in the mouse nicotinamide mononucleotide adenylyltransferase type 1 (Nmnat1) produce two remarkable phenotypes: protection against traumatic axonal degeneration and reduced hypoxic brain injury. Despite intensive efforts, the mechanism of Nmnat1 cytoprotection remains elusive. To develop a new model to define this mechanism, we heterologously expressed a mouse Nmnat1 non-nuclear-localized gain-of-function mutant gene (m-nonN-Nmnat1) in the nematode Caenorhabditis elegans and show that it provides protection from both hypoxia-induced animal death and taxol-induced axonal pathology. Additionally, we find that m-nonN-Nmnat1 significantly lengthens C. elegans lifespan. Using the hypoxia-protective phenotype in C. elegans, we performed a candidate screen for genetic suppressors of m-nonN-Nmnat1 cytoprotection. Loss of function in two genes, haf-1 and dve-1, encoding mitochondrial unfolded protein response (mitoUPR) factors were identified as suppressors. M-nonN-Nmnat1 induced a transcriptional reporter of the mitoUPR gene hsp-6 and provided protection from the mitochondrial proteostasis toxin ethidium bromide. M-nonN-Nmnat1 was also protective against axonal degeneration in C. elegans induced by the chemotherapy drug taxol. Taxol markedly reduced basal expression of a mitoUPR reporter; the expression was restored by m-nonN-Nmnat1. Taken together, these data implicate the mitoUPR as a mechanism whereby Nmnat1 protects from hypoxic and axonal injury. PMID:26913604

  10. Lysosomes and unfolded protein response, determinants of differential resistance of melanoma cells to vinca alkaloids.

    PubMed

    Vincent, Laure-Anais; Attaoua, Chaker; Bellis, Michel; Rozkydalova, Lucie; Hadj-Kaddour, Kamel; Vian, Laurence; Cuq, Pierre

    2015-04-01

    On account of its strong ability to become chemoresistant after a primary response to drugs, malignant melanoma (MM) remains a therapeutic challenge. This study focuses on acquired resistance to vinca alkaloids (VAs) using VA-resistant MM cell lines (CAL1R-VCR, CAL1R-VDS, and CAL1R-VRB), established by long-term continuous exposure of parental CAL1-wt cells to vincristine (VCR), vindesine (VDS), or vinorelbine (VRB), respectively. Transcriptomic profiling using rma and rdam methods led to distinguish two cell groups: CAL1R-VCR and CAL1R-VDS, CAL1R-VRB, and CAL1-wt. mgsa of the specifically altered genes in the first group evidenced the GO terms 'lysosomal lumen' and 'vacuolar lumen' linked to underexpressed genes, and 'endoplasmic reticulum (ER) stress response' associated with overexpressed genes. A specific reduction of lysosomal enzymes, independent of acidic vacuole organelle (AVO) turnover, was observed (LTG probe) in CAL1R-VCR and CAL1R-VDS cells. It was associated with the specific lowering of cathepsin B and L, known to be involved in the lysosomal pathway of apoptosis. Confirming gene profiling, the same groups (CAL1R-VCR and CAL1R-VDS, CAL1-wt and CAL1R-VRB) could be distinguished regarding the VA-mediated changes on mean size areas and on acidic compartment volumes. These two parameters were reduced in CAL1R-VCR and CAL1R-VDS cells, suggesting a smaller AVO accumulation and thus a reduced sensitivity to lysosomal membrane permeabilization-mediated apoptosis. In addition, 'ER stress response' inhibition by tauroursodeoxycholic acid induced a higher VA sensitization of the first cell group. In conclusion, lysosomes and unfolded protein response could be key determinants of the differential resistance of MM to VAs. PMID:25601431

  11. Dominant negative FADD dissipates the proapoptotic signalosome of the unfolded protein response in diabetic embryopathy.

    PubMed

    Wang, Fang; Weng, Hongbo; Quon, Michael J; Yu, Jingwen; Wang, Jian-Ying; Hueber, Anne-Odile; Yang, Peixin

    2015-11-15

    Endoplasmic reticulum (ER) stress and caspase 8-dependent apoptosis are two interlinked causal events in maternal diabetes-induced neural tube defects (NTDs). The inositol-requiring enzyme 1α (IRE1α) signalosome mediates the proapoptotic effect of ER stress. Diabetes increases tumor necrosis factor receptor type 1R-associated death domain (TRADD) expression. Here, we revealed two new unfolded protein response (UPR) regulators, TRADD and Fas-associated protein with death domain (FADD). TRADD interacted with both the IRE1α-TRAF2-ASK1 complex and FADD. In vivo overexpression of a FADD dominant negative (FADD-DN) mutant lacking the death effector domain disrupted diabetes-induced IRE1α signalosome and suppressed ER stress and caspase 8-dependent apoptosis, leading to NTD prevention. FADD-DN abrogated ER stress markers and blocked the JNK1/2-ASK1 pathway. Diabetes-induced mitochondrial translocation of proapoptotic Bcl-2 members mitochondrial dysfunction and caspase cleavage were also alleviated by FADD-DN. In vitro TRADD overexpression triggered UPR and ER stress before manifestation of caspase 3 and caspase 8 cleavage and apoptosis. FADD-DN overexpression repressed high glucose- or TRADD overexpression-induced IRE1α phosphorylation, its downstream proapoptotic kinase activation and endonuclease activities, and apoptosis. FADD-DN also attenuated tunicamycin-induced UPR and ER stress. These findings suggest that TRADD participates in the IRE1α signalosome and induces UPR and ER stress and that the association between TRADD and FADD is essential for diabetes- or high glucose-induced UPR and ER stress. PMID:26419589

  12. Keratin 12 missense mutation induces the unfolded protein response and apoptosis in Meesmann epithelial corneal dystrophy

    PubMed Central

    Allen, Edwin H.A.; Courtney, David G.; Atkinson, Sarah D.; Moore, Johnny E.; Mairs, Laura; Poulsen, Ebbe Toftgaard; Schiroli, Davide; Maurizi, Eleonora; Cole, Christian; Hickerson, Robyn P.; James, John; Murgatroyd, Helen; Smith, Frances J.D.; MacEwen, Carrie; Enghild, Jan J.; Nesbit, M. Andrew; Leslie Pedrioli, Deena M.; McLean, W.H. Irwin; Moore, C.B. Tara

    2016-01-01

    Meesmann epithelial corneal dystrophy (MECD) is a rare autosomal dominant disorder caused by dominant-negative mutations within the KRT3 or KRT12 genes, which encode the cytoskeletal protein keratins K3 and K12, respectively. To investigate the pathomechanism of this disease, we generated and phenotypically characterized a novel knock-in humanized mouse model carrying the severe, MECD-associated, K12-Leu132Pro mutation. Although no overt changes in corneal opacity were detected by slit-lamp examination, the corneas of homozygous mutant mice exhibited histological and ultrastructural epithelial cell fragility phenotypes. An altered keratin expression profile was observed in the cornea of mutant mice, confirmed by western blot, RNA-seq and quantitative real-time polymerase chain reaction. Mass spectrometry (MS) and immunohistochemistry demonstrated a similarly altered keratin profile in corneal tissue from a K12-Leu132Pro MECD patient. The K12-Leu132Pro mutation results in cytoplasmic keratin aggregates. RNA-seq analysis revealed increased chaperone gene expression, and apoptotic unfolded protein response (UPR) markers, CHOP and Caspase 12, were also increased in the MECD mice. Corneal epithelial cell apoptosis was increased 17-fold in the mutant cornea, compared with the wild-type (P < 0.001). This elevation of UPR marker expression was also observed in the human MECD cornea. This is the first reporting of a mouse model for MECD that recapitulates the human disease and is a valuable resource in understanding the pathomechanism of the disease. Although the most severe phenotype is observed in the homozygous mice, this model will still provide a test-bed for therapies not only for corneal dystrophies but also for other keratinopathies caused by similar mutations. PMID:26758872

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

    PubMed

    Carpenter, John E; Grose, Charles

    2014-01-01

    Varicella-zoster virus (VZV) is a human herpesvirus that spreads to children as varicella or chicken pox. The virus then establishes latency in the nervous system and re-emerges, typically decades later, as zoster or shingles. We have reported previously that VZV induces autophagy in infected cells as well as exhibiting evidence of the Unfolded Protein Response (UPR): XBP1 splicing, a greatly expanded Endoplasmic Reticulum (ER) and CHOP expression. Herein we report the results of a UPR specific PCR array that measures the levels of mRNA of 84 different components of the UPR in VZV infected cells as compared to tunicamycin treated cells as a positive control and uninfected, untreated cells as a negative control. Tunicamycin is a mixture of chemicals that inhibits N-linked glycosylation in the ER with resultant protein misfolding and the UPR. We found that VZV differentially induces the UPR when compared to tunicamycin treatment. For example, tunicamycin treatment moderately increased (8-fold) roughly half of the array elements while downregulating only three (one ERAD and two FOLD components). VZV infection on the other hand upregulated 33 components including a little described stress sensor CREB-H (64-fold) as well as ER membrane components INSIG and gp78, which modulate cholesterol synthesis while downregulating over 20 components mostly associated with ERAD and FOLD. We hypothesize that this expression pattern is associated with an expanding ER with downregulation of active degradation by ERAD and apoptosis as the cell attempts to handle abundant viral glycoprotein synthesis. PMID:25071735

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

    PubMed Central

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

    2011-01-01

    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. PMID:22028791

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

    PubMed Central

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

    2014-01-01

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

  16. Maintenance and propagation of a deleterious mitochondrial genome by the mitochondrial unfolded protein response.

    PubMed

    Lin, Yi-Fan; Schulz, Anna M; Pellegrino, Mark W; Lu, Yun; Shaham, Shai; Haynes, Cole M

    2016-05-19

    Mitochondrial genomes (mitochondrial DNA, mtDNA) encode essential oxidative phosphorylation (OXPHOS) components. Because hundreds of mtDNAs exist per cell, a deletion in a single mtDNA has little impact. However, if the deletion genome is enriched, OXPHOS declines, resulting in cellular dysfunction. For example, Kearns-Sayre syndrome is caused by a single heteroplasmic mtDNA deletion. More broadly, mtDNA deletion accumulation has been observed in individual muscle cells and dopaminergic neurons during ageing. It is unclear how mtDNA deletions are tolerated or how they are propagated in somatic cells. One mechanism by which cells respond to OXPHOS dysfunction is by activating the mitochondrial unfolded protein response (UPR(mt)), a transcriptional response mediated by the transcription factor ATFS-1 that promotes the recovery and regeneration of defective mitochondria. Here we investigate the role of ATFS-1 in the maintenance and propagation of a deleterious mtDNA in a heteroplasmic Caenorhabditis elegans strain that stably expresses wild-type mtDNA and mtDNA with a 3.1-kilobase deletion (∆mtDNA) lacking four essential genes. The heteroplasmic strain, which has 60% ∆mtDNA, displays modest mitochondrial dysfunction and constitutive UPR(mt) activation. ATFS-1 impairment reduced the ∆mtDNA nearly tenfold, decreasing the total percentage to 7%. We propose that in the context of mtDNA heteroplasmy, UPR(mt) activation caused by OXPHOS defects propagates or maintains the deleterious mtDNA in an attempt to recover OXPHOS activity by promoting mitochondrial biogenesis and dynamics. PMID:27135930

  17. Endoplasmic Reticulum Stress, Unfolded Protein Response and Altered T Cell Differentiation in Necrotizing Enterocolitis

    PubMed Central

    Lu, Peng; Struijs, Marie-Chantal; Mei, Jiaping; Witte-Bouma, Janneke; Korteland-van Male, Anita M.; de Bruijn, Adrianus C. J. M.; van Goudoever, Johannes B.; Renes, Ingrid B.

    2013-01-01

    Background Endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) play important roles in chronic intestinal inflammation. Necrotizing enterocolitis (NEC) is the most common gastrointestinal emergency in preterm infants and is characterized by acute intestinal inflammation and necrosis. The objective of the study is to investigate the role of ER stress and the UPR in NEC patients. Methods Ileal tissues from NEC and control patients were obtained during surgical resection and/or at stoma closure. Splicing of XBP1 was detected using PCR, and gene expression was quantified using qPCR and Western blot. Results Splicing of XBP1 was only detected in a subset of acute NEC (A-NEC) patients, and not in NEC patients who had undergone reanastomosis (R-NEC). The other ER stress and the UPR pathways, PERK and ATF6, were not activated in NEC patients. A-NEC patients showing XBP1 splicing (A-NEC-XBP1s) had increased mucosal expression of GRP78, CHOP, IL6 and IL8. Similar results were obtained by inducing ER stress and the UPR in vitro. A-NEC-XBP1s patients showed altered T cell differentiation indicated by decreased mucosal expression of RORC, IL17A and FOXP3. A-NEC-XBP1s patients additionally showed more severe morphological damage and a worse surgical outcome. Compared with A-NEC patients, R-NEC patients showed lower mucosal IL6 and IL8 expression and higher mucosal FOXP3 expression. Conclusions XBP1 splicing, ER stress and the UPR in NEC are associated with increased IL6 and IL8 expression levels, altered T cell differentiation and severe epithelial injury. PMID:24194940

  18. Effect of exercise intensity on unfolded protein response in skeletal muscle of rat.

    PubMed

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

    2014-06-01

    Endoplasmic reticulum (ER) stress, unfolded protein response (UPR), and mitochondrial biogenesis were assessed following varying intensities of exercise training. The animals were randomly assigned to receive either low- (LIT, n=7) or high intensity training (HIT, n=7), or were assigned to a control group (n=7). Over 5 weeks, the animals in the LIT were exercised on a treadmill with a 10° incline for 60 min at a speed of 20 m/min group, and in the HIT group at a speed of 34 m/min for 5 days a week. No statistically significant differences were found in the body weight, plasma triglyceride, and total cholesterol levels across the three groups, but fasting glucose and insulin levels were significantly lower in the exercise-trained groups. Additionally, no statistically significant differences were observed in the levels of PERK phosphorylation in skeletal muscles between the three groups. However, compared to the control and LIT groups, the level of BiP was lower in the HIT group. Compared to the control group, the levels of ATF4 in skeletal muscles and CHOP were significantly lower in the HIT group. The HIT group also showed increased PGC-1α mRNA expression in comparison with the control group. Furthermore, both of the trained groups showed higher levels of mitochondrial UCP3 than the control group. In summary, we found that a 5-week high-intensity exercise training routine resulted in increased mitochondrial biogenesis and decreased ER stress and apoptotic signaling in the skeletal muscle tissue of rats. PMID:24976760

  19. Rotavirus Infection Induces the Unfolded Protein Response of the Cell and Controls It through the Nonstructural Protein NSP3▿

    PubMed Central

    Trujillo-Alonso, Vicenta; Maruri-Avidal, Liliana; Arias, Carlos F.; López, Susana

    2011-01-01

    The unfolded protein response (UPR) is a cellular mechanism that is triggered in order to cope with the stress caused by the accumulation of misfolded proteins in the endoplasmic reticulum (ER). This response is initiated by the endoribonuclease inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6), and PKR-like ER kinase, which increase the expression of the genes involved in the folding and degradation processes and decrease the protein input into the ER by inhibiting translation. It has been shown that viruses both induce and manipulate the UPR in order to protect the host cells from an ER stress-mediated death, thus permitting the translation of viral proteins and the efficient replication of the virus. To understand the cellular events that occur during the rotavirus replication cycle, we examined the activation of the three UPR arms following infection, using luciferase reporters driven by promoters of the ER stress-responsive genes and real-time reverse transcription-PCR to determine the levels of the stress-induced mRNAs. Our findings indicated that during rotavirus infection two of the three arms of the UPR (IRE1 and ATF6) become activated; however, these pathways are interrupted at the translational level by the general inhibition of protein synthesis caused by NSP3. This response seems to be triggered by more than one viral protein synthesized during the replication of the virus, but not by the viral double-stranded RNA (dsRNA), since cells transfected with psoralen-inactivated virions, or with naked viral dsRNA, did not induce UPR. PMID:21937647

  20. Nitric Oxide and Protein Disulfide Isomerase Explain the Complexities of Unfolded Protein Response Following Intra-hippocampal Aβ Injection.

    PubMed

    Khodagholi, Fariba; Digaleh, Hadi; Motamedi, Fereshteh; Foolad, Forough; Shaerzadeh, Fatemeh

    2016-08-01

    Several pathways involved in regulation of intracellular protein integrity are known as the protein quality control (PQC) system. Molecular chaperones as the main players are engaged in various aspects of PQC system. According to the importance of these proteins in cell survival, in the present study, we traced endoplasmic reticulum-specific markers and chaperone-mediated autophagy (CMA)-associated factors as two main arms of PQC system in intra-hippocampal amyloid beta (Aβ)-injected rats during 10 days running. Data analysis from Western blot indicated that exposure to Aβ activates immunoglobulin heavy-chain-binding protein (Bip) which is the upstream regulator of unfolded protein responses (UPR). Activation of UPR system eventually led to induction of pro-apoptotic factors like CHOP, calpain, and caspase-12. Moreover, our data revealed that protein disulfide isomerase activity dramatically decreased after Aβ injection, which could be attributed to the increased levels of nitric oxide. Besides, Aβ injection induced levels of 2 members of heat shock proteins (Hsp) 70 and 90. Elevated levels of Hsps family members are accompanied by increased levels of lysosome-associated membrane protein type-2A (Lamp-2A) that are involved in CMA. Despite the reduction in CHOP, calpain, caspase-12, and Lamp-2A protein levels, the levels of molecular chaperones Bip, Hsps70, and 90 increased 10 days after Aβ injection in comparison to the control group. Based on our results, 10 days after Aβ injection, despite the activation of protective chaperones, markers associated with neurotoxicity were still elevated. PMID:26391027

  1. TDP-43 toxicity is mediated by the unfolded protein response-unrelated induction of C/EBP homologous protein expression.

    PubMed

    Suzuki, Hiroaki; Matsuoka, Masaaki

    2012-03-01

    Transactive response DNA-binding protein-43 (TDP-43) neuronal toxicity plays an essential role in the pathogenesis of amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions. In our previous study, we showed that low-grade overexpression of TDP-43, which is thought to mimic the gain-of-function of TDP-43, caused neuronal death, mediated by the upregulation of Bim and the downregulation of Bcl-xL in vitro. In this study, we show that TDP-43 overexpression caused the upregulation of C/EBP-homologous protein (CHOP) and that disruption of the CHOP gene markedly attenuated TDP-43-induced cell death. These results indicate that increases in CHOP expression contribute to TDP-43-induced cell death. We also show that the TDP-43-induced upregulation of CHOP expression is mediated by both the upregulation of the mRNA level of CHOP and the attenuation of thedegradation of CHOP, which is independent on the PERK/eIF2α/ATF4 or other pathway related to the unfolded protein response (UPR) to endoplasmic reticulum stress. This study provides the first example of the CHOP-mediated cell death that is independent of the UPR. © 2011 Wiley Periodicals, Inc. PMID:22057717

  2. Newcastle disease virus NP and P proteins induce autophagy via the endoplasmic reticulum stress-related unfolded protein response

    PubMed Central

    Cheng, Jing-Hua; Sun, Ying-Jie; Zhang, Fan-Qing; Zhang, Xiao-Rong; Qiu, Xv-Sheng; Yu, Li-Ping; Wu, Yan-Tao; Ding, Chan

    2016-01-01

    Newcastle disease virus (NDV) can replicate and trigger autophagy in human tumor cells. Our previous study confirmed the critical role of autophagy in NDV infection. Here we studied the role of NDV structural proteins in the induction of autophagy through endoplasmic reticulum (ER) stress-related unfolded protein response (UPR) pathways. Ectopic expression of the NDV nucleocapsid protein (NP) or phosphoprotein (P) was sufficient to induce autophagy. NP or P expression also altered ER homeostasis. The PERK and ATF6 pathways, but not the XBP1 pathway, all of which are components of the UPR, were activated in both NDV-infected and NP or P-transfected cells. Knockdown of PERK or ATF6 inhibited NDV-induced autophagy and reduced the extent of NDV replication. Collectively, these data suggest not only roles for the NDV NP and P proteins in autophagy, but also offer new insights into the mechanisms of NDV-induced autophagy through activation of the ER stress-related UPR pathway. PMID:27097866

  3. A Novel Link between Fic (Filamentation Induced by cAMP)-mediated Adenylylation/AMPylation and the Unfolded Protein Response*

    PubMed Central

    Sanyal, Anwesha; Chen, Andy J.; Nakayasu, Ernesto S.; Lazar, Cheri S.; Zbornik, Erica A.; Worby, Carolyn A.; Koller, Antonius; Mattoo, Seema

    2015-01-01

    The maintenance of endoplasmic reticulum (ER) homeostasis is a critical aspect of determining cell fate and requires a properly functioning unfolded protein response (UPR). We have discovered a previously unknown role of a post-translational modification termed adenylylation/AMPylation in regulating signal transduction events during UPR induction. A family of enzymes, defined by the presence of a Fic (filamentation induced by cAMP) domain, catalyzes this adenylylation reaction. The human genome encodes a single Fic protein, called HYPE (Huntingtin yeast interacting protein E), with adenylyltransferase activity but unknown physiological target(s). Here, we demonstrate that HYPE localizes to the lumen of the endoplasmic reticulum via its hydrophobic N terminus and adenylylates the ER molecular chaperone, BiP, at Ser-365 and Thr-366. BiP functions as a sentinel for protein misfolding and maintains ER homeostasis. We found that adenylylation enhances BiP's ATPase activity, which is required for refolding misfolded proteins while coping with ER stress. Accordingly, HYPE expression levels increase upon stress. Furthermore, siRNA-mediated knockdown of HYPE prevents the induction of an unfolded protein response. Thus, we identify HYPE as a new UPR regulator and provide the first functional data for Fic-mediated adenylylation in mammalian signaling. PMID:25601083

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

    PubMed

    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

    2011-09-01

    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/eIF2α/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

  5. Unfolded Protein Response (UPR) Regulator Cib1 Controls Expression of Genes Encoding Secreted Virulence Factors in Ustilago maydis.

    PubMed

    Hampel, Martin; Jakobi, Mareike; Schmitz, Lara; Meyer, Ute; Finkernagel, Florian; Doehlemann, Gunther; Heimel, Kai

    2016-01-01

    The unfolded protein response (UPR), a conserved eukaryotic signaling pathway to ensure protein homeostasis in the endoplasmic reticulum (ER), coordinates biotrophic development in the corn smut fungus Ustilago maydis. Exact timing of UPR activation is required for virulence and presumably connected to the elevated expression of secreted effector proteins during infection of the host plant Zea mays. In the baker's yeast Saccharomyces cerevisiae, expression of UPR target genes is induced upon binding of the central regulator Hac1 to unfolded protein response elements (UPREs) in their promoters. While a role of the UPR in effector secretion has been described previously, we investigated a potential UPR-dependent regulation of genes encoding secreted effector proteins. In silico prediction of UPREs in promoter regions identified the previously characterized effector genes pit2 and tin1-1, as bona fide UPR target genes. Furthermore, direct binding of the Hac1-homolog Cib1 to the UPRE containing promoter fragments of both genes was confirmed by quantitative chromatin immunoprecipitation (qChIP) analysis. Targeted deletion of the UPRE abolished Cib1-dependent expression of pit2 and significantly affected virulence. Furthermore, ER stress strongly increased Pit2 expression and secretion. This study expands the role of the UPR as a signal hub in fungal virulence and illustrates, how biotrophic fungi can coordinate cellular physiology, development and regulation of secreted virulence factors. PMID:27093436

  6. Unfolded Protein Response (UPR) Regulator Cib1 Controls Expression of Genes Encoding Secreted Virulence Factors in Ustilago maydis

    PubMed Central

    Hampel, Martin; Jakobi, Mareike; Schmitz, Lara; Meyer, Ute; Finkernagel, Florian; Doehlemann, Gunther; Heimel, Kai

    2016-01-01

    The unfolded protein response (UPR), a conserved eukaryotic signaling pathway to ensure protein homeostasis in the endoplasmic reticulum (ER), coordinates biotrophic development in the corn smut fungus Ustilago maydis. Exact timing of UPR activation is required for virulence and presumably connected to the elevated expression of secreted effector proteins during infection of the host plant Zea mays. In the baker’s yeast Saccharomyces cerevisiae, expression of UPR target genes is induced upon binding of the central regulator Hac1 to unfolded protein response elements (UPREs) in their promoters. While a role of the UPR in effector secretion has been described previously, we investigated a potential UPR-dependent regulation of genes encoding secreted effector proteins. In silico prediction of UPREs in promoter regions identified the previously characterized effector genes pit2 and tin1-1, as bona fide UPR target genes. Furthermore, direct binding of the Hac1-homolog Cib1 to the UPRE containing promoter fragments of both genes was confirmed by quantitative chromatin immunoprecipitation (qChIP) analysis. Targeted deletion of the UPRE abolished Cib1-dependent expression of pit2 and significantly affected virulence. Furthermore, ER stress strongly increased Pit2 expression and secretion. This study expands the role of the UPR as a signal hub in fungal virulence and illustrates, how biotrophic fungi can coordinate cellular physiology, development and regulation of secreted virulence factors. PMID:27093436

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

    PubMed Central

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

    2013-01-01

    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

  8. Activation of the Unfolded Protein Response in Sporadic Inclusion Body Myositis But Not in Hereditary GNE Inclusion Body Myopathy

    PubMed Central

    Nogalska, Anna; D’Agostino, Carla; Engel, W. King; Cacciottolo, Mafalda; Asada, Shinichi; Mori, Kazutoshi; Askanas, Valerie

    2015-01-01

    Muscle fibers in patients with sporadic inclusion-body myositis (s-IBM), the most common age-associated myopathy, are characterized by autophagic vacuoles and accumulation of ubiquitinated and congophilic multiprotein aggregates that contain amyloid-β and phosphorylated tau. Muscle fibers of autosomal-recessive hereditary inclusion-body myopathy due to the GNE mutation (GNE-h-IBM) display similar pathologic features, except with less pronounced congophilia. Accumulation of unfolded/misfolded proteins inside the ER lumen leads to ER stress, which elicits the unfolded protein response (UPR) as a protective mechanism. Here we demonstrate for the first time that UPR is activated in s-IBM muscle biopsies, since there was a) increased ATF4 protein and increased mRNA of its target CHOP, b) cleavage of the ATF6 and increased mRNA of its target GRP78, and c) an increase of the spliced form of XBP-1 and increased mRNA of EDEM, target of heterodimer of cleaved ATF6 and spliced XBP-1. In contrast, we did not find similar evidence of the UPR induction in GNE-h-IBM patient muscle, suggesting that different intracellular mechanisms might lead to the similar pathological phenotypes. Interestingly, cultured GNE-h-IBM muscle fibers had a robust UPR response to experimental ER stress stimuli, suggesting that the GNE mutation per se is not responsible for the lack of UPR in GNE-h-IBM biopsied muscle. PMID:25978849

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

    PubMed Central

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

    2012-01-01

    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

  10. Proteasome inhibition potentiates antitumor effects of photodynamic therapy in mice through induction of ER stress and unfolded protein response

    PubMed Central

    Szokalska, Angelika; Makowski, Marcin; Nowis, Dominika; Wilczyński, Grzegorz M.; Kujawa, Marek; Wójcik, Cezary; Młynarczuk-Biały, Izabela; Salwa, Pawel; Bil, Jacek; Janowska, Sylwia; Agostinis, Patrizia; Verfaillie, Tom; Bugajski, Marek; Gietka, Jan; Issat, Tadeusz; Głodkowska, Eliza; Mrówka, Piotr; Stoklosa, Tomasz; Hamblin, Michael R; Mróz, Paweł; Jakóbisiak, Marek; Golab, Jakub

    2009-01-01

    Photodynamic therapy (PDT) is an approved therapeutic procedure that exerts cytotoxic activity towards tumor cells by inducing production of reactive oxygen species such as singlet oxygen. PDT leads to oxidative damage of cellular macromolecules, including numerous proteins that undergo multiple modifications such as fragmentation, cross-linking and carbonylation that result in protein unfolding and aggregation. Since the major mechanism for elimination of carbonylated proteins is their degradation by proteasomes, we hypothesized that a combination of PDT with proteasome inhibitors might lead to accumulation of carbonylated proteins in endoplasmatic reticulum (ER), aggravated ER stress and potentiated cytotoxicity towards tumor cells. Indeed, we observed that Photofrin-mediated PDT leads to robust carbonylation of cellular proteins and induction of unfolded protein response (UPR). Pre-treatment of tumor cells with three different proteasome inhibitors, including bortezomib, MG132 and PSI gave increased accumulation of carbonylated and ubiquitinated proteins in PDT-treated cells. Proteasome inhibitors effectively sensitized tumor cells of murine (EMT6 and C-26) as well as human (HeLa) origin to PDT-mediated cytotoxicity. Significant retardation of tumor growth with 60-100% complete responses was observed in vivo in two different murine tumor models (EMT6 and C-26) when PDT was combined with either bortezomib or PSI. Altogether these observations indicate that combination of PDT with proteasome inhibitors leads to potentiated antitumor effects. The results of these studies are of immediate clinical application as bortezomib is a clinically approved drug that undergoes extensive clinical evaluations for the treatment of solid tumors. PMID:19435917

  11. Nanomechanics of Protein Unfolding outside Protease Nanopores

    NASA Astrophysics Data System (ADS)

    Luan, Binquan; Zhou, Ruhong

    Protein folding and unfolding have been the subject of active research for decades. Most of previous studies in protein unfolding were focused on temperature, chemical and/or force (such as in AFM) induced denaturations. Recent studies on the functional roles of proteasomes (such as ClpXP) revealed a novel unfolding process in cell, during which a target protein is mechanically unfolded and pulled into a confined, pore-like geometry for degradation. While the proteasome nanomachine has been extensively studied, the mechanism for unfolding proteins with the proteasome pore is still poorly understood. Here, we investigate the mechanical unfolding process of ubiquitin with (or really outside) an idealized proteasome pore, and compare such process with that in the AFM pulling experiment. Unexpectedly, the required force by a proteosome can be much smaller than that by the AFM. Simulation results also unveiled different nanomechanics, tearing fracture vs. shearing friction, in these two distinct types of mechanical unfoldings.

  12. Induction of dsRNA-activated protein kinase links mitochondrial unfolded protein response to the pathogenesis of intestinal inflammation

    PubMed Central

    Messlik, Anja; Nunes, Tiago; Liu, Bo; Kim, Sandy C; Hoogenraad, Nick; Sans, Miquel; Sartor, R Balfour; Haller, Dirk

    2015-01-01

    Objective Inflammatory bowel diseases (IBDs) feature multiple cellular stress responses, including endoplasmic reticulum (ER) unfolded protein responses (UPRs). UPRs represent autoregulatory pathways that adjust organelle capacity to cellular demand. A similar mechanism, mitochondrial UPR (mtUPR), has been described for mitochondria. ER UPR in intestinal epithelial cells (IECs) contributes to the development of intestinal inflammation, and since mitochondrial alterations and dysfunction are implicated in the pathogenesis of IBDs, the authors characterised mtUPR in the context of intestinal inflammation. Methods Truncated ornithine transcarbamylase was used to selectively induce mtUPR in a murine IEC line. Dextran sodium sulphate (DSS) was administered to PKR (double-stranded-RNA-activated protein kinase) knockout mice to induce IEC stress in vivo and to test for their susceptibility to DSS-induced colitis. Expression levels of the mitochondrial chaperone chaperonin 60 (CPN60) and PKR were quantified in IECs from patients with IBDs and from murine models of colitis using immunohistochemistry and Western blot analysis. Results Selective mtUPR induction by truncated ornithine transcarbamylase transfection triggered the phosphorylation of eukaryotic translation initiation factor (eIF) 2α and cJun through the recruitment of PKR. Using pharmacological inhibitors and small inhibitory RNA, the authors identified mtUPR-induced eIF2α phosphorylation and transcription factor activation (cJun/AP1) as being dependent on the activities of the mitochondrial protease ClpP and the cytoplasmic kinase PKR. Pkr−/− mice failed to induce CPN60 in IECs upon DSS treatment at early time points and subsequently showed an almost complete resistance to DSS-induced colitis. Under inflammatory conditions, primary IECs from patients with IBDs and two murine models of colitis exhibited a strong induction of the mtUPR marker protein CPN60 associated with enhanced expression of PKR

  13. Virulence Factors of Pseudomonas aeruginosa Induce Both the Unfolded Protein and Integrated Stress Responses in Airway Epithelial Cells.

    PubMed

    van 't Wout, Emily F A; van Schadewijk, Annemarie; van Boxtel, Ria; Dalton, Lucy E; Clarke, Hanna J; Tommassen, Jan; Marciniak, Stefan J; Hiemstra, Pieter S

    2015-06-01

    Pseudomonas aeruginosa infection can be disastrous in chronic lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease. Its toxic effects are largely mediated by secreted virulence factors including pyocyanin, elastase and alkaline protease (AprA). Efficient functioning of the endoplasmic reticulum (ER) is crucial for cell survival and appropriate immune responses, while an excess of unfolded proteins within the ER leads to "ER stress" and activation of the "unfolded protein response" (UPR). Bacterial infection and Toll-like receptor activation trigger the UPR most likely due to the increased demand for protein folding of inflammatory mediators. In this study, we show that cell-free conditioned medium of the PAO1 strain of P. aeruginosa, containing secreted virulence factors, induces ER stress in primary bronchial epithelial cells as evidenced by splicing of XBP1 mRNA and induction of CHOP, GRP78 and GADD34 expression. Most aspects of the ER stress response were dependent on TAK1 and p38 MAPK, except for the induction of GADD34 mRNA. Using various mutant strains and purified virulence factors, we identified pyocyanin and AprA as inducers of ER stress. However, the induction of GADD34 was mediated by an ER stress-independent integrated stress response (ISR) which was at least partly dependent on the iron-sensing eIF2α kinase HRI. Our data strongly suggest that this increased GADD34 expression served to protect against Pseudomonas-induced, iron-sensitive cell cytotoxicity. In summary, virulence factors from P. aeruginosa induce ER stress in airway epithelial cells and also trigger the ISR to improve cell survival of the host. PMID:26083346

  14. The Unfolded Protein Response Plays a Predominant Homeostatic Role in Response to Mitochondrial Stress in Pancreatic Stellate Cells.

    PubMed

    Su, Hsin-Yuan; Waldron, Richard T; Gong, Raymond; Ramanujan, V Krishnan; Pandol, Stephen J; Lugea, Aurelia

    2016-01-01

    Activated pancreatic stellate cells (PaSC) are key participants in the stroma of pancreatic cancer, secreting extracellular matrix proteins and inflammatory mediators. Tumors are poorly vascularized, creating metabolic stress conditions in cancer and stromal cells that necessitate adaptive homeostatic cellular programs. Activation of autophagy and the endoplasmic reticulum unfolded protein response (UPR) have been described in hepatic stellate cells, but the role of these processes in PaSC responses to metabolic stress is unknown. We reported that the PI3K/mTOR pathway, which AMPK can regulate through multiple inputs, modulates PaSC activation and fibrogenic potential. Here, using primary and immortalized mouse PaSC, we assess the relative contributions of AMPK/mTOR signaling, autophagy and the UPR to cell fate responses during metabolic stress induced by mitochondrial dysfunction. The mitochondrial uncoupler rottlerin at low doses (0.5-2.5 μM) was added to cells cultured in 10% FBS complete media. Mitochondria rapidly depolarized, followed by altered mitochondrial dynamics and decreased cellular ATP levels. This mitochondrial dysfunction elicited rapid, sustained AMPK activation, mTOR pathway inhibition, and blockade of autophagic flux. Rottlerin treatment also induced rapid, sustained PERK/CHOP UPR signaling. Subsequently, high doses (>5 μM) induced loss of cell viability and cell death. Interestingly, AMPK knock-down using siRNA did not prevent rottlerin-induced mTOR inhibition, autophagy, or CHOP upregulation, suggesting that AMPK is dispensable for these responses. Moreover, CHOP genetic deletion, but not AMPK knock-down, prevented rottlerin-induced apoptosis and supported cell survival, suggesting that UPR signaling is a major modulator of cell fate in PaSC during metabolic stress. Further, short-term rottlerin treatment reduced both PaSC fibrogenic potential and IL-6 mRNA expression. In contrast, expression levels of the angiogenic factors HGF and VEGF

  15. The Unfolded Protein Response Plays a Predominant Homeostatic Role in Response to Mitochondrial Stress in Pancreatic Stellate Cells

    PubMed Central

    Su, Hsin-Yuan; Waldron, Richard T.; Gong, Raymond; Ramanujan, V. Krishnan; Pandol, Stephen J.; Lugea, Aurelia

    2016-01-01

    Activated pancreatic stellate cells (PaSC) are key participants in the stroma of pancreatic cancer, secreting extracellular matrix proteins and inflammatory mediators. Tumors are poorly vascularized, creating metabolic stress conditions in cancer and stromal cells that necessitate adaptive homeostatic cellular programs. Activation of autophagy and the endoplasmic reticulum unfolded protein response (UPR) have been described in hepatic stellate cells, but the role of these processes in PaSC responses to metabolic stress is unknown. We reported that the PI3K/mTOR pathway, which AMPK can regulate through multiple inputs, modulates PaSC activation and fibrogenic potential. Here, using primary and immortalized mouse PaSC, we assess the relative contributions of AMPK/mTOR signaling, autophagy and the UPR to cell fate responses during metabolic stress induced by mitochondrial dysfunction. The mitochondrial uncoupler rottlerin at low doses (0.5–2.5 μM) was added to cells cultured in 10% FBS complete media. Mitochondria rapidly depolarized, followed by altered mitochondrial dynamics and decreased cellular ATP levels. This mitochondrial dysfunction elicited rapid, sustained AMPK activation, mTOR pathway inhibition, and blockade of autophagic flux. Rottlerin treatment also induced rapid, sustained PERK/CHOP UPR signaling. Subsequently, high doses (>5 μM) induced loss of cell viability and cell death. Interestingly, AMPK knock-down using siRNA did not prevent rottlerin-induced mTOR inhibition, autophagy, or CHOP upregulation, suggesting that AMPK is dispensable for these responses. Moreover, CHOP genetic deletion, but not AMPK knock-down, prevented rottlerin-induced apoptosis and supported cell survival, suggesting that UPR signaling is a major modulator of cell fate in PaSC during metabolic stress. Further, short-term rottlerin treatment reduced both PaSC fibrogenic potential and IL-6 mRNA expression. In contrast, expression levels of the angiogenic factors HGF and VEGF

  16. Retrotransposon derepression leads to activation of the unfolded protein response and apoptosis in pro-B cells.

    PubMed

    Pasquarella, Alessandra; Ebert, Anja; Pereira de Almeida, Gustavo; Hinterberger, Maria; Kazerani, Maryam; Nuber, Alexander; Ellwart, Joachim; Klein, Ludger; Busslinger, Meinrad; Schotta, Gunnar

    2016-05-15

    The H3K9me3-specific histone methyltransferase Setdb1 impacts on transcriptional regulation by repressing both developmental genes and retrotransposons. How impaired retrotransposon silencing may lead to developmental phenotypes is currently unclear. Here, we show that loss of Setdb1 in pro-B cells completely abrogates B cell development. In pro-B cells, Setdb1 is dispensable for silencing of lineage-inappropriate developmental genes. Instead, we detect strong derepression of endogenous murine leukemia virus (MLV) copies. This activation coincides with an unusual change in chromatin structure, with only partial loss of H3K9me3 and unchanged DNA methylation, but strongly increased H3K4me3. Production of MLV proteins leads to activation of the unfolded protein response pathway and apoptosis. Thus, our data demonstrate that B cell development depends on the proper repression of retrotransposon sequences through Setdb1. PMID:27013243

  17. Chemical Genomics Identifies the PERK-Mediated Unfolded Protein Stress Response as a Cellular Target for Influenza Virus Inhibition

    PubMed Central

    Landeras-Bueno, Sara; Fernández, Yolanda; Falcón, Ana; Oliveros, Juan Carlos

    2016-01-01

    ABSTRACT Influenza A viruses generate annual epidemics and occasional pandemics of respiratory disease with important consequences for human health and the economy. Therefore, a large effort has been devoted to the development of new anti-influenza virus drugs directed to viral targets, as well as to the identification of cellular targets amenable to anti-influenza virus therapy. Here we have addressed the identification of such potential cellular targets by screening collections of drugs approved for human use. We reasoned that screening with a green fluorescent protein-based recombinant replicon system would identify cellular targets involved in virus transcription/replication and/or gene expression and hence address an early stage of virus infection. By using such a strategy, we identified Montelukast (MK) as an inhibitor of virus multiplication. MK inhibited virus gene expression but did not alter viral RNA synthesis in vitro or viral RNA accumulation in vivo. The low selectivity index of MK prevented its use as an antiviral, but it was sufficient to identify a new cellular pathway suitable for anti-influenza virus intervention. By deep sequencing of RNA isolated from mock- and virus-infected human cells, treated with MK or left untreated, we showed that it stimulates the PERK-mediated unfolded protein stress response. The phosphorylation of PERK was partly inhibited in virus-infected cells but stimulated in MK-treated cells. Accordingly, pharmacological inhibition of PERK phosphorylation led to increased viral gene expression, while inhibition of PERK phosphatase reduced viral protein synthesis. These results suggest the PERK-mediated unfolded protein response as a potential cellular target to modulate influenza virus infection. PMID:27094326

  18. Uterine endoplasmic reticulum stress-unfolded protein response regulation of gestational length is caspase-3 and -7-dependent.

    PubMed

    Kyathanahalli, Chandrashekara; Organ, Kenna; Moreci, Rebecca S; Anamthathmakula, Prashanth; Hassan, Sonia S; Caritis, Steve N; Jeyasuria, Pancharatnam; Condon, Jennifer C

    2015-11-10

    We previously identified myometrial caspase-3 (CASP3) as a potential regulator of uterine quiescence. We also determined that during pregnancy, the functional activation of uterine CASP3 is likely governed by an integrated endoplasmic reticulum stress response (ERSR) and is consequently limited by an increased unfolded protein response (UPR). The present study examined the functional relevance of uterine UPR-ERSR in maintaining myometrial quiescence and regulating the timing of parturition. In vitro analysis of the human uterine myocyte hTERT-HM cell line revealed that tunicamycin (TM)-induced ERSR modified uterine myocyte contractile responsiveness. Accordingly, alteration of in vivo uterine UPR-ERSR using a pregnant mouse model significantly modified gestational length. We determined that "normal" gestational activation of the ERSR-induced CASP3 and caspase 7 (CASP7) maintains uterine quiescence through previously unidentified proteolytic targeting of the gap junction protein, alpha 1(GJA1); however, surprisingly, TM-induced uterine ERSR triggered an exaggerated UPR that eliminated uterine CASP3 and 7 tocolytic action precociously. These events allowed for a premature increase in myometrial GJA1 levels, elevated contractile responsiveness, and the onset of preterm labor. Importantly, a successful reversal of the magnified ERSR-induced preterm birth phenotype could be achieved by pretreatment with 4-phenylbutrate, a chaperone protein mimic. PMID:26504199

  19. Computational model for protein unfolding simulation

    NASA Astrophysics Data System (ADS)

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

    2011-06-01

    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 α -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 Φ 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.

  20. Cell Intrinsic and Extrinsic Activators of the Unfolded Protein Response in Cancer: Mechanisms and Targets for Therapy

    PubMed Central

    Tameire, Feven; Verginadis, Ioannis I.; Koumenis, Constantinos

    2015-01-01

    A variety of cell intrinsic or extrinsic stresses evoke perturbations in the folding environment of the endoplasmic reticulum (ER), collectively known as ER stress. Adaptation to stress and reestablishment of ER homeostasis is achieved by activation of an integrated signal transduction pathway called the unfolded protein response (UPR). Both ER stress and UPR activation have been implicated in a variety of human cancers. Although at early stages, or physiological conditions of ER stress, the UPR generally promotes survival, when the stress becomes more stringent or prolonged, its role can switch to a pro-cell death one. Here, we discuss historical and recent evidence supporting an involvement of the UPR in malignancy, describe the main mechanisms by which how tumor cells overcome ER stress to promote their survival, tumor progression and metastasis and discuss the current state of efforts to develop therapeutic approaches of targeting the UPR. PMID:25920797

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

    PubMed

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

    2011-06-01

    Endoplasmic reticulum (ER) stress induced by free fatty acids (FFA) is important to β-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 β 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 β cells. Overall, VO(acac)(2) enhanced stress adaption, thus protecting β cells from palmitate-induced apoptosis. This study provides some new insights into the mechanisms of antidiabetic vanadium compounds. PMID:21512771

  2. Topological determinants of protein unfolding rates.

    PubMed

    Jung, Jaewoon; Lee, Jooyoung; Moon, Hie-Tae

    2005-02-01

    For proteins that fold by two-state kinetics, the folding and unfolding processes are believed to be closely related to their native structures. In particular, folding and unfolding rates are influenced by the native structures of proteins. Thus, we focus on finding important topological quantities from a protein structure that determine its unfolding rate. After constructing graphs from protein native structures, we investigate the relationships between unfolding rates and various topological quantities of the graphs. First, we find that the correlation between the unfolding rate and the contact order is not as prominent as in the case of the folding rate and the contact order. Next, we investigate the correlation between the unfolding rate and the clustering coefficient of the graph of a protein native structure, and observe no correlation between them. Finally, we find that a newly introduced quantity, the impact of edge removal per residue, has a good overall correlation with protein unfolding rates. The impact of edge removal is defined as the ratio of the change of the average path length to the edge removal probability. From these facts, we conclude that the protein unfolding process is closely related to the protein native structure. PMID:15558603

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

    PubMed Central

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

    2013-01-01

    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 μm (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 1α (IRE1α): 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 IRE1α 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

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

    PubMed

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

    2013-06-01

    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

  5. Retention of chimeric Tat2-Gap1 permease in the endoplasmic reticulum induces unfolded protein response in Saccharomyces cerevisiae.

    PubMed

    Mochizuki, Takahiro; Kimata, Yukio; Uemura, Satoshi; Abe, Fumiyoshi

    2015-08-01

    In Saccharomyces cerevisiae, high-affinity tryptophan import is performed by subtle mechanisms involving tryptophan permease Tat2. We have shown that Tat2 requires 15 amino acid residues in the transmembrane domains (TMDs) for its import activity, whereas leucine permease Bap2 requires only seven corresponding residues for its leucine import. For this reason, the structure of Tat2 is elaborately designed to transport the hydrophobic and bulky tryptophan. Newly synthesized cell surface proteins first undergo endoplasmic reticulum (ER)-associated quality check before entering the secretory pathway. In this study, we used domain replacement with general amino acid permease Gap1 to show that Tat2 chimeric proteins were dysfunctional when TMD10 or TMD11 was replaced. These chimeras formed large 270-800-kDa protein complexes and were stably retained in the ER membrane without efficient degradation. In contrast, Tat2 chimeras of TMD9 or TMD12 retained some of their tryptophan import activity and underwent vacuolar degradation as observed with wild-type Tat2. Thus, ours results suggest that TMD10 and TMD11 are essential for the correct folding of Tat2, probably because of their interdomain interactions. Notably, overexpression of Tat2-Gap1 chimera of TMD10 activated the unfolded protein response (UPR) element-lacZ reporter, suggesting that ER retention of the protein aggregates induces the UPR. PMID:26071436

  6. Identification of an old antibiotic clofoctol as a novel activator of unfolded protein response pathways and an inhibitor of prostate cancer

    PubMed Central

    Wang, Minghua; Shim, Joong Sup; Li, Ruo-Jing; Dang, Yongjun; He, Qingli; Das, Manisha; Liu, Jun O

    2014-01-01

    Background and Purpose Finding new indications for existing drugs, also known as drug repositioning or repurposing, is a powerful approach to accelerate drug discovery and development. The unfolded protein response pathways have been proposed to be a viable target for developing new anticancer drugs. Experimental Approach We screened the Johns Hopkins Drug Library for inhibitors of prostate cancer cell proliferation to identify new antiprostate cancer treatments among known drugs. We systematically investigated the mechanism underlying the anticancer activity of a hit and assessed its efficacy in blocking prostate tumour growth in a mouse model. Key Results The antibacterial drug clofoctol was identified as a novel inhibitor of prostate cancer cell proliferation. Morphologically, cells treated with clofoctol were found to undergo massive vacuolization, reminiscent of endoplasmic reticulum stress. Indeed, all three unfolded protein response pathways including inositol requiring enzyme 1, double-stranded RNA-activated PK-like ER kinase and activating transcription factor 6 were found to be activated by clofoctol. Activation of unfolded protein response pathways by clofoctol led to the inhibition of protein translation in cells and the induction of G1 cell cycle arrest in prostate cancer cells. Clofoctol also inhibited prostate cancer xenograft growth in vivo without apparent toxicity. Conclusion and Implications Our findings revealed clofoctol as a novel activator of the unfolded protein response pathways and a promising inhibitor of prostate cancer. As clofoctol has been used in the clinic for years, it is ready for clinical evaluation as a novel antiprostate cancer drug candidate. PMID:24903412

  7. Inhibition of the mitochondrial unfolded protein response by acetylcholine alleviated hypoxia/reoxygenation-induced apoptosis of endothelial cells.

    PubMed

    Xu, Man; Bi, Xueyuan; He, Xi; Yu, Xiaojiang; Zhao, Ming; Zang, Weijin

    2016-05-18

    The mitochondrial unfolded protein response (UPR(mt)) is involved in numerous diseases that have the common feature of mitochondrial dysfunction. However, its pathophysiological relevance in the context of hypoxia/reoxygenation (H/R) in endothelial cells remains elusive. Previous studies have demonstrated that acetylcholine (ACh) protects against cardiomyocyte injury by suppressing generation of mitochondrial reactive oxygen species (mtROS). This study aimed to explore the role of UPR(mt) in endothelial cells during H/R and to clarify the beneficial effects of ACh. Our results demonstrated that H/R triggered UPR(mt) in endothelial cells, as evidenced by the elevation of heat shock protein 60 and LON protease 1 protein levels, and resulted in release of mitochondrial pro-apoptotic proteins, including cytochrome C, Omi/high temperature requirement protein A 2 and second mitochondrial activator of caspases/direct inhibitor of apoptosis-binding protein with low PI, from the mitochondria to cytosol. ACh administration markedly decreased UPR(mt) by inhibiting mtROS and alleviating the mitonuclear protein imbalance. Consequently, ACh alleviated the release of pro-apoptotic proteins and restored mitochondrial ultrastructure and function, thereby reducing the number of terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL)-positive cells. Intriguingly, 4-diphenylacetoxy-N-methylpiperidine methiodide, a type-3 muscarinic ACh receptor (M3AChR) inhibitor, abolished the ACh-elicited attenuation of UPR(mt) and TUNEL positive cells, indicating that the salutary effects of ACh were likely mediated by M3AChR in endothelial cells. In conclusion, our studies demonstrated that UPR(mt) might be essential for triggering the mitochondrion-associated apoptotic pathway during H/R. ACh markedly suppressed UPR(mt) by inhibiting mtROS and alleviating the mitonuclear protein imbalance, presumably through M3AChR. PMID:27111378

  8. Unfolded protein response in hypothalamic cultures of wild-type and ATF6α-knockout mice.

    PubMed

    Lu, Wenjun; Hagiwara, Daisuke; Morishita, Yoshiaki; Tochiya, Masayoshi; Azuma, Yoshinori; Suga, Hidetaka; Goto, Motomitsu; Banno, Ryoichi; Sugimura, Yoshihisa; Oyadomari, Seiichi; Mori, Kazutoshi; Arima, Hiroshi

    2016-01-26

    Recent studies suggest that endoplasmic reticulum (ER) stress in the hypothalamus could affect systemic homeostatic regulation in areas such as energy and water balance. Activating transcription factor 6α (ATF6α) is an ER stress transducer which increases the expression of ER chaperones and ER-associated degradation (ERAD) components under ER stress. In the present study, we examined the regulation of the unfolding protein response (UPR) in mouse hypothalamic cultures of wild-type (WT) and ATF6α(-/-) mice. Thapsigargin (TG), an ER stressor, significantly increased the mRNA expression of immunoglobulin heavy chain binding protein (BiP), spliced X-box binding protein 1 (XBP1), activating transcription factor 4 (ATF4), C/EBP homologous protein (CHOP), and ERAD components, in hypothalamic cultures of WT mice with the same threshold (0.1μM) and similar time courses. On the other hand, TG-induced upregulation of BiP and CHOP as well as most ERAD-related genes, but not spliced XBP1 or ATF4, was attenuated in ATF6α(-/-) mice compared with WT mice. Our data suggest that all the UPR arms are activated similarly in the mouse hypothalamus under ER stress conditions, where ATF6α regulates the expression of ER chaperones, CHOP, and ERAD components. PMID:26708632

  9. Activated AMPK boosts the Nrf2/HO-1 signaling axis—A role for the unfolded protein response

    PubMed Central

    Zimmermann, Kristin; Baldinger, Johannes; Mayerhofer, Barbara; Atanasov, Atanas G.; Dirsch, Verena M.; Heiss, Elke H.

    2015-01-01

    In light of the emerging interplay between redox and metabolic signaling pathways we investigated the potential cross talk between nuclear factor E2-related factor 2 (Nrf2) and AMP-activated kinase (AMPK), central regulators of the cellular redox and energy balance, respectively. Making use of xanthohumol (XN) as an activator of both the AMPK and the Nrf2 signaling pathway we show that AMPK exerts a positive influence on Nrf2/heme oxygenase (HO)-1 signaling in mouse embryonic fibroblasts. Genetic ablation and pharmacological inhibition of AMPK blunts Nrf2-dependent HO-1 expression by XN already at the mRNA level. XN leads to AMPK activation via interference with mitochondrial function and activation of liver kinase B1 as upstream AMPK kinase. The subsequent AMPK-mediated enhancement of the Nrf2/HO-1 response does not depend on inhibition of the mammalian target of rapamycin, inhibition of glycogen synthase kinase 3β, or altered abundance of Nrf2 (total and nuclear). However, reduced endoplasmic reticulum stress was identified and elaborated as a step in the AMPK-augmented Nrf2/HO-1 response. Overall, we shed more light on the hitherto incompletely understood cross talk between the LKB1/AMPK and the Nrf2/HO-1 axis revealing for the first time involvement of the unfolded protein response as an additional player and suggesting tight cooperation between signaling pathways controlling cellular redox, energy, or protein homeostasis. PMID:25843659

  10. Targeting the unfolded protein response, XBP1, and the NLRP3 inflammasome in fibrosis and cancer

    PubMed Central

    Overley-Adamson, Beth; Artlett, Carol M; Stephens, Connie; Sassi-Gaha, Sihem; Weis, Ransome D; Thacker, James D

    2014-01-01

    Increasing health care costs in the US are due in a large part to the increasing prevalence of chronic diseases in an aging population. Current therapeutic strategies for treating chronic diseases alleviate symptoms allowing patients to live longer with these diseases, but they do little, however, to alter the underlying disease course. Recent advances in molecular biology are revealing new drug targets that may significantly alter the course of these diseases and, as a result, offer economic relief from burgeoning health care costs. Endoplasmic reticulum (ER) stress has been implicated as an underlying pathology in many chronic diseases, and, therefore, the development of therapies designed to ameliorate ER stress may yield novel, effective treatment strategies. Herein, we report that X-box binding protein 1 (XBP1) may be one of the earliest proteins engaged in response to ER stress. We show that a new signaling peptide derived from the ER-embedded transient receptor potential calcium channel protein 1 (TRPC1) engages XBP1 upstream of NLRP3 inflammasome-mediated maturation and secretion of IL-1β/IL-18. Moreover, we show that a synthetic homolog of this signaling peptide (Naclynamide™) administered intravenously twice weekly over a 4-week treatment course induced suppuration and evoked partial or complete resolution of lesions associated with a fibrotic granuloma, a lymphosarcoma, and a colo-rectal carcinoma in canine patients. The mode of action for Naclynamide™ as a first-in-class anti-cancer drug candidate is discussed. PMID:24496016

  11. A Reevaluation of the Role of the Unfolded Protein Response in Islet Dysfunction: Maladaptation or a Failure to Adapt?

    PubMed

    Herbert, Terence P; Laybutt, D Ross

    2016-06-01

    Endoplasmic reticulum (ER) stress caused by perturbations in ER homeostasis activates an adaptive response termed the unfolded protein response (UPR) whose function is to resolve ER stress. If unsuccessful, the UPR initiates a proapoptotic program to eliminate the malfunctioning cells from the organism. It is the activation of this proapoptotic UPR in pancreatic β-cells that has been implicated in the onset of type 2 diabetes and thus, in this context, is considered a maladaptive response. However, there is growing evidence that β-cell death in type 2 diabetes may not be caused by a maladaptive UPR but by the inhibition of the adaptive UPR. In this review, we discuss the evidence for a role of the UPR in β-cell dysfunction and death in the development of type 2 diabetes and ask the following question: Is β-cell dysfunction the result of a maladaptive UPR or a failure of the UPR to adequately adapt? The answer to this question is critically important in defining potential therapeutic strategies for the treatment and prevention of type 2 diabetes. In addition, we discuss the potential role of the adaptive UPR in staving off type 2 diabetes by enhancing β-cell mass and function in response to insulin resistance. PMID:27222391

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

    PubMed Central

    Lee, Dong Yun

    2008-01-01

    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 2α (eIF2α). We have found that LMP1 activates PERK to induce phosphorylation of eIF2α, 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. PMID:18042799

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

    PubMed Central

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

    2013-01-01

    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

  14. The Role of Endoplasmic Reticulum Stress and Unfolded Protein Response in Atherosclerosis

    PubMed Central

    Ivanova, Ekaterina A.; Orekhov, Alexander N.

    2016-01-01

    Pathogenesis of atherosclerosis is a complex process involving several metabolic and signalling pathways. Accumulating evidence demonstrates that endoplasmic reticulum stress and associated apoptosis can be induced in the pathological conditions of atherosclerotic lesions and contribute to the disease progression. Notably, they may play a role in the development of vulnerable plaques that induce thrombosis and are therefore especially dangerous. Endoplasmic reticulum stress response is regulated by several signaling mechanisms that involve protein kinases and transcription factors. Some of these molecules can be regarded as potential therapeutic targets to improve treatment of atherosclerosis. In this review we will discuss the role of endoplasmic reticulum stress and apoptosis in atherosclerosis development in different cell types and summarize the current knowledge on potential therapeutic agents targeting molecules regulating these pathways and their possible use for anti-atherosclerotic therapy. PMID:26840309

  15. Alpha-fetoprotein is a biomarker of unfolded protein response and altered proteostasis in hepatocellular carcinoma cells exposed to sorafenib.

    PubMed

    Houessinon, Aline; Gicquel, Albane; Bochereau, Flora; Louandre, Christophe; Nyga, Rémy; Godin, Corinne; Degonville, James; Fournier, Emma; Saidak, Zuzana; Drullion, Claire; Barbare, Jean-Claude; Chauffert, Bruno; François, Catherine; Pluquet, Olivier; Galmiche, Antoine

    2016-01-28

    Sorafenib is the treatment of reference for advanced hepatocellular carcinoma (HCC). A decrease in the serum levels of Alpha-fetoprotein (AFP) is reported to be the biological parameter that is best associated with disease control by sorafenib. In order to provide a biological rationale for the variations of AFP, we analyzed the various steps of AFP production in human HCC cell lines exposed to sorafenib. Sorafenib dramatically reduced the levels of AFP produced by HCC cells independently of its effect on cell viability. The mRNA levels of AFP decreased upon sorafenib treatment, while the AFP protein remained localized in the Golgi apparatus. Sorafenib activated the Regulated Inositol-Requiring Enzyme-1α (IRE-1α) and the PKR-like ER Kinase (PERK)-dependent arms of the Unfolded Protein Response (UPR). The inhibition of IRE-1α partially restored the mRNA levels of AFP upon treatment with sorafenib. The inhibition of both pathways partially prevented the drop in the production of AFP induced by sorafenib. The findings provide new insights on the regulation of AFP, and identify it as a biomarker suitable for the exploration of HCC cell proteostasis in the context of therapeutic targeting. PMID:26546044

  16. The ATF6 branch of unfolded protein response and apoptosis are activated to promote African swine fever virus infection

    PubMed Central

    Galindo, I; Hernáez, B; Muñoz-Moreno, R; Cuesta-Geijo, M A; Dalmau-Mena, I; Alonso, C

    2012-01-01

    African swine fever virus (ASFV) infection induces apoptosis in the infected cell; however, the consequences of this activation on virus replication have not been defined. In order to identify the role of apoptosis in ASFV infection, we analyzed caspase induction during the infection and the impact of caspase inhibition on viral production. Caspases 3, 9 and 12 were activated from 16 h post-infection, but not caspase 8. Indeed, caspase 3 activation during the early stages of the infection appeared to be crucial for efficient virus exit. In addition, the inhibition of membrane blebbing reduced the release of virus particles from the cell. ASFV uses the endoplasmic reticulum (ER) as a site of replication and this process can trigger ER stress and the unfolded protein response (UPR) of the host cell. In addition to caspase 12 activation, indicators of ER stress include the upregulation of the chaperones calnexin and calreticulin upon virus infection. Moreover, ASFV induces transcription factor 6 signaling pathway of the UPR, but not the protein kinase-like ER kinase or the inositol-requiring enzyme 1 pathways. Thus, the capacity of ASFV to regulate the UPR may prevent early apoptosis and ensure viral replication. PMID:22764100

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

    PubMed

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

    2013-11-01

    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 eIF2α, in Oca2-null melanocytes, eIF2α was rapidly dephosphorylated upon treatment. Dephosphorylation was mediated by the Gadd34-PP1α phosphatase complex. Gadd34-complex inhibition blocked eIF2α 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 eIF2α dephosphorylation as an adaptive mechanism to ER stress. PMID:23962237

  18. RTCB-1 mediates neuroprotection via XBP-1 mRNA splicing in the unfolded protein response pathway.

    PubMed

    Ray, Arpita; Zhang, Siyuan; Rentas, Courtney; Caldwell, Kim A; Caldwell, Guy A

    2014-11-26

    Parkinson's disease (PD), the second most prevalent neurodegenerative disorder, is characterized by the degeneration of dopamine (DA) neurons and age-dependent formation of protein inclusions that contain the α-synuclein (α-syn) protein. RNA interference (RNAi) screening using Caenorhabditis elegans identified RTCB-1, an uncharacterized gene product, as one of several significant modifiers of α-syn protein misfolding. RTCB-1 is the worm ortholog of the human HSPC117 protein, a component of RNA trafficking granules in mammalian neurons. Here we show that RTCB-1 protects C. elegans DA neurons from age-dependent degeneration induced by human α-syn. Moreover, neuronal-specific RNAi depletion of rtcb-1 enhanced α-syn-induced degeneration. Similar results were obtained when worms were exposed to the DA neurotoxin 6-hydroxydopamine. HSPC117 has been characterized recently as an essential subunit of the human tRNA splicing ligase complex. tRNA ligases have alternative functions in RNA repair and nonconventional mRNA splicing events. For example, in yeast, unconventional splicing of HAC1, a transcription factor that controls the unfolded protein response (UPR), is mediated by a tRNA ligase. In C. elegans, we demonstrate that RTCB-1 is necessary for xbp-1 (worm homolog of HAC1) mRNA splicing. Moreover, using a RNA ligase-dead mutant, we determine that the ligase activity of worm RTCB-1 is required for its neuroprotective role, which, in turn, is mediated through XBP-1 in the UPR pathway. Collectively, these studies highlight the mechanistic intersection of RNA processing and proteostasis in mediating neuroprotection. PMID:25429148

  19. Cavities determine the pressure unfolding of proteins

    PubMed Central

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

    2012-01-01

    It has been known for nearly 100 years that pressure unfolds proteins, yet the physical basis of this effect is not understood. Unfolding by pressure implies that the molar volume of the unfolded state of a protein is smaller than that of the folded state. This decrease in volume has been proposed to arise from differences between the density of bulk water and water associated with the protein, from pressure-dependent changes in the structure of bulk water, from the loss of internal cavities in the folded states of proteins, or from some combination of these three factors. Here, using 10 cavity-containing variants of staphylococcal nuclease, we demonstrate that pressure unfolds proteins primarily as a result of cavities that are present in the folded state and absent in the unfolded one. High-pressure NMR spectroscopy and simulations constrained by the NMR data were used to describe structural and energetic details of the folding landscape of staphylococcal nuclease that are usually inaccessible with existing experimental approaches using harsher denaturants. Besides solving a 100-year-old conundrum concerning the detailed structural origins of pressure unfolding of proteins, these studies illustrate the promise of pressure perturbation as a unique tool for examining the roles of packing, conformational fluctuations, and water penetration as determinants of solution properties of proteins, and for detecting folding intermediates and other structural details of protein-folding landscapes that are invisible to standard experimental approaches. PMID:22496593

  20. Cavities determine the pressure unfolding of proteins.

    PubMed

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

    2012-05-01

    It has been known for nearly 100 years that pressure unfolds proteins, yet the physical basis of this effect is not understood. Unfolding by pressure implies that the molar volume of the unfolded state of a protein is smaller than that of the folded state. This decrease in volume has been proposed to arise from differences between the density of bulk water and water associated with the protein, from pressure-dependent changes in the structure of bulk water, from the loss of internal cavities in the folded states of proteins, or from some combination of these three factors. Here, using 10 cavity-containing variants of staphylococcal nuclease, we demonstrate that pressure unfolds proteins primarily as a result of cavities that are present in the folded state and absent in the unfolded one. High-pressure NMR spectroscopy and simulations constrained by the NMR data were used to describe structural and energetic details of the folding landscape of staphylococcal nuclease that are usually inaccessible with existing experimental approaches using harsher denaturants. Besides solving a 100-year-old conundrum concerning the detailed structural origins of pressure unfolding of proteins, these studies illustrate the promise of pressure perturbation as a unique tool for examining the roles of packing, conformational fluctuations, and water penetration as determinants of solution properties of proteins, and for detecting folding intermediates and other structural details of protein-folding landscapes that are invisible to standard experimental approaches. PMID:22496593

  1. KAT2B Is Required for Pancreatic Beta Cell Adaptation to Metabolic Stress by Controlling the Unfolded Protein Response.

    PubMed

    Rabhi, Nabil; Denechaud, Pierre-Damien; Gromada, Xavier; Hannou, Sarah Anissa; Zhang, Hongbo; Rashid, Talha; Salas, Elisabet; Durand, Emmanuelle; Sand, Olivier; Bonnefond, Amélie; Yengo, Loic; Chavey, Carine; Bonner, Caroline; Kerr-Conte, Julie; Abderrahmani, Amar; Auwerx, Johan; Fajas, Lluis; Froguel, Philippe; Annicotte, Jean-Sébastien

    2016-05-01

    The endoplasmic reticulum (ER) unfolded protein response (UPR(er)) pathway plays an important role in helping pancreatic β cells to adapt their cellular responses to environmental cues and metabolic stress. Although altered UPR(er) gene expression appears in rodent and human type 2 diabetic (T2D) islets, the underlying molecular mechanisms remain unknown. We show here that germline and β cell-specific disruption of the lysine acetyltransferase 2B (Kat2b) gene in mice leads to impaired insulin secretion and glucose intolerance. Genome-wide analysis of Kat2b-regulated genes and functional assays reveal a critical role for Kat2b in maintaining UPR(er) gene expression and subsequent β cell function. Importantly, Kat2b expression is decreased in mouse and human diabetic β cells and correlates with UPR(er) gene expression in normal human islets. In conclusion, Kat2b is a crucial transcriptional regulator for adaptive β cell function during metabolic stress by controlling UPR(er) and represents a promising target for T2D prevention and treatment. PMID:27117420

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

    PubMed

    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

    2013-10-24

    To study central nervous system airborne PM related subchronic toxicity, SD male rats were exposed for eight weeks to either coarse (32 μg/m³), fine (178 μg/m³) or ultrafine (107 μg/m³) 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-1β and TNFα), 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-κB, 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-κB and increased IL-1β and TNFα 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

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

    PubMed Central

    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.

    2014-01-01

    To study central nervous system airborne PM related subchronic toxicity, SD male rats were exposed for eight weeks to either coarse (32 µg/m3), fine (178 µg/m3) or ultrafine (107 µg/m3) 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-1β and TNFα), 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-κB, 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-κB and increased IL-1β and TNFα 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

  4. Ceapins are a new class of unfolded protein response inhibitors, selectively targeting the ATF6α branch

    PubMed Central

    Gallagher, Ciara M; Garri, Carolina; Cain, Erica L; Ang, Kenny Kean-Hooi; Wilson, Christopher G; Chen, Steven; Hearn, Brian R; Jaishankar, Priyadarshini; Aranda-Diaz, Andres; Arkin, Michelle R; Renslo, Adam R; Walter, Peter

    2016-01-01

    The membrane-bound transcription factor ATF6α plays a cytoprotective role in the unfolded protein response (UPR), required for cells to survive ER stress. Activation of ATF6α promotes cell survival in cancer models. We used cell-based screens to discover and develop Ceapins, a class of pyrazole amides, that block ATF6α signaling in response to ER stress. Ceapins sensitize cells to ER stress without impacting viability of unstressed cells. Ceapins are highly specific inhibitors of ATF6α signaling, not affecting signaling through the other branches of the UPR, or proteolytic processing of its close homolog ATF6β or SREBP (a cholesterol-regulated transcription factor), both activated by the same proteases. Ceapins are first-in-class inhibitors that can be used to explore both the mechanism of activation of ATF6α and its role in pathological settings. The discovery of Ceapins now enables pharmacological modulation all three UPR branches either singly or in combination. DOI: http://dx.doi.org/10.7554/eLife.11878.001 PMID:27435960

  5. Ceapins are a new class of unfolded protein response inhibitors, selectively targeting the ATF6α branch.

    PubMed

    Gallagher, Ciara M; Garri, Carolina; Cain, Erica L; Ang, Kenny Kean-Hooi; Wilson, Christopher G; Chen, Steven; Hearn, Brian R; Jaishankar, Priyadarshini; Aranda-Diaz, Andres; Arkin, Michelle R; Renslo, Adam R; Walter, Peter

    2016-01-01

    The membrane-bound transcription factor ATF6α plays a cytoprotective role in the unfolded protein response (UPR), required for cells to survive ER stress. Activation of ATF6α promotes cell survival in cancer models. We used cell-based screens to discover and develop Ceapins, a class of pyrazole amides, that block ATF6α signaling in response to ER stress. Ceapins sensitize cells to ER stress without impacting viability of unstressed cells. Ceapins are highly specific inhibitors of ATF6α signaling, not affecting signaling through the other branches of the UPR, or proteolytic processing of its close homolog ATF6β or SREBP (a cholesterol-regulated transcription factor), both activated by the same proteases. Ceapins are first-in-class inhibitors that can be used to explore both the mechanism of activation of ATF6α and its role in pathological settings. The discovery of Ceapins now enables pharmacological modulation all three UPR branches either singly or in combination. PMID:27435960

  6. Chibby drives β catenin cytoplasmic accumulation leading to activation of the unfolded protein response in BCR-ABL1+ cells.

    PubMed

    Mancini, Manuela; Leo, Elisa; Takemaru, Ken-Ichi; Campi, Virginia; Borsi, Enrica; Castagnetti, Fausto; Gugliotta, Gabriele; Santucci, Maria Alessandra; Martinelli, Giovanni

    2013-09-01

    Chronic myeloid leukemia (CML) is a myeloproliferative disease caused by the constitutive tyrosine kinase (TK) activity of the BCR-ABL fusion protein. However, the phenotype of leukemic stem cells (LSC) is sustained by β catenin rather than by the BCR-ABL TK. β catenin activity in CML is contingent upon its stabilization proceeding from the BCR-ABL-induced phosphorylation at critical residues for interaction with the Adenomatous polyposis coli (APC)/Axin/glycogen synthase kinase 3 (GSK3) destruction complex or GSK3 inactivating mutations. Here we studied the impact of β catenin antagonist Chibby (CBY) on β catenin signaling in BCR-ABL1+ cells. CBY is a small conserved protein which interacts with β catenin and impairs β catenin-mediated transcriptional activation through two distinct molecular mechanisms: 1) competition with T cell factor (TCF) or lymphoid enhancer factor (LEF) for β catenin binding; and 2) nuclear export of β catenin via interaction with 14-3-3. We found that its enforced expression in K562 cell line promoted β catenin cytoplasmic translocation resulting in inhibition of target gene transcription. Moreover, cytoplasmic accumulation of β catenin activated the endoplasmic reticulum (ER) stress-associated pathway known as unfolded protein response (UPR). CBY-driven cytoplasmic accumulation of β catenin is also a component of BCR-ABL1+ cell response to the TK inhibitor Imatinib (IM). It evoked the UPR activation leading to the induction of BCL2-interacting mediator of cell death (BIM) by UPR sensors. BIM, in turn, contributed to the execution phase of apoptosis in the activation of ER resident caspase 12 and mobilization of Ca(2+) stores. PMID:23707389

  7. Autophagosome Formation during Varicella-Zoster Virus Infection following Endoplasmic Reticulum Stress and the Unfolded Protein Response ▿ † ‡

    PubMed Central

    Carpenter, John E.; Jackson, Wallen; Benetti, Luca; Grose, Charles

    2011-01-01

    Autophagy is a recently recognized component of the life cycle of varicella-zoster virus (VZV). We have documented abundant autophagosome formation in skin vesicles (final site of virion assembly) from randomly selected cases of varicella and zoster. The fact that autophagy was an early event in the VZV replication cycle was documented by finding infected vesicle cells with the VZV IE62 protein confined to the nucleus. Next, we pursued studies in VZV-infected cultured cells to define whether autophagy was preceded by endoplasmic reticulum (ER) stress and the unfolded protein response (UPR). First, we demonstrated that autophagosome formation in infected cells closely resembled that seen after treatment of cells with tunicamycin, a potent initiator of ER stress. Second, we demonstrated a marked expansion of ER size in both VZV-infected cells and cells transfected with the predominant VZV glycoprotein complex gE/gI. An enlarged ER is critical evidence of ER stress, which in turn is relieved by the UPR. To this end, we documented the UPR by detecting the alternatively spliced form of the XBP1 protein as well as CHOP (C/EBP homologous protein), both transcriptional activators of other UPR genes in an ER stress-dependent manner. Because VZV does not encode inhibitors of autophagy, the above results suggested that autophagy was a common event in VZV-infected cells and that it was provoked at least in part by ER stress secondary to overly abundant VZV glycoprotein biosynthesis, which led to UPR activation in an attempt to maintain cellular homeostasis. PMID:21752906

  8. Endoplasmic reticulum stress-independent activation of unfolded protein response kinases by a small molecule ATP-mimic.

    PubMed

    Mendez, Aaron S; Alfaro, Jennifer; Morales-Soto, Marisol A; Dar, Arvin C; McCullagh, Emma; Gotthardt, Katja; Li, Han; Acosta-Alvear, Diego; Sidrauski, Carmela; Korennykh, Alexei V; Bernales, Sebastian; Shokat, Kevan M; Walter, Peter

    2015-01-01

    Two ER membrane-resident transmembrane kinases, IRE1 and PERK, function as stress sensors in the unfolded protein response. IRE1 also has an endoribonuclease activity, which initiates a non-conventional mRNA splicing reaction, while PERK phosphorylates eIF2α. We engineered a potent small molecule, IPA, that binds to IRE1's ATP-binding pocket and predisposes the kinase domain to oligomerization, activating its RNase. IPA also inhibits PERK but, paradoxically, activates it at low concentrations, resulting in a bell-shaped activation profile. We reconstituted IPA-activation of PERK-mediated eIF2α phosphorylation from purified components. We estimate that under conditions of maximal activation less than 15% of PERK molecules in the reaction are occupied by IPA. We propose that IPA binding biases the PERK kinase towards its active conformation, which trans-activates apo-PERK molecules. The mechanism by which partial occupancy with an inhibitor can activate kinases may be wide-spread and carries major implications for design and therapeutic application of kinase inhibitors. PMID:25986605

  9. Low glucose under hypoxic conditions induces unfolded protein response and produces reactive oxygen species in lens epithelial cells.

    PubMed

    Elanchezhian, R; Palsamy, P; Madson, C J; Mulhern, M L; Lynch, D W; Troia, A M; Usukura, J; Shinohara, T

    2012-01-01

    Aging is enhanced by hypoxia and oxidative stress. As the lens is located in the hypoglycemic environment under hypoxia, aging lens with diabetes might aggravate these stresses. This study was designed to examine whether low glucose under hypoxic conditions induces the unfolded protein response (UPR), and also if the UPR then generates the reactive oxygen species (ROS) in lens epithelial cells (LECs). The UPR was activated within 1 h by culturing the human LECs (HLECs) and rat LECs in <1.5 mM glucose under hypoxic conditions. These conditions also induced the Nrf2-dependent antioxidant-protective UPR, production of ROS, and apoptosis. The rat LECs located in the anterior center region were the least susceptible to the UPR, whereas the proliferating LECs in the germinative zone were the most susceptible. Because the cortical lens fiber cells are differentiated from the LECs after the onset of diabetes, we suggest that these newly formed cortical fibers have lower levels of Nrf2, and are then oxidized resulting in cortical cataracts. Thus, low glucose and oxygen conditions induce the UPR, generation of ROS, and expressed the Nrf2 and Nrf2-dependent antioxidant enzymes at normal levels. But these cells eventually lose reduced glutathione (GSH) and induce apoptosis. The results indicate a new link between hypoglycemia under hypoxia and impairment of HLEC functions. PMID:22513875

  10. Low glucose under hypoxic conditions induces unfolded protein response and produces reactive oxygen species in lens epithelial cells

    PubMed Central

    Elanchezhian, R; Palsamy, P; Madson, C J; Mulhern, M L; Lynch, D W; Troia, A M; Usukura, J; Shinohara, T

    2012-01-01

    Aging is enhanced by hypoxia and oxidative stress. As the lens is located in the hypoglycemic environment under hypoxia, aging lens with diabetes might aggravate these stresses. This study was designed to examine whether low glucose under hypoxic conditions induces the unfolded protein response (UPR), and also if the UPR then generates the reactive oxygen species (ROS) in lens epithelial cells (LECs). The UPR was activated within 1 h by culturing the human LECs (HLECs) and rat LECs in <1.5 mM glucose under hypoxic conditions. These conditions also induced the Nrf2-dependent antioxidant-protective UPR, production of ROS, and apoptosis. The rat LECs located in the anterior center region were the least susceptible to the UPR, whereas the proliferating LECs in the germinative zone were the most susceptible. Because the cortical lens fiber cells are differentiated from the LECs after the onset of diabetes, we suggest that these newly formed cortical fibers have lower levels of Nrf2, and are then oxidized resulting in cortical cataracts. Thus, low glucose and oxygen conditions induce the UPR, generation of ROS, and expressed the Nrf2 and Nrf2-dependent antioxidant enzymes at normal levels. But these cells eventually lose reduced glutathione (GSH) and induce apoptosis. The results indicate a new link between hypoglycemia under hypoxia and impairment of HLEC functions. PMID:22513875

  11. Endoplasmic reticulum stress-independent activation of unfolded protein response kinases by a small molecule ATP-mimic

    PubMed Central

    Mendez, Aaron S; Alfaro, Jennifer; Morales-Soto, Marisol A; Dar, Arvin C; McCullagh, Emma; Gotthardt, Katja; Li, Han; Acosta-Alvear, Diego; Sidrauski, Carmela; Korennykh, Alexei V; Bernales, Sebastian; Shokat, Kevan M; Walter, Peter

    2015-01-01

    Two ER membrane-resident transmembrane kinases, IRE1 and PERK, function as stress sensors in the unfolded protein response. IRE1 also has an endoribonuclease activity, which initiates a non-conventional mRNA splicing reaction, while PERK phosphorylates eIF2α. We engineered a potent small molecule, IPA, that binds to IRE1's ATP-binding pocket and predisposes the kinase domain to oligomerization, activating its RNase. IPA also inhibits PERK but, paradoxically, activates it at low concentrations, resulting in a bell-shaped activation profile. We reconstituted IPA-activation of PERK-mediated eIF2α phosphorylation from purified components. We estimate that under conditions of maximal activation less than 15% of PERK molecules in the reaction are occupied by IPA. We propose that IPA binding biases the PERK kinase towards its active conformation, which trans-activates apo-PERK molecules. The mechanism by which partial occupancy with an inhibitor can activate kinases may be wide-spread and carries major implications for design and therapeutic application of kinase inhibitors. DOI: http://dx.doi.org/10.7554/eLife.05434.001 PMID:25986605

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

    PubMed Central

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

    2013-01-01

    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 μmol/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

  13. Multistep protein unfolding during nanopore translocation

    NASA Astrophysics Data System (ADS)

    Rodriguez-Larrea, David; Bayley, Hagan

    2013-04-01

    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.

  14. Disseminated Tumor Cells Persist in the Bone Marrow of Breast Cancer Patients through Sustained Activation of the Unfolded Protein Response.

    PubMed

    Bartkowiak, Kai; Kwiatkowski, Marcel; Buck, Friedrich; Gorges, Tobias M; Nilse, Lars; Assmann, Volker; Andreas, Antje; Müller, Volkmar; Wikman, Harriet; Riethdorf, Sabine; Schlüter, Hartmut; Pantel, Klaus

    2015-12-15

    Disseminated tumor cells (DTC), which share mesenchymal and epithelial properties, are considered to be metastasis-initiating cells in breast cancer. However, the mechanisms supporting DTC survival are poorly understood. DTC extravasation into the bone marrow may be encouraged by low oxygen concentrations that trigger metabolic and molecular alterations contributing to DTC survival. Here, we investigated how the unfolded protein response (UPR), an important cytoprotective program induced by hypoxia, affects the behavior of stressed cancer cells. DTC cell lines established from the bone marrow of patients with breast cancer (BC-M1), lung cancer, (LC-M1), and prostate cancer (PC-E1) were subjected to hypoxic and hypoglycemic conditions. BC-M1 and LC-M1 exhibiting mesenchymal and epithelial properties adapted readily to hypoxia and glucose starvation. Upregulation of UPR proteins, such as the glucose-regulated protein Grp78, induced the formation of filamentous networks, resulting in proliferative advantages and sustained survival under total glucose deprivation. High Grp78 expression correlated with mesenchymal attributes of breast and lung cancer cells and with poor differentiation in clinical samples of primary breast and lung carcinomas. In DTCs isolated from bone marrow specimens from breast cancer patients, Grp78-positive stress granules were observed, consistent with the likelihood these cells were exposed to acute cell stress. Overall, our findings provide the first evidence that the UPR is activated in DTC in the bone marrow from cancer patients, warranting further study of this cell stress pathway as a predictive biomarker for recurrent metastatic disease. PMID:26573792

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

    PubMed

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

    2012-02-01

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

  16. DEFECTIVE TRAFFICKING OF CONE PHOTORECEPTOR CNG CHANNELS INDUCES THE UNFOLDED PROTEIN RESPONSE AND ER STRESS-ASSOCIATED CELL DEATH

    PubMed Central

    Duricka, Deborah L.; Brown, R. Lane; Varnum, Michael D.

    2011-01-01

    SYNOPSIS Mutations that perturb the function of photoreceptor cyclic nucleotide-gated (CNG) channels are associated with several human retinal disorders, but the molecular and cellular mechanisms leading to photoreceptor dysfunction and degeneration remain unclear. Many loss-of-function mutations result in intracellular accumulation of CNG channel subunits. Accumulation of proteins in the endoplasmic reticulum (ER) is known to cause ER stress and trigger the unfolded protein response (UPR), an evolutionarily conserved cellular program that results in either adaptation via increased protein processing capacity or apoptotic cell death. We hypothesize that defective trafficking of cone photoreceptor CNG channels can induce UPR-mediated cell death. To test this idea, CNGA3 subunits bearing the R563H and Q655X mutations were expressed in photoreceptor-derived 661W cells with CNGB3 subunits. Compared to wild type, R563H and Q655X subunits displayed altered degradation rates and/or were retained in the ER. ER retention was associated with increased expression of UPR-related markers of ER stress and with decreased cell viability. Chemical and pharmacological chaperones (TUDCA, 4PBA, and the cGMP analog CPT-cGMP) differentially reduced degradation and/or promoted plasma-membrane localization of defective subunits. Improved subunit maturation was concordant with reduced expression of ER stress markers and improved viability of cells expressing localization-defective channels. These results indicate that ER stress can arise from expression of localization defective CNG channels, and may represent a contributing factor for photoreceptor degeneration. PMID:21992067

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

    PubMed

    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

    2010-06-01

    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

  18. Unfolded protein response gene GADD34 is overexpressed in rheumatoid arthritis and related to the presence of circulating anti-citrullinated protein antibodies.

    PubMed

    Clavarino, Giovanna; Adriouach, Souad; Quesada, Jean-Louis; Clay, Marine; Chevreau, Maxime; Trocmé, Candice; Grange, Laurent; Gaudin, Philippe; Gatti, Evelina; Pierre, Philippe; Cesbron, Jean-Yves; Dumestre-Pérard, Chantal

    2016-05-01

    Growth arrest and DNA damage-inducible gene 34 (GADD34) is an inducible cofactor of protein phosphatase 1, which has an important role in the unfolded protein response. GADD34 has been shown to be necessary for type I interferon and proinflammatory cytokine production in response to viral infection in murine models. We investigate the expression of GADD34 in rheumatoid arthritis (RA), in which proinflammatory cytokines have an important pathogenic role. The objective of this study was to evaluate the potential of GADD34 expression as a biomarker in RA patients. We report a case-control study on GADD34 gene expression in peripheral blood mononuclear cells of patients (n = 75) with RA and age- and sex-matched healthy controls (n = 25). The study was approved by the relevant local ethics committees. GADD34 gene expression level in peripheral blood mononuclear cells was measured by quantitative PCR and analyzed with Mann-Whitney test. The relation between GADD34 gene overexpression and clinical or biological characteristics was analyzed with univariate and multivariate analysis. GADD34 gene expression was significantly higher in RA patients compared with healthy controls (p ≤ 0.001). Interestingly, GADD34 overexpression in PBMC of patients was related to the presence of circulating anti-citrullinated protein antibodies (p = 0.030). Data of this study strengthen the evidence of an unfolded protein response during the course of RA and provide an insight of the potential interest in GADD34 as a relevant marker for RA. PMID:26829377

  19. Reduced endoplasmic reticulum stress-induced apoptosis and impaired unfolded protein response in TRPC3-deficient M1 macrophages

    PubMed Central

    Solanki, Sumeet; Dube, Prabhatchandra R.; Tano, Jean-Yves; Birnbaumer, Lutz

    2014-01-01

    Endoplasmic reticulum (ER) stress is a prominent mechanism of macrophage apoptosis in advanced atherosclerotic lesions. Recent studies from our laboratory showed that advanced atherosclerotic plaques in Apoe−/− mice with bone marrow deficiency of the calcium-permeable channel Transient Receptor Potential Canonical 3 (TRPC3) are characterized by reduced areas of necrosis and fewer apoptotic macrophages than animals transplanted with Trpc3+/+ bone marrow. In vitro, proinflammatory M1 but not anti-inflammatory M2 macrophages derived from Trpc3−/−Apoe−/− animals exhibited reduced ER stress-induced apoptosis. However, whether this was due to a specific effect of TRPC3 deficiency on macrophage ER stress signaling remained to be determined. In the present work we used polarized macrophages derived from mice with macrophage-specific deficiency of TRPC3 to examine the expression level of ER stress markers and the activation status of some typical mediators of macrophage apoptosis. We found that the reduced susceptibility of TRPC3-deficient M1 macrophages to ER stress-induced apoptosis correlates with an impaired unfolded protein response (UPR), reduced mitochondrion-dependent apoptosis, and reduced activation of the proapoptotic molecules calmodulin-dependent protein kinase II and signal transducer and activator of transcription 1. Notably, none of these pathways was altered in TRPC3-deficient M2 macrophages. These findings show for the first time an obligatory requirement for a member of the TRPC family of cation channels in ER stress-induced apoptosis in macrophages, underscoring a rather selective role of the TRPC3 channel on mechanisms related to the UPR signaling in M1 macrophages. PMID:25031020

  20. Unfolded protein response-induced dysregulation of calcium homeostasis promotes retinal degeneration in rat models of autosomal dominant retinitis pigmentosa

    PubMed Central

    Shinde, V; Kotla, P; Strang, C; Gorbatyuk, M

    2015-01-01

    The molecular mechanism of autosomal dominant retinitis pigmentosa (ADRP) in rats is closely associated with a persistently activated unfolded protein response (UPR). If unchecked, the UPR might trigger apoptosis, leading to photoreceptor death. One of the UPR-activated cellular signaling culminating in apoptotic photoreceptor cell death is linked to an increase in intracellular Ca2+. Therefore, we validated whether ADRP retinas experience a cytosolic Ca2+ overload, and whether sustained UPR in the wild-type retina could promote retinal degeneration through Ca2+-mediated calpain activation. We performed an ex vivo experiment to measure intracellular Ca2+ in ADRP retinas as well as to detect the expression levels of proteins that act as Ca2+ sensors. In separate experiments with the subretinal injection of tunicamycin (UPR inducer) and a mixture of calcium ionophore (A231278) and thapsigargin (SERCA2b inhibitor) we assessed the consequences of a sustained UPR activation and increased intracellular Ca2+ in the wild-type retina, respectively, by performing scotopic ERG, histological, and western blot analyses. Results of the study revealed that induced UPR in the retina activates calpain-mediated signaling, and increased intracellular Ca2+ is capable of promoting retinal degeneration. A significant decline in ERG amplitudes at 6 weeks post treatment was associated with photoreceptor cell loss that occurred through calpain-activated CDK5-pJNK-Csp3/7 pathway. Similar calpain activation was found in ADRP rat retinas. A twofold increase in intracellular Ca2+ and up- and downregulations of ER membrane-associated Ca2+-regulated IP3R channels and SERCA2b transporters were detected. Therefore, sustained UPR activation in the ADRP rat retinas could promote retinal degeneration through increased intracellular Ca2+ and calpain-mediated apoptosis.

  1. Unfolded protein response-induced dysregulation of calcium homeostasis promotes retinal degeneration in rat models of autosomal dominant retinitis pigmentosa

    PubMed Central

    Shinde, V; Kotla, P; Strang, C; Gorbatyuk, M

    2016-01-01

    The molecular mechanism of autosomal dominant retinitis pigmentosa (ADRP) in rats is closely associated with a persistently activated unfolded protein response (UPR). If unchecked, the UPR might trigger apoptosis, leading to photoreceptor death. One of the UPR-activated cellular signaling culminating in apoptotic photoreceptor cell death is linked to an increase in intracellular Ca2+. Therefore, we validated whether ADRP retinas experience a cytosolic Ca2+ overload, and whether sustained UPR in the wild-type retina could promote retinal degeneration through Ca2+-mediated calpain activation. We performed an ex vivo experiment to measure intracellular Ca2+ in ADRP retinas as well as to detect the expression levels of proteins that act as Ca2+ sensors. In separate experiments with the subretinal injection of tunicamycin (UPR inducer) and a mixture of calcium ionophore (A231278) and thapsigargin (SERCA2b inhibitor) we assessed the consequences of a sustained UPR activation and increased intracellular Ca2+ in the wild-type retina, respectively, by performing scotopic ERG, histological, and western blot analyses. Results of the study revealed that induced UPR in the retina activates calpain-mediated signaling, and increased intracellular Ca2+ is capable of promoting retinal degeneration. A significant decline in ERG amplitudes at 6 weeks post treatment was associated with photoreceptor cell loss that occurred through calpain-activated CDK5-pJNK-Csp3/7 pathway. Similar calpain activation was found in ADRP rat retinas. A twofold increase in intracellular Ca2+ and up- and downregulations of ER membrane-associated Ca2+-regulated IP3R channels and SERCA2b transporters were detected. Therefore, sustained UPR activation in the ADRP rat retinas could promote retinal degeneration through increased intracellular Ca2+ and calpain-mediated apoptosis. PMID:26844699

  2. HCV induces transforming growth factor β1 through activation of endoplasmic reticulum stress and the unfolded protein response

    PubMed Central

    Chusri, Pattranuch; Kumthip, Kattareeya; Hong, Jian; Zhu, Chuanlong; Duan, Xiaoqiong; Jilg, Nikolaus; Fusco, Dahlene N.; Brisac, Cynthia; Schaefer, Esperance A.; Cai, Dachuan; Peng, Lee F.; Maneekarn, Niwat; Lin, Wenyu; Chung, Raymond T.

    2016-01-01

    HCV replication disrupts normal endoplasmic reticulum (ER) function and activates a signaling network called the unfolded protein response (UPR). UPR is directed by three ER transmembrane proteins including ATF6, IRE1, and PERK. HCV increases TGF-β1 and oxidative stress, which play important roles in liver fibrogenesis. HCV has been shown to induce TGF-β1 through the generation of reactive oxygen species (ROS) and p38 MAPK, JNK, ERK1/2, and NFκB-dependent pathways. However, the relationship between HCV-induced ER stress and UPR activation with TGF-β1 production has not been fully characterized. In this study, we found that ROS and JNK inhibitors block HCV up-regulation of ER stress and UPR activation. ROS, JNK and IRE1 inhibitors blocked HCV-activated NFκB and TGF-β1 expression. ROS, ER stress, NFκB, and TGF-β1 signaling were blocked by JNK specific siRNA. Knockdown IRE1 inhibited JFH1-activated NFκB and TGF-β1 activity. Knockdown of JNK and IRE1 blunted JFH1 HCV up-regulation of NFκB and TGF-β1 activation. We conclude that HCV activates NFκB and TGF-β1 through ROS production and induction of JNK and the IRE1 pathway. HCV infection induces ER stress and the UPR in a JNK-dependent manner. ER stress and UPR activation partially contribute to HCV-induced NF-κB activation and enhancement of TGF-β1. PMID:26927933

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

    PubMed

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

    2014-01-01

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

  4. The unfolded protein response and its potential role in Huntington's disease elucidated by a systems biology approach

    PubMed Central

    Kalathur, Ravi Kiran Reddy; Giner-Lamia, Joaquin; Machado, Susana; Barata, Tania; Ayasolla, Kameshwar R S; Futschik, Matthias E.

    2016-01-01

    Huntington ´s disease (HD) is a progressive, neurodegenerative disease with a fatal outcome. Although the disease-causing gene (huntingtin) has been known for over 20 years, the exact mechanisms leading to neuronal cell death are still controversial. One potential mechanism contributing to the massive loss of neurons observed in the brain of HD patients could be the unfolded protein response (UPR) activated by accumulation of misfolded proteins in the endoplasmic reticulum (ER). As an adaptive response to counter-balance accumulation of un- or misfolded proteins, the UPR upregulates transcription of chaperones, temporarily attenuates new translation, and activates protein degradation via the proteasome. However, persistent ER stress and an activated UPR can also cause apoptotic cell death. Although different studies have indicated a role for the UPR in HD, the evidence remains inconclusive. Here, we present extensive bioinformatic analyses that revealed UPR activation in different experimental HD models based on transcriptomic data. Accordingly, we have identified 53 genes, including RAB5A, HMGB1, CTNNB1, DNM1, TUBB, TSG101, EEF2, DYNC1H1, SLC12A5, ATG5, AKT1, CASP7 and SYVN1 that provide a potential link between UPR and HD. To further elucidate the potential role of UPR as a disease-relevant process, we examined its connection to apoptosis based on molecular interaction data, and identified a set of 40 genes including ADD1, HSP90B1, IKBKB, IKBKG, RPS3A and LMNB1, which seem to be at the crossroads between these two important cellular processes. Remarkably, we also found strong correlation of UPR gene expression with the length of the polyglutamine tract of Huntingtin, which is a critical determinant of age of disease onset in human HD patients pointing to the UPR as a promising target for therapeutic intervention. The study is complemented by a newly developed web-portal called UPR-HD (http://uprhd.sysbiolab.eu) that enables visualization and interactive analysis

  5. Transcriptome analysis of the unfolded protein response in hemocytes of Litopenaeus vannamei.

    PubMed

    Chen, Yi-Hong; Yuan, Feng-Hua; Bi, Hai-Tao; Zhang, Ze-Zhi; Yue, Hai-Tao; Yuan, Kai; Chen, Yong-Gui; Wen, Shao-Ping; He, Jian-Guo

    2016-07-01

    In this study, Litopenaeus vannamei was injected with double-stranded RNA (dsRNA) against L. vannamei immunoglobulin heavy chain binding protein (LvBip) to activating UPR in the hemocytes, shirmps injected dsRNA against enhanced green fluorescence protein (eGFP) as control group. And genes expression in hemocytes of then were analyzed using Illumina Hiseq 2500 (PE100). By comparing the analyzed results, 1418 unigenes were significantly upregulated, and 596 unigenes were significantly down-regulated upon UPR. Analysis of the differentially expressed genes against known databases indicated that the distribution of gene pathways between the upregulated and down-regulated genes were substantially different. A total of 208 genes of UPR system were obtained, and 69 of them were differentially expressed between the two groups. Results also showed that L. vannamei UPR was involved in various metabolic processes, such as glycometabolism, lipid metabolism, amino acid metabolism, and nucleic acid metabolism. In addition, UPR was emgaged in immune-assicoated signaling pathways, such as NF-κB signaling pathway, NOD-like receptor signaling pathway, Hippo signaling pathway, p38 MAPK signaling pathway and Wnt signaling pathway in L. vannamei. These results improved our current understanding of the L. vannamei UPR, and highlighted its importance in cell homeostasis upon environmental stress. PMID:26497095

  6. Bortezomib-induced unfolded protein response increases oncolytic HSV-1 replication resulting in synergistic, anti-tumor effects

    PubMed Central

    Yoo, Ji Young; Hurwitz, Brian S; Bolyard, Chelsea; Yu, Jun-Ge; Zhang, Jianying; Selvendiran, Karuppaiyah; Rath, Kellie S; He, Shun; Bailey, Zachary; Eaves, David; Cripe, Timothy P; Parris, Deborah S.; Caligiuri, Michael A.; Yu, Jianhua; Old, Matthew; Kaur, Balveen

    2014-01-01

    Background Bortezomib is an FDA-approved proteasome inhibitor, and oncolytic HSV-1 (oHSV) is a promising therapeutic approach for cancer. We tested the impact of combining bortezomib with oHSV for anti-tumor efficacy. Methods The synergistic interaction between oHSV and bortezomib was calculated using Chou-Talalay analysis. Viral replication was evaluated using plaque assay and immune fluorescence. Western-blot assays were used to evaluate induction of ER stress and unfolded protein response (UPR). Inhibitors targeting Hsp90 were utilized to investigate the mechanism of cell killing. Anti-tumor efficacy in vivo was evaluated using subcutaneous and intracranial tumor xenografts of glioma and head and neck cancer. Survival was analyzed by Kaplan-Meier curves and two-sided log rank test. Results Combination treatment with bortezomib and oHSV, 34.5ENVE, displayed strong synergistic interaction in ovarian cancer, head & neck cancer, glioma, and malignant peripheral nerve sheath tumor (MPNST) cells. Bortezomib treatment induced ER stress, evident by strong induction of Grp78, CHOP, PERK and IRE1α (western blot analysis) and the UPR (induction of hsp40, 70 and 90). Bortezomib treatment of cells at both sublethal and lethal doses increased viral replication (p value <0.001), but inhibition of Hsp90 ablated this response, reducing viral replication and synergistic cell killing. The combination of bortezomib and 34.5ENVE significantly enhanced anti-tumor efficacy in multiple different tumor models in vivo. Conclusions The dramatic synergy of bortezomib and 34.5ENVE is mediated by bortezomib- induced UPR and warrants future clinical testing in patients. PMID:24815720

  7. Divergent forms of endoplasmic reticulum stress trigger a robust unfolded protein response in honey bees.

    PubMed

    Johnston, Brittany A; Hooks, Katarzyna B; McKinstry, Mia; Snow, Jonathan W

    2016-03-01

    Honey bee colonies in the United States have suffered from an increased rate of die-off in recent years, stemming from a complex set of interacting stresses that remain poorly described. While we have some understanding of the physiological stress responses in the honey bee, our molecular understanding of honey bee cellular stress responses is incomplete. Thus, we sought to identify and began functional characterization of the components of the UPR in honey bees. The IRE1-dependent splicing of the mRNA for the transcription factor Xbp1, leading to translation of an isoform with more transactivation potential, represents the most conserved of the UPR pathways. Honey bees and other Apoidea possess unique features in the Xbp1 mRNA splice site, which we reasoned could have functional consequences for the IRE1 pathway. However, we find robust induction of target genes upon UPR stimulation. In addition, the IRE1 pathway activation, as assessed by splicing of Xbp1 mRNA upon UPR, is conserved. By providing foundational knowledge about the UPR in the honey bee and the relative sensitivity of this species to divergent stresses, this work stands to improve our understanding of the mechanistic underpinnings of honey bee health and disease. PMID:26699660

  8. Effects of ER stress on unfolded protein responses, cell survival, and viral replication in primary effusion lymphoma.

    PubMed

    Shigemi, Zenpei; Baba, Yusuke; Hara, Naoko; Matsuhiro, Jumpei; Kagawa, Hiroki; Watanabe, Tadashi; Fujimuro, Masahiro

    2016-01-15

    Primary effusion lymphoma (PEL), a subtype of non-Hodgkin's B-lymphoma, is an aggressive neoplasm caused by Kaposi's sarcoma-associated herpesvirus (KSHV) in immunosuppressed patients. Endoplasmic reticulum (ER) stress induces activation of the unfolded protein response (UPR), which induces expression of ER chaperones, which in turn decrease ER stress, leading to ER homeostasis. The UPR is necessary for not only ER homeostasis but also persistent infection by, and replication of, many viruses. However, the precise roles and regulation of the UPR in KSHV infection remain poorly understood. Here, we found that IRE1α and PERK were significantly downregulated in PEL cells cultured under normal conditions, compared with KSHV-uninfected B-lymphoma cells. IRE1α and PERK mRNA levels were decreased in PEL cells, and KSHV-encoded LANA and v-cyclin D led to suppressed IRE1α transcription. Thapsigargin-induced ER stress activated the UPR and increased the mRNA levels of UPR-related molecules, including IRE1α and PERK, in PEL cells. However the IRE1α and PERK mRNA levels in PEL cells were lower than those in KSHV-uninfected cells. Furthermore, ER stress induced by brefeldin A and thapsigargin dramatically reduced the viability of PEL cells, compared with KSHV-uninfected cells, and induced apoptosis of PEL cells via the pro-apoptotic UPR through expression of CHOP and activation of caspase-9. In addition to the pro-apoptotic UPR, thapsigargin-induced ER stress enhanced transcription of lytic genes, including RTA, K-bZIP and K8.1, and viral production in PEL cells resulted in induction of the lytic cycle. Thus, we demonstrated downregulation of IRE1α and PERK in PEL cells, transcriptional suppression of IRE1α by LANA and v-cyclin D, apoptosis induction in PEL cells by ER stress, and potentiation of lytic replication by ER stress. PMID:26692493

  9. Microarray analysis of nemorosone-induced cytotoxic effects on pancreatic cancer cells reveals activation of the unfolded protein response (UPR)

    PubMed Central

    Holtrup, Frank; Bauer, Andrea; Fellenberg, Kurt; Hilger, Ralf A; Wink, Michael; Hoheisel, Jörg D

    2011-01-01

    BACKGROUND AND PURPOSE Pancreatic cancer is one of the leading cancer-related causes of death due to high chemo-resistance and fast metastasation. Nemorosone, a polycyclic polyprenylated acylphloroglucinol, has recently been identified as a promising anticancer agent. Here, we examine its growth-inhibitory effects on pancreatic cancer cells. Based on transcription profiling, a molecular mode of action is proposed. EXPERIMENTAL APPROACH Nemorosone cytotoxicity was assessed by the resazurin proliferation assay on pancreatic cancer cells and fibroblasts. Apoptosis was determined by Annexin V/propidium iodide staining as well as cytochrome c and caspase activation assays. Staining with the voltage-dependent dye JC-1 and fluorescence microscopy were used to detect effects on mitochondrial membrane potential. Total RNA was isolated from treated cell lines and subjected to microarray analysis, subsequent pathway identification and modelling. Gene expression data were validated by quantitative polymerase chain reaction and siRNA-mediated gene knock-down. KEY RESULTS Nemorosone significantly inhibited cancer cell growth, induced cytochrome c release and subsequent caspase-dependent apoptosis, rapidly abolished mitochondrial membrane potential and elevated cytosolic calcium levels, while fibroblasts were largely unaffected. Expression profiling revealed 336 genes to be affected by nemorosone. A total of 75 genes were altered in all three cell lines, many of which were within the unfolded protein response (UPR) network. DNA damage inducible transcript 3 was identified as a key regulator in UPR-mediated cell death. CONCLUSIONS AND IMPLICATIONS Nemorosone could be a lead compound for the development of novel anticancer drugs amplifying the already elevated UPR level in solid tumours, thus driving them into apoptosis. This study forms the basis for further investigations identifying nemorosone's direct molecular target(s). PMID:21091652

  10. The unfolded protein response regulator GRP78/BiP as a novel target for increasing chemosensitivity in malignant gliomas.

    PubMed

    Pyrko, Peter; Schönthal, Axel H; Hofman, Florence M; Chen, Thomas C; Lee, Amy S

    2007-10-15

    Poor chemosensitivity and the development of chemoresistance remain major obstacles to successful chemotherapy of malignant gliomas. GRP78 is a key regulator of the unfolded protein response (UPR). As a Ca2+-binding molecular chaperone in the endoplasmic reticulum (ER), GRP78 maintains ER homeostasis, suppresses stress-induced apoptosis, and controls UPR signaling. We report here that GRP78 is expressed at low levels in normal adult brain, but is significantly elevated in malignant glioma specimens and human malignant glioma cell lines, correlating with their rate of proliferation. Down-regulation of GRP78 by small interfering RNA leads to a slowdown in glioma cell growth. Our studies further reveal that temozolomide, the chemotherapeutic agent of choice for treatment of malignant gliomas, leads to induction of CHOP, a major proapoptotic arm of the UPR. Knockdown of GRP78 in glioblastoma cell lines induces CHOP and activates caspase-7 in temozolomide-treated cells. Colony survival assays further establish that knockdown of GRP78 lowers resistance of glioma cells to temozolomide, and, conversely, overexpression of GRP78 confers higher resistance. Knockdown of GRP78 also sensitizes glioma cells to 5-fluorouracil and CPT-11. Treatment of glioma cells with (-)-epigallocatechin gallate, which targets the ATP-binding domain of GRP78 and blocks its protective function, sensitizes glioma cells to temozolomide. These results identify a novel chemoresistance mechanism in malignant gliomas and show that combination of drugs capable of suppressing GRP78 with conventional agents such as temozolomide might represent a novel approach to eliminate residual tumor cells after surgery and increase the effectiveness of malignant glioma chemotherapy. PMID:17942911

  11. An ER-directed transcriptional response to unfolded protein stress in the absence of conserved sensor-transducer proteins in Giardia lamblia.

    PubMed

    Spycher, Cornelia; Herman, Emily K; Morf, Laura; Qi, Weihong; Rehrauer, Hubert; Aquino Fournier, Catharine; Dacks, Joel B; Hehl, Adrian B

    2013-05-01

    The protozoan Giardia lamblia has a minimized organelle repertoire, and most strikingly lacks a classical stacked Golgi apparatus. Nevertheless, Giardia trophozoites constitutively secrete variant surface proteins, and dramatically increase the volume of protein secretion during differentiation to cysts. Eukaryotic cells have evolved an elaborate system for quality control (QC) of protein folding and capacity in the endoplasmic reticulum (ER). Upon ER-overload, an unfolded protein response (UPR) is triggered on transcriptional/translational level aiming at alleviating ER stress. In Giardia, a minimized secretory machinery and absence of glycan-dependent QC suggests that a genetically conserved UPR (or functional equivalent) to cope with insults to the secretory system has been eliminated. We tested this hypothesis of UPR elimination by profiling the transcriptional response during induced ER-folding stress. We show that on the contrary, ER-folding stress triggers a stressor-specific, ER-directed response with upregulation of only ~ 30 genes, with different kinetics and scope compared with the UPR of other eukaryotes. Computational genomics revealed conserved cis-acting motifs in upstream regions of responder genes capable of stressor-specific gene regulation in transfected cells. Interestingly, the sensors/transducers of folding stress, well conserved in model eukaryotes, are absent in Giardia suggesting the presence of a novel version of this essential eukaryotic function. PMID:23617761

  12. Proteins Take up Water Before Unfolding.

    PubMed

    Groot, Carien C M; Bakker, Huib J

    2016-05-19

    Proteins perform specific biological functions that strongly depend on their three-dimensional structure. This three-dimensional structure, i.e. the way the protein folds, is strongly determined by the interaction between the protein and the water solvent. We study the dynamics of water in aqueous solutions of several globular proteins at different degrees of urea-induced unfolding, using polarization-resolved femtosecond infrared spectroscopy. We observe that a fraction of the water molecules is strongly slowed down by their interaction with the protein surface. By monitoring the slow water fraction we can directly probe the amount of water-exposed protein surface. We find that at mild denaturing conditions, the water-exposed surface increases by almost 50%, while the secondary structure is still intact. This finding indicates that protein unfolding starts with the protein structure becoming less tight, thereby allowing water to enter. PMID:27120433

  13. Nuclear Matrix Protein 4 Is a Novel Regulator of Ribosome Biogenesis and Controls the Unfolded Protein Response via Repression of Gadd34 Expression.

    PubMed

    Young, Sara K; Shao, Yu; Bidwell, Joseph P; Wek, Ronald C

    2016-06-24

    The unfolded protein response (UPR) maintains protein homeostasis by governing the processing capacity of the endoplasmic reticulum (ER) to manage ER client loads; however, key regulators within the UPR remain to be identified. Activation of the UPR sensor PERK (EIFAK3/PEK) results in the phosphorylation of the α subunit of eIF2 (eIF2α-P), which represses translation initiation and reduces influx of newly synthesized proteins into the overloaded ER. As part of this adaptive response, eIF2α-P also induces a feedback mechanism through enhanced transcriptional and translational expression of Gadd34 (Ppp1r15A),which targets type 1 protein phosphatase for dephosphorylation of eIF2α-P to restore protein synthesis. Here we describe a novel mechanism by which Gadd34 expression is regulated through the activity of the zinc finger transcription factor NMP4 (ZNF384, CIZ). NMP4 functions to suppress bone anabolism, and we suggest that this occurs due to decreased protein synthesis of factors involved in bone formation through NMP4-mediated dampening of Gadd34 and c-Myc expression. Loss of Nmp4 resulted in an increase in c-Myc and Gadd34 expression that facilitated enhanced ribosome biogenesis and global protein synthesis. Importantly, protein synthesis was sustained during pharmacological induction of the UPR through a mechanism suggested to involve GADD34-mediated dephosphorylation of eIF2α-P. Sustained protein synthesis sensitized cells to pharmacological induction of the UPR, and the observed decrease in cell viability was restored upon inhibition of GADD34 activity. We conclude that NMP4 is a key regulator of ribosome biogenesis and the UPR, which together play a central role in determining cell viability during endoplasmic reticulum stress. PMID:27129771

  14. BH3-only proteins are part of a regulatory network that control the sustained signalling of the unfolded protein response sensor IRE1α

    PubMed Central

    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-Muiños, Felipe X; Kroemer, Guido; Hetz, Claudio

    2012-01-01

    Adaptation to endoplasmic reticulum (ER) stress depends on the activation of the unfolded protein response (UPR) stress sensor inositol-requiring enzyme 1α (IRE1α), 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 IRE1α interactor. Immun oprecipitation experiments confirmed this interaction and further detected the association of IRE1α 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 IRE1α, 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 IRE1α 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. PMID:22510886

  15. Genetic evidence for a role of BiP/Kar2 that regulates Ire1 in response to accumulation of unfolded proteins.

    PubMed

    Kimata, Yukio; Kimata, Yuki I; Shimizu, Yusuke; Abe, Hiroshi; Farcasanu, Ileana C; Takeuchi, Masato; Rose, Mark D; Kohno, Kenji

    2003-06-01

    In the unfolded protein response (UPR) signaling pathway, accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates a transmembrane kinase/ribonuclease Ire1, which causes the transcriptional induction of ER-resident chaperones, including BiP/Kar2. It was previously hypothesized that BiP/Kar2 plays a direct role in the signaling mechanism. In this model, association of BiP/Kar2 with Ire1 represses the UPR pathway while under conditions of ER stress, BiP/Kar2 dissociation leads to activation. To test this model, we analyzed five temperature-sensitive alleles of the yeast KAR2 gene. When cells carrying a mutation in the Kar2 substrate-binding domain were incubated at the restrictive temperature, association of Kar2 to Ire1 was disrupted, and the UPR pathway was activated even in the absence of extrinsic ER stress. Conversely, cells carrying a mutation in the Kar2 ATPase domain, in which Kar2 poorly dissociated from Ire1 even in the presence of tunicamycin, a potent inducer of ER stress, were unable to activate the pathway. Our findings provide strong evidence in support of BiP/Kar2-dependent Ire1 regulation model and suggest that Ire1 associates with Kar2 as a chaperone substrate. We speculate that recognition of unfolded proteins is based on their competition with Ire1 for binding with BiP/Kar2. PMID:12808051

  16. Genetic Evidence for a Role of BiP/Kar2 That Regulates Ire1 in Response to Accumulation of Unfolded Proteins

    PubMed Central

    Kimata, Yukio; Kimata, Yuki I.; Shimizu, Yusuke; Abe, Hiroshi; Farcasanu, Ileana C.; Takeuchi, Masato; Rose, Mark D.; Kohno, Kenji

    2003-01-01

    In the unfolded protein response (UPR) signaling pathway, accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates a transmembrane kinase/ribonuclease Ire1, which causes the transcriptional induction of ER-resident chaperones, including BiP/Kar2. It was previously hypothesized that BiP/Kar2 plays a direct role in the signaling mechanism. In this model, association of BiP/Kar2 with Ire1 represses the UPR pathway while under conditions of ER stress, BiP/Kar2 dissociation leads to activation. To test this model, we analyzed five temperature-sensitive alleles of the yeast KAR2 gene. When cells carrying a mutation in the Kar2 substrate-binding domain were incubated at the restrictive temperature, association of Kar2 to Ire1 was disrupted, and the UPR pathway was activated even in the absence of extrinsic ER stress. Conversely, cells carrying a mutation in the Kar2 ATPase domain, in which Kar2 poorly dissociated from Ire1 even in the presence of tunicamycin, a potent inducer of ER stress, were unable to activate the pathway. Our findings provide strong evidence in support of BiP/Kar2-dependent Ire1 regulation model and suggest that Ire1 associates with Kar2 as a chaperone substrate. We speculate that recognition of unfolded proteins is based on their competition with Ire1 for binding with BiP/Kar2. PMID:12808051

  17. Role of the Unfolded Protein Response in Regulating the Mucin-Dependent Filamentous-Growth Mitogen-Activated Protein Kinase Pathway

    PubMed Central

    Adhikari, Hema; Vadaie, Nadia; Chow, Jacky; Caccamise, Lauren M.; Chavel, Colin A.; Li, Boyang; Bowitch, Alexander; Stefan, Christopher J.

    2015-01-01

    Signaling mucins are evolutionarily conserved regulators of signal transduction pathways. The signaling mucin Msb2p regulates the Cdc42p-dependent mitogen-activated protein kinase (MAPK) pathway that controls filamentous growth in yeast. The cleavage and release of the glycosylated inhibitory domain of Msb2p is required for MAPK activation. We show here that proteolytic processing of Msb2p was induced by underglycosylation of its extracellular domain. Cleavage of underglycosylated Msb2p required the unfolded protein response (UPR), a quality control (QC) pathway that operates in the endoplasmic reticulum (ER). The UPR regulator Ire1p, which detects misfolded/underglycosylated proteins in the ER, controlled Msb2p cleavage by regulating transcriptional induction of Yps1p, the major protease that processes Msb2p. Accordingly, the UPR was required for differentiation to the filamentous cell type. Cleavage of Msb2p occurred in conditional trafficking mutants that trap secretory cargo in the endomembrane system. Processed Msb2p was delivered to the plasma membrane, and its turnover by the ubiquitin ligase Rsp5p and ESCRT attenuated the filamentous-growth pathway. We speculate that the QC pathways broadly regulate signaling glycoproteins and their cognate pathways by recognizing altered glycosylation patterns that can occur in response to extrinsic cues. PMID:25666509

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

    PubMed

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

    2013-06-15

    Dehydroxymethylepoxyquinomicin (DHMEQ) is a low-m.w. compound that strongly inhibits NF-κB. Previous reports showed that DHMEQ directly binds to specific cysteine residues of NF-κB 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-κB. We found that sustained exposure of renal tubular cells to DHMEQ blocked TNF-α- and IL-1β-induced TGF-β-activated kinase 1 (TAK1) phosphorylation, a crucial event for NF-κB activation upstream of IκB 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-κB activation; and 3) scavenging of ROS attenuated the suppressive effects of DHMEQ on TAK1 and NF-κB. 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-κB. The UPR-mediated inhibition of NF-κB occurred downstream of degradation of IκBα and phosphorylation of p65. Subsequent experiments revealed the following: 1) DHMEQ caused selective induction of C/EBPβ through the UPR; 2) overexpression of C/EBPβ suppressed activation of NF-κB; 3) knockdown of C/EBPβ attenuated the inhibitory effect of DHMEQ; and 4) DHMEQ-induced expression of C/EBPβ did not affect TNF-α-triggered degradation of IκBα and phosphorylation of p65. These results suggest that, in addition to its known effect on nuclear translocation of NF-κB, DHMEQ interferes with the cytokine-induced NF-κB signaling via generation of ROS at both upstream and downstream of the IκB kinase-IκB level. PMID:23690471

  19. The Unfolded Protein Response Element IRE1α Senses Bacterial Proteins Invading the ER to Activate RIG-I and Innate Immune Signaling

    PubMed Central

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

    2013-01-01

    SUMMARY The plasma membrane and all membrane-bound organelles except for the Golgi and endoplasmic reticulum (ER) are equipped with pattern-recognition molecules to sense microbes or their products and induce innate immunity for host defense. Here, we report that inositol-requiring-1α (IRE1α), an ER protein that signals in the unfolded protein response (UPR), is activated to induce inflammation by binding a portion of cholera toxin as it co-opts the ER to cause disease. Other known UPR transducers, including the IRE1α-dependent transcription factor XBP1, are dispensable for this signaling. The inflammatory response depends instead on the RNase activity of IRE1α to degrade endogenous mRNA, a process termed regulated IRE1α-dependent decay (RIDD) of mRNA. The mRNA fragments produced engage retinoic-acid inducible gene 1 (RIG-I), a cyto-solic sensor of RNA viruses, to activate NF-κB and interferon pathways. We propose IRE1α provides for a generalized mechanism of innate immune surveillance originating within the ER lumen. PMID:23684307

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

    SciTech Connect

    Hayakawa, Kunihiro; Hiramatsu, Nobuhiko; Okamura, Maro; Yao, Jian; Paton, Adrienne W.; Paton, James C.; Kitamura, Masanori

    2008-01-04

    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.

  1. Unfolding Kinetics of Egg Protein

    NASA Astrophysics Data System (ADS)

    Sharma, Dipti

    2011-03-01

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

  2. Methionine Sulfoxide Reductases Preferentially Reduce Unfolded Oxidized Proteins and Protect Cells from Oxidative Protein Unfolding*

    PubMed Central

    Tarrago, Lionel; Kaya, Alaattin; Weerapana, Eranthie; Marino, Stefano M.; Gladyshev, Vadim N.

    2012-01-01

    Reduction of methionine sulfoxide (MetO) residues in proteins is catalyzed by methionine sulfoxide reductases A (MSRA) and B (MSRB), which act in a stereospecific manner. Catalytic properties of these enzymes were previously established mostly using low molecular weight MetO-containing compounds, whereas little is known about the catalysis of MetO reduction in proteins, the physiological substrates of MSRA and MSRB. In this work we exploited an NADPH-dependent thioredoxin system and determined the kinetic parameters of yeast MSRA and MSRB using three different MetO-containing proteins. Both enzymes showed Michaelis-Menten kinetics with the Km lower for protein than for small MetO-containing substrates. MSRA reduced both oxidized proteins and low molecular weight MetO-containing compounds with similar catalytic efficiencies, whereas MSRB was specialized for the reduction of MetO in proteins. Using oxidized glutathione S-transferase as a model substrate, we showed that both MSR types were more efficient in reducing MetO in unfolded than in folded proteins and that their activities increased with the unfolding state. Biochemical quantification and identification of MetO reduced in the substrates by mass spectrometry revealed that the increased activity was due to better access to oxidized MetO in unfolded proteins; it also showed that MSRA was intrinsically more active with unfolded proteins regardless of MetO availability. Moreover, MSRs most efficiently protected cells from oxidative stress that was accompanied by protein unfolding. Overall, this study indicates that MSRs serve a critical function in the folding process by repairing oxidatively damaged nascent polypeptides and unfolded proteins. PMID:22628550

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

    SciTech Connect

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

    2010-03-08

    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.

  4. CHAC1/MGC4504 is a novel proapoptotic component of the unfolded protein response, downstream of the ATF4-ATF3-CHOP cascade.

    PubMed

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

    2009-01-01

    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. PMID:19109178

  5. 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

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

    2001-01-01

    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. PMID:11256944

  6. Cross-talk unfolded: MARCKS proteins.

    PubMed Central

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

    2002-01-01

    The proteins of the MARCKS (myristoylated alanine-rich C kinase substrate) family were first identified as prominent substrates of protein kinase C (PKC). Since then, these proteins have been implicated in the regulation of brain development and postnatal survival, cellular migration and adhesion, as well as endo-, exo- and phago-cytosis, and neurosecretion. The effector domain of MARCKS proteins is phosphorylated by PKC, binds to calmodulin and contributes to membrane binding. This multitude of mutually exclusive interactions allows cross-talk between the signal transduction pathways involving PKC and calmodulin. This review focuses on recent, mostly biophysical and biochemical results renewing interest in this protein family. MARCKS membrane binding is now understood at the molecular level. From a structural point of view, there is a consensus emerging that MARCKS proteins are "natively unfolded". Interestingly, domains similar to the effector domain have been discovered in other proteins. Furthermore, since the effector domain enhances the polymerization of actin in vitro, MARCKS proteins have been proposed to mediate regulation of the actin cytoskeleton. However, the recent observations that MARCKS might serve to sequester phosphatidylinositol 4,5-bisphosphate in the plasma membrane of unstimulated cells suggest an alternative model for the control of the actin cytoskeleton. While myristoylation is classically considered to be a co-translational, irreversible event, new reports on MARCKS proteins suggest a more dynamic picture of this protein modification. Finally, studies with mice lacking MARCKS proteins have investigated the functions of these proteins during embryonic development in the intact organism. PMID:11829734

  7. The unfolded protein response is activated in disease-affected brain regions in progressive supranuclear palsy and Alzheimer’s disease

    PubMed Central

    2013-01-01

    Background Progressive supranuclear palsy (PSP) is a neurodegenerative disorder pathologically characterized by intracellular tangles of hyperphosphorylated tau protein distributed throughout the neocortex, basal ganglia, and brainstem. A genome-wide association study identified EIF2AK3 as a risk factor for PSP. EIF2AK3 encodes PERK, part of the endoplasmic reticulum’s (ER) unfolded protein response (UPR). PERK is an ER membrane protein that senses unfolded protein accumulation within the ER lumen. Recently, several groups noted UPR activation in Alzheimer’s disease (AD), Parkinson’s disease (PD), amyotrophic lateral sclerosis, multiple system atrophy, and in the hippocampus and substantia nigra of PSP subjects. Here, we evaluate UPR PERK activation in the pons, medulla, midbrain, hippocampus, frontal cortex and cerebellum in subjects with PSP, AD, and in normal controls. Results We found UPR activation primarily in disease-affected brain regions in both disorders. In PSP, the UPR was primarily activated in the pons and medulla and to a much lesser extent in the hippocampus. In AD, the UPR was extensively activated in the hippocampus. We also observed UPR activation in the hippocampus of some elderly normal controls, severity of which positively correlated with both age and tau pathology but not with Aβ plaque burden. Finally, we evaluated EIF2AK3 coding variants that influence PERK activation. We show that a haplotype associated with increased PERK activation is genetically associated with increased PSP risk. Conclusions The UPR is activated in disease affected regions in PSP and the genetic evidence shows that this activation increases risk for PSP and is not a protective response. PMID:24252572

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

    PubMed Central

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

    2014-01-01

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

  9. Electrospray Ionization-Induced Protein Unfolding

    NASA Astrophysics Data System (ADS)

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

    2012-12-01

    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.

  10. 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

    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

    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

  11. Unfolding of β-Sheet Proteins in SDS

    PubMed Central

    Nielsen, Mette M.; Andersen, Kell K.; Westh, Peter; Otzen, Daniel E.

    2007-01-01

    β-Sheet proteins are particularly resistant to denaturation by sodium dodecyl sulfate (SDS). Here we compare unfolding of two β-sandwich proteins TNfn3 and TII27 in SDS. The two proteins show different surface electrostatic potential. Correspondingly, TII27 unfolds below the critical micelle concentration via the formation of hemimicelles on the protein surface, whereas TNfn3 only unfolds around the critical micelle concentration. Isothermal titration calorimetry confirms that unfolding of TII27 sets in at lower SDS concentrations, although the total number of bound SDS molecules is similar at the end of unfolding. In mixed micelles with the nonionic detergent dodecyl maltoside, where the concentration of monomeric SDS is insignificant, the behavior of the two proteins converges. TII27 unfolds more slowly than TNfn3 in SDS and follows a two-mode behavior. Additionally TNfn3 shows inhibition of SDS unfolding at intermediate SDS concentrations. Mutagenic analysis suggests that the overall unfolding mechanism is similar to that observed in denaturant for both proteins. Our data confirm the kinetic robustness of β-sheet proteins toward SDS. We suggest this is related to the inability of SDS to induce significant amounts of α-helix structure in these proteins as part of the denaturation process, forcing the protein to denature by global rather than local unfolding. PMID:17351005

  12. Treatment with the HIV protease inhibitor nelfinavir triggers the unfolded protein response and may overcome proteasome inhibitor resistance of multiple myeloma in combination with bortezomib: a phase I trial (SAKK 65/08)

    PubMed Central

    Driessen, Christoph; Kraus, Marianne; Joerger, Markus; Rosing, Hilde; Bader, Jürgen; Hitz, Felicitas; Berset, Catherine; Xyrafas, Alexandros; Hawle, Hanne; Berthod, Gregoire; Overkleeft, Hermann S.; Sessa, Christiana; Huitema, Alwin; Pabst, Thomas; von Moos, Roger; Hess, Dagmar; Mey, Ulrich J.M.

    2016-01-01

    Downregulation of the unfolded protein response mediates proteasome inhibitor resistance in multiple myeloma. The Human Immunodeficieny Virus protease inhibitor nelfinavir activates the unfolded protein response in vitro. We determined dose-limiting toxicity and recommended dose for phase II of nelfinavir in combination with the proteasome inhibitor bortezomib. Twelve patients with advanced hematologic malignancies were treated with nelfinavir (2500–5000 mg/day p.o., days 1–14, 3+3 dose escalation) and bortezomib (1.3 mg/m2, days 1, 4, 8, 11; 21-day cycles). A run in phase with nelfinavir monotherapy allowed pharmakokinetic/pharmakodynamic assessment of nelfinavir in the presence or absence of concomittant bortezomib. End points included dose-limiting toxicity, activation of the unfolded protein response, proteasome activity, toxicity and response to trial treatment. Nelfinavir 2×2500 mg was the recommended phase II dose identified. Nelfinavir alone significantly up-regulated expression of proteins related to the unfolded protein response in peripheral blood mononuclear cells and inhibited proteasome activity. Of 10 evaluable patients in the dose escalation cohort, 3 achieved a partial response, 4 stable disease for 2 cycles or more, while 3 had progressive disease as best response. In an exploratory extension cohort with 6 relapsed, bortezomib-refractory, lenalidomide-resistant myeloma patients treated at the recommended phase II dose, 3 reached a partial response, 2 a minor response, and one progressive disease. The combination of nelfinavir with bortezomib is safe and shows promising activity in advanced, bortezomib-refractory multiple myeloma. Induction of the unfolded protein response by nelfinavir may overcome the biological features of proteasome inhibitor resistance. PMID:26659919

  13. Autism-associated R451C mutation in neuroligin3 leads to activation of the unfolded protein response in a PC12 Tet-On inducible system.

    PubMed

    Ulbrich, Lisa; Favaloro, Flores Lietta; Trobiani, Laura; Marchetti, Valentina; Patel, Vruti; Pascucci, Tiziana; Comoletti, Davide; Marciniak, Stefan J; De Jaco, Antonella

    2016-02-15

    Several forms of monogenic heritable autism spectrum disorders are associated with mutations in the neuroligin genes. The autism-linked substitution R451C in neuroligin3 induces local misfolding of its extracellular domain, causing partial retention in the ER (endoplasmic reticulum) of expressing cells. We have generated a PC12 Tet-On cell model system with inducible expression of wild-type or R451C neuroligin3 to investigate whether there is activation of the UPR (unfolded protein response) as a result of misfolded protein retention. As a positive control for protein misfolding, we also expressed the mutant G221R neuroligin3, which is known to be completely retained within the ER. Our data show that overexpression of either R451C or G221R mutant proteins leads to the activation of all three signalling branches of the UPR downstream of the stress sensors ATF6 (activating transcription factor 6), IRE1 (inositol-requiring enzyme 1) and PERK [PKR (dsRNA-dependent protein kinase)-like endoplasmic reticulum kinase]. Each branch displayed different activation profiles that partially correlated with the degree of misfolding caused by each mutation. We also show that up-regulation of BiP (immunoglobulin heavy-chain-binding protein) and CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein] was induced by both mutant proteins but not by wild-type neuroligin3, both in proliferative cells and cells differentiated to a neuron-like phenotype. Collectively, our data show that mutant R451C neuroligin3 activates the UPR in a novel cell model system, suggesting that this cellular response may have a role in monogenic forms of autism characterized by misfolding mutations. PMID:26621873

  14. Autism-associated R451C mutation in neuroligin3 leads to activation of the unfolded protein response in a PC12 Tet-On inducible system

    PubMed Central

    Ulbrich, Lisa; Favaloro, Flores Lietta; Trobiani, Laura; Marchetti, Valentina; Patel, Vruti; Pascucci, Tiziana; Comoletti, Davide; Marciniak, Stefan J.; De Jaco, Antonella

    2015-01-01

    Several forms of monogenic heritable autism spectrum disorders are associated with mutations in the neuroligin genes. The autism-linked substitution R451C in neuroligin3 induces local misfolding of its extracellular domain, causing partial retention in the ER (endoplasmic reticulum) of expressing cells. We have generated a PC12 Tet-On cell model system with inducible expression of wild-type or R451C neuroligin3 to investigate whether there is activation of the UPR (unfolded protein response) as a result of misfolded protein retention. As a positive control for protein misfolding, we also expressed the mutant G221R neuroligin3, which is known to be completely retained within the ER. Our data show that overexpression of either R451C or G221R mutant proteins leads to the activation of all three signalling branches of the UPR downstream of the stress sensors ATF6 (activating transcription factor 6), IRE1 (inositol-requiring enzyme 1) and PERK [PKR (dsRNA-dependent protein kinase)-like endoplasmic reticulum kinase]. Each branch displayed different activation profiles that partially correlated with the degree of misfolding caused by each mutation. We also show that up-regulation of BiP (immunoglobulin heavy-chain-binding protein) and CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein] was induced by both mutant proteins but not by wild-type neuroligin3, both in proliferative cells and cells differentiated to a neuron-like phenotype. Collectively, our data show that mutant R451C neuroligin3 activates the UPR in a novel cell model system, suggesting that this cellular response may have a role in monogenic forms of autism characterized by misfolding mutations. PMID:26621873

  15. Sestrin2 is induced by glucose starvation via the unfolded protein response and protects cells from non-canonical necroptotic cell death

    PubMed Central

    Ding, Boxiao; Parmigiani, Anita; Divakaruni, Ajit S.; Archer, Kellie; Murphy, Anne N.; Budanov, Andrei V.

    2016-01-01

    Sestrin2 is a member of a family of stress responsive proteins, which controls cell viability via antioxidant activity and regulation of the mammalian target of rapamycin protein kinase (mTOR). Sestrin2 is induced by different stress insults, which diminish ATP production and induce energetic stress in the cells. Glucose is a critical substrate for ATP production utilized via glycolysis and mitochondrial respiration as well as for glycosylation of newly synthesized proteins in the endoplasmic reticulum (ER) and Golgi. Thus, glucose starvation causes both energy deficiency and activation of ER stress followed by the unfolding protein response (UPR). Here, we show that UPR induces Sestrin2 via ATF4 and NRF2 transcription factors and demonstrate that Sestrin2 protects cells from glucose starvation-induced cell death. Sestrin2 inactivation sensitizes cells to necroptotic cell death that is associated with a decline in ATP levels and can be suppressed by Necrostatin 7. We propose that Sestrin2 protects cells from glucose starvation-induced cell death via regulation of mitochondrial homeostasis. PMID:26932729

  16. Oxygen-Glucose Deprivation (OGD) Modulates the Unfolded Protein Response (UPR) and Inflicts Autophagy in a PC12 Hypoxia Cell Line Model.

    PubMed

    Vavilis, Theofanis; Delivanoglou, Nikoleta; Aggelidou, Eleni; Stamoula, Eleni; Mellidis, Kyriakos; Kaidoglou, Aikaterini; Cheva, Angeliki; Pourzitaki, Chryssa; Chatzimeletiou, Katerina; Lazou, Antigone; Albani, Maria; Kritis, Aristeidis

    2016-07-01

    Hypoxia is the lack of sufficient oxygenation of tissue, imposing severe stress upon cells. It is a major feature of many pathological conditions such as stroke, traumatic brain injury, cerebral hemorrhage, perinatal asphyxia and can lead to cell death due to energy depletion and increased free radical generation. The present study investigates the effect of hypoxia on the unfolded protein response of the cell (UPR), utilizing a 16-h oxygen-glucose deprivation protocol (OGD) in a PC12 cell line model. Expression of glucose-regulated protein 78 (GRP78) and glucose-regulated protein 94 (GRP94), key players of the UPR, was studied along with the expression of glucose-regulated protein 75 (GRP75), heat shock cognate 70 (HSC70), and glyceraldehyde 3-phosphate dehydrogenase, all with respect to the cell death mechanism(s). Cells subjected to OGD displayed upregulation of GRP78 and GRP94 and concurrent downregulation of GRP75. These findings were accompanied with minimal apoptotic cell death and induction of autophagy. The above observation warrants further investigation to elucidate whether autophagy acts as a pro-survival mechanism that upon severe and prolonged hypoxia acts as a concerted cell response leading to cell death. In our OGD model, hypoxia modulates UPR and induces autophagy. PMID:26239244

  17. Transcription factor ATF4 directs basal and stress-induced gene expression in the unfolded protein response and cholesterol metabolism in the liver

    PubMed Central

    Fusakio, Michael E.; Willy, Jeffrey A.; Wang, Yongping; Mirek, Emily T.; Al Baghdadi, Rana J. T.; Adams, Christopher M.; Anthony, Tracy G.; Wek, Ronald C.

    2016-01-01

    Disturbances in protein folding and membrane compositions in the endoplasmic reticulum (ER) elicit the unfolded protein response (UPR). Each of three UPR sensory proteins—PERK (PEK/EIF2AK3), IRE1, and ATF6—is activated by ER stress. PERK phosphorylation of eIF2 represses global protein synthesis, lowering influx of nascent polypeptides into the stressed ER, coincident with preferential translation of ATF4 (CREB2). In cultured cells, ATF4 induces transcriptional expression of genes directed by the PERK arm of the UPR, including genes involved in amino acid metabolism, resistance to oxidative stress, and the proapoptotic transcription factor CHOP (GADD153/DDIT3). In this study, we characterize whole-body and tissue-specific ATF4-knockout mice and show in liver exposed to ER stress that ATF4 is not required for CHOP expression, but instead ATF6 is a primary inducer. RNA-Seq analysis indicates that ATF4 is responsible for a small portion of the PERK-dependent UPR genes and reveals a requirement for expression of ATF4 for expression of genes involved in oxidative stress response basally and cholesterol metabolism both basally and under stress. Consistent with this pattern of gene expression, loss of ATF4 resulted in enhanced oxidative damage, and increased free cholesterol in liver under stress accompanied by lowered cholesterol in sera. PMID:26960794

  18. Differential unfolded protein response during Chikungunya and Sindbis virus infection: CHIKV nsP4 suppresses eIF2α phosphorylation

    PubMed Central

    2013-01-01

    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 (eIF2α), 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

  19. Assessment of the effect of sphingosine kinase inhibitors on apoptosis,unfolded protein response and autophagy of T-cell acute lymphoblastic leukemia cells; indications for novel therapeutics.

    PubMed

    Evangelisti, Cecilia; Evangelisti, Camilla; Teti, Gabriella; Chiarini, Francesca; Falconi, Mirella; Melchionda, Fraia; Pession, Andrea; Bertaina, Alice; Locatelli, Franco; McCubrey, James A; Beak, Dong Jae; Bittman, Robert; Pyne, Susan; Pyne, Nigel J; Martelli, Alberto M

    2014-09-15

    Sphingosine 1-phosphate (S1P) is a bioactive lipid that is formed by the phosphorylation of sphingosine and catalysed by sphingosine kinase 1 (SK1) or sphingosine kinase 2 (SK2). Sphingosine kinases play a fundamental role in many signaling pathways associated with cancer, suggesting that proteins belonging to this signaling network represent potential therapeutic targets. Over the last years, many improvements have been made in the treatment of T-cell acute lymphoblastic leukemia (T-ALL); however, novel and less toxic therapies are still needed, especially for relapsing and chemo-resistant patients. Here, we analyzed the therapeutic potential of SKi and ROMe, a sphingosine kinase 1 and 2 inhibitor and SK2-selective inhibitor, respectively. While SKi induced apoptosis, ROMe initiated an autophagic cell death in our in vitro cell models. SKi treatment induced an increase in SK1 protein levels in Molt-4 cells, whereas it activated the endoplasmic reticulum (ER) stress/unfolded protein response (UPR) pathway in Jurkat and CEM-R cells as protective mechanisms in a sub-population of T-ALL cells. Interestingly, we observed a synergistic effect of SKi with the classical chemotherapeutic drug vincristine. In addition, we reported that SKi affected signaling cascades implicated in survival, proliferation and stress response of cells. These findings indicate that SK1 or SK2 represent potential targets for treating T-ALL. PMID:25226616

  20. The unfolded protein response and cellular senescence. A review in the theme: cellular mechanisms of endoplasmic reticulum stress signaling in health and disease.

    PubMed

    Pluquet, Olivier; Pourtier, Albin; Abbadie, Corinne

    2015-03-15

    The endoplasmic reticulum (ER) is a multifunctional organelle critical for the proper folding and assembly of secreted and transmembrane proteins. Perturbations of ER functions cause ER stress, which activates a coordinated system of transcriptional and translational controls called the unfolded protein response (UPR), to cope with accumulation of misfolded proteins and proteotoxicity. It results in ER homeostasis restoration or in cell death. Senescence is a complex cell phenotype induced by several stresses such as telomere attrition, DNA damage, oxidative stress, and activation of some oncogenes. It is mainly characterized by a cell enlargement, a permanent cell-cycle arrest, and the production of a secretome enriched in proinflammatory cytokines and components of the extracellular matrix. Senescent cells accumulate with age in tissues and are suspected to play a role in age-associated diseases. Since senescence is a stress response, the question arises of whether an ER stress could occur concomitantly with senescence and participate in the onset or maintenance of the senescent features. Here, we described the interconnections between the UPR signaling and the different aspects of the cellular senescence programs and discuss the implication of UPR modulations in this context. PMID:25540175

  1. The unfolded protein response and programmed cell death are induced by expression of Garlic virus X p11 in Nicotiana benthamiana.

    PubMed

    Lu, Yuwen; Yin, Mingyuan; Wang, Xiaodan; Chen, Binghua; Yang, Xue; Peng, Jiejun; Zheng, Hongying; Zhao, Jinping; Lin, Lin; Yu, Chulang; MacFarlane, Stuart; He, Jianqing; Liu, Yong; Chen, Jianping; Dai, Liangying; Yan, Fei

    2016-06-01

    Garlic virus X (GarVX) ORF3 encodes a p11 protein, which contributes to virus cell-to-cell movement and forms granules on the endoplasmic reticulum (ER) in Nicotiana benthamiana. Expression of p11 either from a binary vector, PVX or TMV induced ER stress and the unfolded protein response (UPR), as demonstrated by an increase in transcription of the ER luminal binding protein (BiP) and bZIP60 genes. UPR-related programmed cell death (PCD) was elicited by PVX : p11 or TMV : p11 in systemic infected leaves. Examination of p11 mutants with deletions of two transmembrane domains (TM) revealed that both were required for generating granules and for inducing necrosis. TRV-based VIGS was used to investigate the correlation between bZIP60 expression and p11-induced UPR-related PCD. Less necrosis was observed on local and systemic leaves of bZIP60 knockdown plants when infected with PVXp11, suggesting that bZIP60 plays an important role in the UPR-related PCD response to p11 in N. benthamiana. PMID:27011387

  2. Interferon-γ-Induced Unfolded Protein Response in Conjunctival Goblet Cells as a Cause of Mucin Deficiency in Sjögren Syndrome.

    PubMed

    Coursey, Terry G; Tukler Henriksson, Johanna; Barbosa, Flavia L; de Paiva, Cintia S; Pflugfelder, Stephen C

    2016-06-01

    Goblet cells (GCs) are specialized secretory cells that produce mucins and a variety of other proteins. Significant conjunctival GC loss occurs in both experimental dry eye models and patients with keratoconjunctivitis sicca due to the induction of interferon (IFN)-γ. With the use of a primary murine culture model, we found that GCs are highly sensitive to IFN-γ with significantly reduced proliferation and altered structure with low concentrations. GC cultures treated with IFN-γ have increased gene expression of Muc2 and Muc5AC but do not express these mucin glycoproteins. We hypothesized that IFN-γ induces endoplasmic reticulum stress and the unfolded protein response (UPR) in GCs. Cultures treated with IFN-γ increased expression of UPR-associated genes and proteins. Increased GRP78 and sXBP1 expression was found in experimental dry eye and Sjögren syndrome models and was GC specific. Increased GRP78 was also found in the conjunctiva of patients with Sjögren syndrome at the gene and protein levels. Treatment with dexamethasone inhibited expression of UPR-associated genes and increased mucin production. These results indicate that induction of UPR by IFN-γ is an important cause of GC-associated mucin deficiency observed in aqueous-deficient dry eye. Therapies to block the effects of IFN-γ on the metabolically active endoplasmic reticulum in these cells might enhance synthesis and secretion of the protective GC mucins on the ocular surface. PMID:27085137

  3. Human βA3/A1-crystallin splicing mutation causes cataracts by activating the unfolded protein response and inducing apoptosis in differentiating lens fiber cells.

    PubMed

    Ma, Zhiwei; Yao, Wenliang; Chan, Chi-Chao; Kannabiran, Chitra; Wawrousek, Eric; Hejtmancik, J Fielding

    2016-06-01

    βγ-Crystallins, having a uniquely stable two domain four Greek key structure, are crucial for transparency of the eye lens,. Mutations in lens crystallins have been proposed to cause cataract formation by a variety of mechanisms most of which involve destabilization of the protein fold. The underlying molecular mechanism for autosomal dominant zonular cataracts with sutural opacities in an Indian family caused by a c.215+1G>A splice mutation in the βA3/A1-crystallin gene CRYBA1 was elucidated using three transgenic mice models. This mutation causes a splice defect in which the mutant mRNA escapes nonsense mediated decay by skipping both exons 3 and 4. Skipping these exons results in an in-frame deletion of the mRNA and synthesis of an unstable p.Ile33_Ala119del mutant βA3/A1-crystallin protein. Transgenic expression of mutant βA3/A1-crystallin but not the wild type protein results in toxicity and abnormalities in the maturation and orientation of differentiating lens fibers in c.97_357del CRYBA1 transgenic mice, leading to a small spherical lens, cataract, and often lens capsule rupture. On a cellular level, the lenses accumulated p.Ile33_Ala119del βA3/A1-crystallin with resultant activation of the stress signaling pathway - unfolded protein response (UPR) and inhibition of normal protein synthesis, culminating in apoptosis. This highlights the mechanistic contrast between mild mutations that destabilize crystallins and other proteins, resulting in their being bound by the α-crystallins that buffer lens cells against damage by denatured proteins, and severely misfolded proteins that are not bound by α-crystallin but accumulate and have a direct toxic effect on lens cells, resulting in early onset cataracts. PMID:26851658

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

    PubMed Central

    2013-01-01

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

  5. A Mouse Model Suggests Two Mechanisms for Thyroid Alterations in Infantile Cystinosis: Decreased Thyroglobulin Synthesis Due to Endoplasmic Reticulum Stress/Unfolded Protein Response and Impaired Lysosomal Processing

    PubMed Central

    Gaide Chevronnay, H. P.; Janssens, V.; Van Der Smissen, P.; Liao, X. H.; Abid, Y.; Nevo, N.; Antignac, C.; Refetoff, S.; Cherqui, S.; Pierreux, C. E.

    2015-01-01

    Thyroid hormones are released from thyroglobulin (Tg) in lysosomes, which are impaired in infantile/nephropathic cystinosis. Cystinosis is a lysosomal cystine storage disease due to defective cystine exporter, cystinosin. Cystinotic children develop subclinical and then overt hypothyroidism. Why hypothyroidism is the most frequent and earliest endocrine complication of cystinosis is unknown. We here defined early alterations in Ctns−/− mice thyroid and identified subcellular and molecular mechanisms. At 9 months, T4 and T3 plasma levels were normal and TSH was moderately increased (∼4-fold). By histology, hyperplasia and hypertrophy of most follicles preceded colloid exhaustion. Increased immunolabeling for thyrocyte proliferation and apoptotic shedding indicated accelerated cell turnover. Electron microscopy revealed endoplasmic reticulum (ER) dilation, apical lamellipodia indicating macropinocytic colloid uptake, and lysosomal cystine crystals. Tg accumulation in dilated ER contrasted with mRNA down-regulation. Increased expression of ER chaperones, glucose-regulated protein of 78 kDa and protein disulfide isomerase, associated with alternative X-box binding protein-1 splicing, revealed unfolded protein response (UPR) activation by ER stress. Decreased Tg mRNA and ER stress suggested reduced Tg synthesis. Coordinated increase of UPR markers, activating transcription factor-4 and C/EBP homologous protein, linked ER stress to apoptosis. Hormonogenic cathepsins were not altered, but lysosome-associated membrane protein-1 immunolabeling disclosed enlarged vesicles containing iodo-Tg and impaired lysosomal fusion. Isopycnic fractionation showed iodo-Tg accumulation in denser lysosomes, suggesting defective lysosomal processing and hormone release. In conclusion, Ctns−/− mice showed the following alterations: 1) compensated primary hypothyroidism and accelerated thyrocyte turnover; 2) impaired Tg production linked to ER stress/UPR response; and 3) altered

  6. Anticipatory estrogen activation of the unfolded protein response is linked to cell proliferation and poor survival in estrogen receptor α-positive breast cancer.

    PubMed

    Andruska, N; Zheng, X; Yang, X; Helferich, W G; Shapiro, D J

    2015-07-01

    In response to cell stress, cancer cells often activate the endoplasmic reticulum (EnR) stress sensor, the unfolded protein response (UPR). Little was known about the potential role in cancer of a different mode of UPR activation, anticipatory activation of the UPR prior to accumulation of unfolded protein or cell stress. We show that estrogen, acting via estrogen receptor α (ERα), induces rapid anticipatory activation of the UPR, resulting in increased production of the antiapoptotic chaperone BiP/GRP78, preparing cancer cells for the increased protein production required for subsequent estrogen-ERα-induced cell proliferation. In ERα-containing cancer cells, the estrogen, 17β-estradiol (E2) activates the UPR through a phospholipase C γ (PLCγ)-mediated opening of EnR IP3R calcium channels, enabling passage of calcium from the lumen of the EnR into the cytosol. siRNA knockdown of ERα blocked the estrogen-mediated increase in cytosol calcium and UPR activation. Knockdown or inhibition of PLCγ, or of IP3R, strongly inhibited the estrogen-mediated increases in cytosol calcium, UPR activation and cell proliferation. E2-ERα activates all three arms of the UPR in breast and ovarian cancer cells in culture and in a mouse xenograft. Knockdown of ATF6α, which regulates UPR chaperones, blocked estrogen induction of BiP and strongly inhibited E2-ERα-stimulated cell proliferation. Mild and transient UPR activation by estrogen promotes an adaptive UPR response that protects cells against subsequent UPR-mediated apoptosis. Analysis of data from ERα(+) breast cancers demonstrates elevated expression of a UPR gene signature that is a powerful new prognostic marker tightly correlated with subsequent resistance to tamoxifen therapy, reduced time to recurrence and poor survival. Thus, as an early component of the E2-ERα proliferation program, the mitogen estrogen, drives rapid anticipatory activation of the UPR. Anticipatory activation of the UPR is a new role for

  7. IRE-1/XBP-1 pathway of the unfolded protein response is required for properly localizing neuronal UNC-6/Netrin for axon guidance in C. elegans.

    PubMed

    Asakura, Taro; Ogura, Ken-ichi; Goshima, Yoshio

    2015-03-01

    During developing nervous system, neurons project axons to their targets precisely. In this process, axon guidance molecules provide positional information to the axons. Therefore, the spatially and temporally controlled localization of the axon guidance molecules is required for the proper structure formation of the complex nervous system. In C. elegans, UNC-6/Netrin is a secreted protein that elicits both attractive and repulsive response in axon guidance. UNC-6/Netrin secreted from ventral cells may establish a concentration gradient from the ventral to the dorsal side of the animal, thus providing dorso-ventral positional information. However, the mechanisms specifying positional information of UNC-6/Netrin are largely unknown. Here, we show that the ire-1/xbp-1 pathway of the unfolded protein response (UPR) is required for axonal distribution of UNC-6/Netrin in the ventral neurons. In addition, the ire-1/xbp-1 pathway is also required for dorso-ventral axon guidance mediated by UNC-6/Netrin. Our results suggest that the ire-1/xbp-1 pathway of the UPR is crucial for establishing positional information of UNC-6/Netrin. We propose that the proper secretion of UNC-6/Netrin from the ventral neurons requires the activity of IRE-1. PMID:25469499

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

    ERIC Educational Resources Information Center

    Roberts, James S.; Laughlin, James E.

    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…

  9. Bovine viral diarrhea virus 2 infection activates the unfolded protein response in MDBK cells, leading to apoptosis.

    PubMed

    Maeda, Kouji; Fujihara, Masatoshi; Harasawa, Ryô

    2009-06-01

    Bovine viral diarrhea virus 2 (BVDV-2) strains are divided into cytopathic and non-cytopathic biotypes based on the ablity to induce cytopathic effects in cultured cells. The mechanism of cytopathogenicity of BVDV-2 is not well understood. We examined cytopathogenesis in MDBK cells resulting from BVDV-2 infections by microscopic examinations and microarray analysis. We found that BVDV-2 activates endoplasmic reticulum (ER) stress signaling pathways that contribute to apoptosis of infected cells. We also monitored the expression of ER stress marker gene by RT-PCR during BVDV-2 infection and demonstrated that infection of MDBK cells with a cytopathic strain of BVDV-2 induces glucose-regulated protein 78 expression. Infection with BVDV-2 also induces DNA-damage-inducible transcript 3 expression and downregulates the lectin-galactoside-binding soluble 1 level. These results show that cytopathic strains of BVDV-2 induce an ER stress response resulting in apoptosis. PMID:19578292

  10. The virulence of the opportunistic fungal pathogen Aspergillus fumigatus requires cooperation between the endoplasmic reticulum-associated degradation pathway (ERAD) and the unfolded protein response (UPR)

    PubMed Central

    Richie, Daryl L; Feng, Xizhi; Hartl, Lukas; Aimanianda, Vishukumar; Krishnan, Karthik; Powers-Fletcher, Margaret V; Watson, Douglas S; Galande, Amit K; White, Stephanie M; Willett, Taryn; Latgé, Jean-Paul; Rhodes, Judith C

    2011-01-01

    The filamentous fungal pathogen Aspergillus fumigatus secretes hydrolytic enzymes to acquire nutrients from host tissues. The production of these enzymes exerts stress on the endoplasmic reticulum (ER), which is alleviated by two stress responses: the unfolded protein response (UPR), which adjusts the protein folding capacity of the ER, and ER-associated degradation (ERAD), which disposes of proteins that fail to fold correctly. In this study, we examined the contribution of these integrated pathways to the growth and virulence of A. fumigatus, focusing on the ERAD protein DerA and the master regulator of the UPR, HAcA. A ΔderA mutant grew normally and showed no increase in sensitivity to ER stress. However, expression of the UPR target gene bipA was constitutively elevated in this strain, suggesting that the UPR was compensating for the absence of DerA function. To test this, the UPR was disrupted by deleting the hacA gene. The combined loss of derA and hacA caused a more severe reduction in hyphal growth, antifungal drug resistance and protease secretion than the loss of either gene alone, suggesting that DerA and HacA cooperate to support these functions. Moreover, the ΔderA/ΔhacA mutant was avirulent in a mouse model of invasive aspergillosis, which contrasted the wild-type virulence of ΔderA and the reduced virulence of the ΔhacA mutant. Taken together, these data demonstrate that DerA cooperates with the UPR to support the expression of virulence-related attributes of A. fumigatus. PMID:21217201

  11. Unfolded-protein response-associated stabilization of p27(Cdkn1b) interferes with lens fiber cell denucleation, leading to cataract.

    PubMed

    Lyu, Lei; Whitcomb, Elizabeth A; Jiang, Shuhong; Chang, Min-Lee; Gu, Yumei; Duncan, Melinda K; Cvekl, Ales; Wang, Wei-Lin; Limi, Saima; Reneker, Lixing W; Shang, Fu; Du, Linfang; Taylor, Allen

    2016-03-01

    Failure of lens fiber cell denucleation (LFCD) is associated with congenital cataracts, but the pathobiology awaits elucidation. Recent work has suggested that mechanisms that direct the unidirectional process of LFCD are analogous to the cyclic processes associated with mitosis. We found that lens-specific mutations that elicit an unfolded-protein response (UPR) in vivo accumulate p27(Cdkn1b), show cyclin-dependent kinase (Cdk)-1 inhibition, retain their LFC nuclei, and are cataractous. Although a UPR was not detected in lenses expressing K6W-Ub, they also accumulated p27 and showed failed LFCD. Induction of a UPR in human lens epithelial cells (HLECs) also induced accumulation of p27 associated with decreased levels of S-phase kinase-associated protein (Skp)-2, a ubiquitin ligase that regulates mitosis. These cells also showed decreased lamin A/C phosphorylation and metaphase arrest. The suppression of lamin A/C phosphorylation and metaphase transition induced by the UPR was rescued by knockdown of p27. Taken together, these data indicate that accumulation of p27, whether related to the UPR or not, prevents the phosphorylation of lamin A/C and LFCD in maturing LFCs in vivo, as well as in dividing HLECs. The former leads to cataract and the latter to metaphase arrest. These results suggest that accumulation of p27 is a common mechanism underlying retention of LFC nuclei. PMID:26590164

  12. Ask1 Gene Deletion Blocks Maternal Diabetes–Induced Endoplasmic Reticulum Stress in the Developing Embryo by Disrupting the Unfolded Protein Response Signalosome

    PubMed Central

    Wang, Fang; Wu, Yanqing; Gu, Hui; Reece, E. Albert; Fang, Shengyun; Gabbay-Benziv, Rinat; Aberdeen, Graham

    2015-01-01

    Apoptosis signal–regulating kinase 1 (ASK1) is activated by various stresses. The link between ASK1 activation and endoplasmic reticulum (ER) stress, two causal events in diabetic embryopathy, has not been determined. We sought to investigate whether ASK1 is involved in the unfolded protein response (UPR) that leads to ER stress. Deleting Ask1 abrogated diabetes-induced UPR by suppressing phosphorylation of inositol-requiring enzyme 1α (IRE1α), and double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK) blocked the mitochondrial translocation of proapoptotic Bcl-2 members and ER stress. ASK1 participated in the IRE1α signalosome, and removing ASK1 abrogated the proapoptotic kinase activity of IRE1α. Ask1 deletion suppressed diabetes-induced IRE1α endoriboneclease activities, which led to X-box binding protein 1 mRNA cleavage, an ER stress marker, decreased expression of microRNAs, and increased expression of a miR-17 target, thioredoxin-interacting protein (Txnip), a thioredoxin binding protein, which enhanced ASK1 activation by disrupting the thioredoxin-ASK1 complexes. ASK1 is essential for the assembly and function of the IRE1α signalosome, which forms a positive feedback loop with ASK1 through Txnip. ASK1 knockdown in C17.2 neural stem cells diminished high glucose– or tunicamycin-induced IRE1α activation, which further supports our hypothesis that ASK1 plays a causal role in diabetes-induced ER stress and apoptosis. PMID:25249581

  13. Endoplasmic reticulum vacuolation and unfolded protein response leading to paraptosis like cell death in cyclosporine A treated cancer cervix cells is mediated by cyclophilin B inhibition.

    PubMed

    Ram, Babul Moni; Ramakrishna, Gayatri

    2014-11-01

    Cyclosporine A (CsA), a widely used immunosuppressant shows cytotoxic effects by either inducing apoptosis or redirecting the cell towards non-apoptotic cell death. However, there still remains a lacuna in understanding the mechanism of CsA induced non-apoptotic cell death. In the present study we investigated calcineurin dependent or independent cytotoxic effects of CsA, a calcineurin inhibitor, in cervical cancerous SiHa cells. Decreased cell viability and massive cytoplasmic vacuolations were observed in CsA treated SiHa cells, having increased calcineurin activity. Endoplasmic reticulum (ER) stress and unfolded protein response (UPR), accompanied by a decrease in cyclophilin B (ER resident PPIase), preceded the formation of the vacuoles. These vacuoles stained positive for many ER resident markers confirming their ER origin; but the absence of autophagosomal marker, LC3II, ruled out autophagy. Extensively vacuolated cells eventually undergo cell death which lacked the typical apoptotic features, but showed significant decrease in AIP (ALG2 interacting protein) as seen in paraptosis. ER-vacuolation was prevented by cycloheximide and salubrinal thereby indicating requirement of active protein synthesis. Inhibiting calcineurin activity by either Tacrolimus (FK506) or by knockdown of calcineurin B subunit did not result in either ER-stress or cellular vacuolation. However, knockdown of cyclophilin B by siRNA resulted in increased expression of Bip and IRE1α, together with cytoplasmic vacuolation. In conclusion, we report that persistent ER stress due to cyclophilin B inhibition in CsA treated cervical cancer cells caused cellular vacuolation which culminated in a non-apoptotic cell death response similar to paraptosis. Additionally, the paraptotic effects of CsA are independent of calcineurin inhibition. PMID:25003316

  14. Monocrotaline pyrrole-induced megalocytosis of lung and breast epithelial cells: Disruption of plasma membrane and Golgi dynamics and an enhanced unfolded protein response.

    PubMed

    Mukhopadhyay, Somshuvra; Shah, Mehul; Patel, Kirit; Sehgal, Pravin B

    2006-03-15

    The pyrrolizidine alkaloid monocrotaline (MCT) initiates pulmonary hypertension by inducing a "megalocytosis" phenotype in target pulmonary arterial endothelial, smooth muscle and Type II alveolar epithelial cells. In cultured endothelial cells, a single exposure to the pyrrolic derivative of monocrotaline (MCTP) results in large cells with enlarged endoplasmic reticulum (ER) and Golgi and increased vacuoles. However, these cells fail to enter mitosis. Largely based upon data from endothelial cells, we proposed earlier that a disruption of the trafficking and mitosis-sensor functions of the Golgi (the "Golgi blockade" hypothesis) may represent the subcellular mechanism leading to MCTP-induced megalocytosis. In the present study, we investigated the applicability of the Golgi blockade hypothesis to epithelial cells. MCTP induced marked megalocytosis in cultures of lung A549 and breast MCF-7 cells. This was associated with a change in the distribution of the cis-Golgi scaffolding protein GM130 from a discrete juxtanuclear localization to a circumnuclear distribution consistent with an anterograde block of GM130 trafficking to/through the Golgi. There was also a loss of plasma membrane caveolin-1 and E-cadherin, cortical actin together with a circumnuclear accumulation of clathrin heavy chain (CHC) and alpha-tubulin. Flotation analyses revealed losses/alterations in the association of caveolin-1, E-cadherin and CHC with raft microdomains. Moreover, megalocytosis was accompanied by an enhanced unfolded protein response (UPR) as evidenced by nuclear translocation of Ire1alpha and glucose regulated protein 58 (GRP58/ER-60/ERp57) and a circumnuclear accumulation of PERK kinase and protein disulfide isomerase (PDI). These data further support the hypothesis that an MCTP-induced Golgi blockade and enhanced UPR may represent the subcellular mechanism leading to enlargement of ER and Golgi and subsequent megalocytosis. PMID:16000202

  15. The bZIP Transcription Factor HAC-1 Is Involved in the Unfolded Protein Response and Is Necessary for Growth on Cellulose in Neurospora crassa

    PubMed Central

    Larrondo, Luis F.

    2015-01-01

    High protein secretion capacity in filamentous fungi requires an extremely efficient system for protein synthesis, folding and transport. When the folding capacity of the endoplasmic reticulum (ER) is exceeded, a pathway known as the unfolded protein response (UPR) is triggered, allowing cells to mitigate and cope with this stress. In yeast, this pathway relies on the transcription factor Hac1, which mediates the up-regulation of several genes required under these stressful conditions. In this work, we identified and characterized the ortholog of the yeast HAC1 gene in the filamentous fungus Neurospora crassa. We show that its mRNA undergoes an ER stress-dependent splicing reaction, which in N. crassa removes a 23 nt intron and leads to a change in the open reading frame. By disrupting the N. crassa hac-1 gene, we determined it to be crucial for activating UPR and for proper growth in the presence of ER stress-inducing chemical agents. Neurospora is naturally found growing on dead plant material, composed primarily by lignocellulose, and is a model organism for the study of plant cell wall deconstruction. Notably, we found that growth on cellulose, a substrate that requires secretion of numerous enzymes, imposes major demands on ER function and is dramatically impaired in the absence of hac-1, thus broadening the range of physiological functions of the UPR in filamentous fungi. Growth on hemicellulose however, another carbon source that necessitates the secretion of various enzymes for its deconstruction, is not impaired in the mutant nor is the amount of proteins secreted on this substrate, suggesting that secretion, as a whole, is unaltered in the absence of hac-1. The characterization of this signaling pathway in N. crassa will help in the study of plant cell wall deconstruction by fungi and its manipulation may result in important industrial biotechnological applications. PMID:26132395

  16. Monocrotaline pyrrole-induced megalocytosis of lung and breast epithelial cells: Disruption of plasma membrane and Golgi dynamics and an enhanced unfolded protein response

    SciTech Connect

    Mukhopadhyay, Somshuvra; Shah, Mehul; Patel, Kirit; Sehgal, Pravin B. . E-mail: pravin_sehgal@nymc.edu

    2006-03-15

    The pyrrolizidine alkaloid monocrotaline (MCT) initiates pulmonary hypertension by inducing a 'megalocytosis' phenotype in target pulmonary arterial endothelial, smooth muscle and Type II alveolar epithelial cells. In cultured endothelial cells, a single exposure to the pyrrolic derivative of monocrotaline (MCTP) results in large cells with enlarged endoplasmic reticulum (ER) and Golgi and increased vacuoles. However, these cells fail to enter mitosis. Largely based upon data from endothelial cells, we proposed earlier that a disruption of the trafficking and mitosis-sensor functions of the Golgi (the 'Golgi blockade' hypothesis) may represent the subcellular mechanism leading to MCTP-induced megalocytosis. In the present study, we investigated the applicability of the Golgi blockade hypothesis to epithelial cells. MCTP induced marked megalocytosis in cultures of lung A549 and breast MCF-7 cells. This was associated with a change in the distribution of the cis-Golgi scaffolding protein GM130 from a discrete juxtanuclear localization to a circumnuclear distribution consistent with an anterograde block of GM130 trafficking to/through the Golgi. There was also a loss of plasma membrane caveolin-1 and E-cadherin, cortical actin together with a circumnuclear accumulation of clathrin heavy chain (CHC) and {alpha}-tubulin. Flotation analyses revealed losses/alterations in the association of caveolin-1, E-cadherin and CHC with raft microdomains. Moreover, megalocytosis was accompanied by an enhanced unfolded protein response (UPR) as evidenced by nuclear translocation of Ire1{alpha} and glucose regulated protein 58 (GRP58/ER-60/ERp57) and a circumnuclear accumulation of PERK kinase and protein disulfide isomerase (PDI). These data further support the hypothesis that an MCTP-induced Golgi blockade and enhanced UPR may represent the subcellular mechanism leading to enlargement of ER and Golgi and subsequent megalocytosis.

  17. Expression and splicing of the unfolded protein response gene XBP-1 are significantly associated with clinical outcome of endocrine-treated breast cancer.

    PubMed

    Davies, Michael P A; Barraclough, Dong Liu; Stewart, Ceri; Joyce, Kathryn A; Eccles, Richard M; Barraclough, Roger; Rudland, Philip S; Sibson, David Ross

    2008-07-01

    X-box binding protein 1 (XBP-1) is stimulated by endoplasmic reticulum stress as part of the unfolded protein response (UPR), which can promote apoptosis or cell survival. Non-conventional splicing, stimulated during the UPR, converts mRNA for "unspliced" XBP-1U to "spliced" XBP-1S mRNA. XBP-1 mRNA is oestrogen-responsive, but XBP-1S confers oestrogen independence and anti-oestrogen resistance to breast cancer cell lines. We therefore evaluated XBP-1 mRNA splicing as a factor in response of breast cancer patients to endocrine treatment. XBP-1 isoforms were measured by quantitative RT-PCR in 100 primary breast cancer patients treated with adjuvant tamoxifen (including 30 ER alpha-negative cases). In ER alpha-positive cases, levels of XBP-1U mRNA correlated with ER alpha mRNA levels and were lower in grade 3 tumors. Higher levels of XBP-1U mRNA were significantly associated with breast cancer survival (Log-rank p = 0.002; Cox hazard ratio (HR) 0.2, p = 0.005), independent of grade, size, nodal status and progesterone receptor status. However, in the full cohort, higher ratios of XBP-1S/XBP-1U mRNA (indicating enhanced splicing) were associated with poor survival (Log-rank p = 0.03; Cox HR 2.3, p = 0.03) and related factors: ER alpha-negative status, progesterone receptor negative status, grade 3 tumors and greater proliferation. Significant associations with poor outcome were also seen for XBP-1 splicing in ER alpha-positive cases. Our findings, that XBP-1 isoforms are differently associated with outcome of endocrine therapy for patients, can be explained by higher levels of dominant-negative XBP-1U favouring apoptosis of tumor cells and higher levels of XBP-1S increasing tumor survival. PMID:18386815

  18. Yip1A, a Novel Host Factor for the Activation of the IRE1 Pathway of the Unfolded Protein Response during Brucella Infection

    PubMed Central

    Taguchi, Yuki; Imaoka, Koichi; Kataoka, Michiyo; Uda, Akihiko; Nakatsu, Daiki; Horii-Okazaki, Sakuya; Kunishige, Rina; Kano, Fumi; Murata, Masayuki

    2015-01-01

    Brucella species replicate within host cells in the form of endoplasmic reticulum (ER)-derived vacuoles. The mechanisms by which the bacteria are sequestered into such vacuoles and obtain a continuous membrane supply for their replication remain to be elucidated. In the present study, we provided several lines of evidence that demonstrate the mechanism by which B. abortus acquires the ER-derived membrane. First, during Brucella infection, the IRE1 pathway, but not the PERK and ATF6 pathways, of the unfolded protein response (UPR) was activated in a time-dependent manner, and the COPII vesicle components Sar1, Sec23, and Sec24D were upregulated. Second, a marked accretion of ER-derived vacuoles was observed around replicating bacteria using fluorescent microscopy and electron microscopy. Third, we identified a novel host factor, Yip1A, for the activation of the IRE1 pathway in response to both tunicamycin treatment and infection with B. abortus. We found that Yip1A is responsible for the phosphorylation of IRE1 through high-order assembly of Ire1 molecules at ER exit sites (ERES) under the UPR conditions. In Yip1A-knockdown cells, B. abortus failed to generate the ER-derived vacuoles, and remained in endosomal/lysosomal compartments. These results indicate that the activation of the IRE1 pathway and the subsequent formation of ER-derived vacuoles are critical for B. abortus to establish a safe replication niche, and that Yip1A is indispensable for these processes. Furthermore, we showed that the autophagy-related proteins Atg9 and WIPI1, but not DFCP1, were required for the biogenesis of the ER-derived membrane compartments.  On the basis of our findings, we propose a model for intracellular Brucella replication that exploits the host UPR and ER-derived vacuole formation machineries, both of which depend on Yip1A-mediated IRE1 activation. PMID:25742138

  19. Endogenous catecholamine enhances the dysfunction of unfolded protein response and alpha-synuclein oligomerization in PC12 cells overexpressing human alpha-synuclein.

    PubMed

    Ito, Satoru; Nakaso, Kazuhiro; Imamura, Keiko; Takeshima, Takao; Nakashima, Kenji

    2010-01-01

    Parkinson's disease (PD) is a neurodegenerative disorder characterized by the selective loss of dopaminergic neurons and the presence of Lewy bodies. alpha-Synuclein is a major component of Lewy bodies. Recently, many studies have focused on the interaction between alpha-synuclein and catecholamine in the pathogenesis of PD. However, no detailed relationship between cathecholamine and alpha-synuclein cytotoxicity has been elucidated. Therefore, this study established PC12 cell lines which overexpress human alpha-synuclein in a tetracycline-inducible manner. The overexpression of human alpha-synuclein increased the number of apoptotic cells in a long-term culture. Moreover, human alpha-synuclein expressing PC12 cells demonstrated an increased vulnerability to several stressors in a short culture period. Thapsigargin increased the SDS soluble oligomers of alpha-synuclein associated with catecholamine-quinone. The unfolded protein response (UPR) study showed that thapsigargin increased eIF2alpha phosphorylation and nuclear GADD153/CHOP induction under alpha-synuclein overexpressed conditions. The activities of the ATF6alpha and IRE1alpha pathways decreased. These findings suggest that an overexpression of alpha-synuclein partly inactivates the UPR. alpha-Methyltyrosine inhibited the dysfunction of the UPR caused by an overexpression of human alpha-synuclein. Therefore, these findings suggest that the coexistence of human alpha-synuclein with catecholamine enhances the endoplasmic reticulum stress-related toxicity in PD pathogenesis. PMID:19835916

  20. A synthetic chalcone, 2'-hydroxy-2,3,5'-trimethoxychalcone triggers unfolded protein response-mediated apoptosis in breast cancer cells.

    PubMed

    Lee, Da Hyun; Jung Jung, You; Koh, Dongsoo; Lim, Yoongho; Lee, Young Han; Shin, Soon Young

    2016-03-01

    The primary aim of this study was to find novel chemopreventive agents effective against breast cancer. Endoplasmic reticulum (ER) stress can induce apoptosis through the unfolded protein response (UPR). 2'-Hydroxy-2,3,5'-trimethoxychalcone (DK143) is a synthetic flavonoid derivative. The present study provides evidence supporting the role of the UPR in mediating the apoptotic effect of DK143. Treatment with DK143 triggered apoptosis through the activation of the caspase pathway in MDA-MB-231 breast cancer cells without affecting viability of MCF10A non-transformed breast epithelial cells. Further analysis revealed that DK143 produced reactive oxygen species (ROS) in MDA-MB-231 cells, but not in MCF10A cells, and upregulated the expression of ER stress sensors, including GRP78/BiP, IRE1α, CHOP, and Bim in MDA-MB-231 cells. In addition, UPR-related transcription factors, XBP-1 and CHOP, were activated by DK143. Moreover, silencing of IRE1α or CHOP by corresponding siRNA molecules attenuated DK143-induced apoptosis. Furthermore, DK143 suppressed mouse tumor growth in vivo. These results demonstrate that promoting ER stress in breast cancer cells via UPR induction might be a promising strategy for developing new chemotherapeutic or chemopreventive agents for breast cancer. PMID:26742460

  1. Targeting the IRE1α/XBP1 and ATF6 Arms of the Unfolded Protein Response Enhances VEGF Blockade to Prevent Retinal and Choroidal Neovascularization

    PubMed Central

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

    2014-01-01

    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) α and activating transcription factor 6 (ATF6) arms of the UPR compared with untreated cells. This was associated with increased expression in α-basic crystallin (CRYAB), which has previously bound VEGF. siRNA knockdown or pharmacological blockade of IRE1α, ATF6, or CRYAB increased intracellular VEGF degradation and decreased full-length intracellular VEGF. Inhibition of IRE1α, 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 IRE1α 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 IRE1α/ATF6 arms of the UPR offer a potential therapeutic target in the treatment of pathological angiogenesis. PMID:23395094

  2. The aqueous extract of Glycyrrhiza inflata can upregulate unfolded protein response-mediated chaperones to reduce tau misfolding in cell models of Alzheimer’s disease

    PubMed Central

    Chang, Kuo-Hsuan; Chen, I-Cheng; Lin, Hsuan-Yuan; Chen, Hsuan-Chiang; Lin, Chih-Hsin; Lin, Te-Hsien; Weng, Yu-Ting; Chao, Chih-Ying; Wu, Yih-Ru; Lin, Jung-Yaw; Lee-Chen, Guey-Jen; Chen, Chiung-Mei

    2016-01-01

    Background Alzheimer’s disease (AD) and several neurodegenerative disorders known as tauopathies are characterized by misfolding and aggregation of tau protein. Although several studies have suggested the potential of traditional Chinese medicine (TCM) as treatment for neurodegenerative diseases, the role of TCM in treating AD and tauopathies have not been well explored. Materials and methods Tau protein was coupled to the DsRed fluorophore by fusing a pro-aggregation mutant of repeat domain of tau (ΔK280 tauRD) with DsRed. The ΔK280 tauRD-DsRed fusion gene was then used to generate Tet-On 293 and SH-SY5Y cell clones as platforms to test the efficacy of 39 aqueous extracts of TCM in reducing tau misfolding and in neuroprotection. Results Seven TCM extracts demonstrated a significant reduction in tau misfolding and reactive oxidative species with low cytotoxicity in the ΔK280 tauRD-DsRed 293 cell model. Glycyrrhiza inflata and Panax ginseng also demonstrated the potential to improve neurite outgrowth in the ΔK280 tauRD-DsRed SH-SY5Y neuronal cell model. G. inflata further rescued the upregulation of ERN2 (pro-apoptotic) and downregulation of unfolded-protein-response-mediated chaperones ERP44, DNAJC3, and SERP1 in ΔK280 tauRD-DsRed 293 cells. Conclusion This in vitro study provides evidence that G. inflata may be a novel therapeutic for AD and tauopathies. Future applications of G. inflata on animal models of AD and tauopathies are warranted to corroborate its effect of reducing misfolding and potential disease modification. PMID:27013866

  3. Distinct roles of activating transcription factor 6 (ATF6) and double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK) in transcription during the mammalian unfolded protein response.

    PubMed Central

    Okada, Tetsuya; Yoshida, Hiderou; Akazawa, Rieko; Negishi, Manabu; Mori, Kazutoshi

    2002-01-01

    In response to accumulation of unfolded proteins in the endoplasmic reticulum (ER), a homoeostatic response, termed the unfolded protein response (UPR), is activated in all eukaryotic cells. The UPR involves only transcriptional regulation in yeast, and approx. 6% of all yeast genes, encoding not only proteins to augment the folding capacity in the ER, but also proteins working at various stages of secretion, are induced by ER stress [Travers, Patil, Wodicka, Lockhart, Weissman and Walter (2000) Cell (Cambridge, Mass.) 101, 249-258]. In the present study, we conducted microarray analysis of HeLa cells, although our analysis covered only a small fraction of the human genome. A great majority of human ER stress-inducible genes (approx. 1% of 1800 genes examined) were classified into two groups. One group consisted of genes encoding ER-resident molecular chaperones and folding enzymes, and these genes were directly regulated by the ER-membrane-bound transcription factor activating transcription factor (ATF) 6. The ER-membrane-bound protein kinase double-stranded RNA-activated protein kinase-like ER kinase (PERK)-mediated signalling pathway appeared to be responsible for induction of the remaining genes, which are not involved in secretion, but may be important after cellular recovery from ER stress. In higher eukaryotes, the PERK-mediated translational-attenuation system is known to operate in concert with the transcriptional-induction system. Thus we propose that mammalian cells have evolved a strategy to cope with ER stress different from that of yeast cells. PMID:12014989

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

    PubMed Central

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

    2012-01-01

    Protein-folding occurs in several intracellular locations including the endoplasmic reticulum and mitochondria. In normal conditions there is a balance between the levels of unfolded proteins and protein folding machinery. Disruption of homeostasis and an accumulation of unfolded proteins trigger stress responses, or unfolded protein responses (UPR), in these organelles. These pathways signal to increase the folding capacity, inhibit protein import or expression, increase protein degradation, and potentially trigger cell death. Many aging-related neurodegenerative diseases involve the accumulation of misfolded proteins in both the endoplasmic reticulum and mitochondria. The exact participation of the UPRs in the onset of neurodegeneration is unclear, but there is significant evidence for the alteration of these pathways in the endoplasmic reticulum and mitochondria. Here we will discuss the involvement of endoplasmic reticulum and mitochondrial stress and the possible contributions of the UPR in these organelles to the development of two neurodegenerative diseases, Parkinson's disease (PD) and Alzheimer's disease (AD). PMID:22539924

  5. Protein unfolding and degradation by the AAA+ Lon protease.

    PubMed

    Gur, Eyal; Vishkautzan, Marina; Sauer, Robert T

    2012-02-01

    AAA+ proteases employ a hexameric ring that harnesses the energy of ATP binding and hydrolysis to unfold native substrates and translocate the unfolded polypeptide into an interior compartment for degradation. What determines the ability of different AAA+ enzymes to unfold and thus degrade different native protein substrates is currently uncertain. Here, we explore the ability of the E. coli Lon protease to unfold and degrade model protein substrates beginning at N-terminal, C-terminal, or internal degrons. Lon has historically been viewed as a weak unfoldase, but we demonstrate robust and processive unfolding/degradation of some substrates with very stable protein domains, including mDHFR and titin(I27) . For some native substrates, Lon is a more active unfoldase than related AAA+ proteases, including ClpXP and ClpAP. For other substrates, this relationship is reversed. Thus, unfolding activity does not appear to be an intrinsic enzymatic property. Instead, it depends on the specific protease and substrate, suggesting that evolution has diversified rather than optimized the protein unfolding activities of different AAA+ proteases. PMID:22162032

  6. Employing Multiple Spectroscopic Techniques Simultaneously to Observe Protein Unfolding

    NASA Astrophysics Data System (ADS)

    Crowe, Michael; Kelty, Ben; Link, Justin

    2015-03-01

    A protein's function is directly related to its native, folded structure. In order to study the structure of proteins, the unfolding process may be characterized. In our study, by using the spectroscopic techniques of circular dichroism (CD), absorption, and fluorescence simultaneously, we examined the unfolding of horse heart cytochrome c, a well-studied, model protein by gradually increasing the concentration of the chemical denaturant, guanidine hydrochloride. The signal changes from these modalities over the course of the unfolding reaction provides some of the thermodynamic properties like Gibbs free energy for insight into the stability of the protein. This allows us to compare the three techniques under the exact same conditions. The objective of this session is to present recent work in developing a protocol to observe the unfolding of cytochrome c using fluorescence, absorbance, and CD simultaneously.

  7. Protein Folding and Unfolding Under Force

    PubMed Central

    Jagannathan, Bharat; Marqusee, Susan

    2014-01-01

    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. PMID:23784721

  8. Sequential protein unfolding through a carbon nanotube pore

    NASA Astrophysics Data System (ADS)

    Xu, Zhonghe; Zhang, Shuang; Weber, Jeffrey K.; Luan, Binquan; Zhou, Ruhong; Li, Jingyuan

    2016-06-01

    An assortment of biological processes, like protein degradation and the transport of proteins across membranes, depend on protein unfolding events mediated by nanopore interfaces. In this work, we exploit fully atomistic simulations of an artificial, CNT-based nanopore to investigate the nature of ubiquitin unfolding. With one end of the protein subjected to an external force, we observe non-canonical unfolding behaviour as ubiquitin is pulled through the pore opening. Secondary structural elements are sequentially detached from the protein and threaded into the nanotube, interestingly, the remaining part maintains native-like characteristics. The constraints of the nanopore interface thus facilitate the formation of stable ``unfoldon'' motifs above the nanotube aperture that can exist in the absence of specific native contacts with the other secondary structure. Destruction of these unfoldons gives rise to distinct force peaks in our simulations, providing us with a sensitive probe for studying the kinetics of serial unfolding events. Our detailed analysis of nanopore-mediated protein unfolding events not only provides insight into how related processes might proceed in the cell, but also serves to deepen our understanding of structural arrangements which form the basis for protein conformational stability.An assortment of biological processes, like protein degradation and the transport of proteins across membranes, depend on protein unfolding events mediated by nanopore interfaces. In this work, we exploit fully atomistic simulations of an artificial, CNT-based nanopore to investigate the nature of ubiquitin unfolding. With one end of the protein subjected to an external force, we observe non-canonical unfolding behaviour as ubiquitin is pulled through the pore opening. Secondary structural elements are sequentially detached from the protein and threaded into the nanotube, interestingly, the remaining part maintains native-like characteristics. The constraints of

  9. Phenotypic assays identify azoramide as a small-molecule modulator of the unfolded protein response with antidiabetic activity.

    PubMed

    Fu, Suneng; Yalcin, Abdullah; Lee, Grace Y; Li, Ping; Fan, Jason; Arruda, Ana Paula; Pers, Benedicte M; Yilmaz, Mustafa; Eguchi, Kosei; Hotamisligil, Gökhan S

    2015-06-17

    The endoplasmic reticulum (ER) plays a critical role in protein, lipid, and glucose metabolism as well as cellular calcium signaling and homeostasis. Perturbation of ER function and chronic ER stress are associated with many pathologies ranging from diabetes and neurodegenerative diseases to cancer and inflammation. Although ER targeting shows therapeutic promise in preclinical models of obesity and other pathologies, the available chemical entities generally lack the specificity and other pharmacological properties required for effective clinical translation. To overcome these challenges and identify new potential therapeutic candidates, we first designed and chemically and genetically validated two high-throughput functional screening systems that independently measure the free chaperone content and protein-folding capacity of the ER. With these quantitative platforms, we characterized a small-molecule compound, azoramide, that improves ER protein-folding ability and activates ER chaperone capacity to protect cells against ER stress in multiple systems. This compound also exhibited potent antidiabetic efficacy in two independent mouse models of obesity by improving insulin sensitivity and pancreatic β cell function. Together, these results demonstrate the utility of this functional, phenotypic assay platform for ER-targeted drug discovery and provide proof of principle for the notion that specific ER modulators can be potential drug candidates for type 2 diabetes. PMID:26084805

  10. Protein unfolding in crowded milieu: what crowding can do to a protein undergoing unfolding?

    PubMed

    Stepanenko, Olga V; Povarova, Olga I; Sulatskaya, Anna I; Ferreira, Luisa A; Zaslavsky, Boris Y; Kuznetsova, Irina M; Turoverov, Konstantin K; Uversky, Vladimir N

    2016-10-01

    The natural environment of a protein inside a cell is characterized by the almost complete lack of unoccupied space, limited amount of free water, and the tightly packed crowd of various biological macromolecules, such as proteins, nucleic acids, polysaccharides, and complexes thereof. This extremely crowded natural milieu is poorly mimicked by slightly salted aqueous solutions containing low concentrations of a protein of interest. The accepted practice is to model crowded environments by adding high concentrations of various polymers that serve as model "crowding agents" to the solution of a protein of interest. Although studies performed under these model conditions revealed that macromolecular crowding might have noticeable influence on various aspects related to the protein structure, function, folding, conformational stability, and aggregation propensity, the complete picture describing conformational behavior of a protein under these conditions is missing as of yet. Furthermore, there is an accepted belief that the conformational stability of globular proteins increases in the presence crowding agents due to the excluded volume effects. The goal of this study was to conduct a systematic analysis of the effect of high concentrations of PEG-8000 and Dextran-70 on the unfolding behavior of eleven globular proteins belonging to different structural classes. PMID:26474212

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

    PubMed

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

    2013-09-28

    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

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

    NASA Astrophysics Data System (ADS)

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

    2013-09-01

    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

  13. BAX inhibitor-1 regulates autophagy by controlling the IRE1α branch of the unfolded protein response

    PubMed Central

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

    2011-01-01

    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 α (IRE1α). 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 IRE1α expression, possibly due to a displacement of TNF-receptor associated factor-2 (TRAF2) from IRE1α. 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. PMID:21926971

  14. Cell Surface Relocalization of the Endoplasmic Reticulum Chaperone and Unfolded Protein Response Regulator GRP78/BiP*

    PubMed Central

    Zhang, Yi; Liu, Ren; Ni, Min; Gill, Parkash; Lee, Amy S.

    2010-01-01

    The recent discovery that GRP78/BiP, a typical endoplasmic reticulum (ER) lumenal chaperone, can be expressed on the cell surface, interacting with an increasing repertoire of surface proteins and acting as receptor in signaling pathways, represents a paradigm shift in its biological function. However, the mechanism of GRP78 trafficking from the ER to the cell surface is not well understood. Using a combination of cellular, biochemical, and mutational approaches, we tested multiple hypotheses. Here we report that ER stress actively promotes GRP78 localization on the cell surface, whereas ectopic expression of GRP78 is also able to cause cell surface relocation in the absence of ER stress. Moreover, deletion of the C-terminal ER retention motif in GRP78 alters its cell surface presentation in a dose-dependent manner; however, mutation of the putative O-linked glycosylation site Thr648 of human GRP78 is without effect. We also identified the exposure of multiple domains of GRP78 on the cell surface and determined that binding of extracellular GRP78 to the cell surface is unlikely. A new topology model for cell surface GRP78 is presented. PMID:20208072

  15. Up-regulation of endoplasmic reticulum stress induced genes of the unfolded protein response in the liver of periparturient dairy cows

    PubMed Central

    2014-01-01

    Background In dairy cows, the periparturient phase is a stressful period, which is commonly associated with strong metabolic adaptations and the development of pathophysiologic conditions and disorders. Some of the symptoms occurring in the liver, such as the development of fatty liver, are similar to those observed under the condition of endoplasmic reticulum (ER) stress. Therefore, we hypothesized, that in the liver of dairy cows ER stress is induced during the periparturient phase, which in turn leads to an induction of the unfolded protein response (UPR). In order to investigate this hypothesis, we determined relative mRNA concentrations of 14 genes of the ER stress-induced UPR in liver biopsy samples of 13 dairy cows at 3 wk antepartum and 1, 5 and 14 wk postpartum. Results We found, that the mRNA concentrations of 13 out of the 14 genes involved in the UPR in the liver were significantly increased (1.9 to 4.0 fold) at 1 wk postpartum compared to 3 wk antepartum. From 1 wk postpartum to later lactation, mRNA concentrations of all the genes considered were declining. Moreover, at 1 wk postpartum, mRNA concentration of the spliced variant of XBP1 was increased in comparison to 3 wk antepartum, indicating that splicing of XBP1 – a hallmark of ER stress - was induced following the onset of lactation. Conclusion The present study reveals, that ER stress might be induced during the periparturient phase in the liver of dairy cows. We assume that the ER stress-induced UPR might contribute to the pathophysiologic conditions commonly observed in the liver of periparturient cows, such as the development of fatty liver, ketosis or inflammation. PMID:24555446

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

    PubMed Central

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

    2014-01-01

    Aim N-acyl dopamines (NADD) are gaining attention in the field of inflammatory and neurological disorders. Due to their hydrophobicity, NADD may have access to the endoplasmic reticulum (ER). We therefore investigated if NADD induce the unfolded protein response (UPR) and if this in turn influences cell behaviour. Methods Genome wide gene expression profiling, confirmatory qPCR and reporter assays were employed on human umbilical vein endothelial cells (HUVEC) to validate induction of UPR target genes and UPR sensor activation by N-octanoyl dopamine (NOD). Intracellular ATP, apoptosis and induction of thermotolerance were used as functional parameters to assess adaptation of HUVEC. Results NOD, but not dopamine dose dependently induces the UPR. This was also found for other synthetic NADD. Induction of the UPR was dependent on the redox activity of NADD and was not caused by selective activation of a particular UPR sensor. UPR induction did not result in cell apoptosis, yet NOD strongly impaired cell proliferation by attenuation of cells in the S-G2/M phase. Long-term treatment of HUVEC with low NOD concentration showed decreased intracellular ATP concentration paralleled with activation of AMPK. These cells were significantly more resistant to cold inflicted injury. Conclusions We provide for the first time evidence that NADD induce the UPR in vitro. It remains to be assessed if UPR induction is causally associated with hypometabolism and thermotolerance. Further pharmacokinetic studies are warranted to address if the NADD concentrations used in vitro can be obtained in vivo and if this in turn shows therapeutic efficacy. PMID:24926788

  17. Exclusion of the Unfolded Protein Response in Light-Induced Retinal Degeneration in the Canine T4R RHO Model of Autosomal Dominant Retinitis Pigmentosa

    PubMed Central

    Marsili, Stefania; Genini, Sem; Sudharsan, Raghavi; Gingrich, Jeremy; Aguirre, Gustavo D.; Beltran, William A.

    2015-01-01

    Purpose To examine the occurrence of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) following acute light damage in the naturally-occurring canine model of RHO-adRP (T4R RHO dog). Methods The left eyes of T4R RHO dogs were briefly light-exposed and retinas collected 3, 6 and 24 hours later. The contra-lateral eyes were shielded and used as controls. To evaluate the time course of cell death, histology and TUNEL assays were performed. Electron microscopy was used to examine ultrastructural alterations in photoreceptors at 15 min, 1 hour, and 6 hours after light exposure. Gene expression of markers of ER stress and UPR were assessed by RT-PCR, qRT-PCR and western blot at the 6 hour time-point. Calpain and caspase-3 activation were assessed at 1, 3 and 6 hours after exposure. Results A brief exposure to clinically-relevant levels of white light causes within minutes acute disruption of the rod outer segment disc membranes, followed by prominent ultrastructural alterations in the inner segments and the initiation of cell death by 6 hours. Activation of the PERK and IRE1 pathways, and downstream targets (BIP, CHOP) of the UPR was not observed. However increased transcription of caspase-12 and hsp70 occurred, as well as calpain activation, but not that of caspase-3. Conclusion The UPR is not activated in the early phase of light-induced photoreceptor cell death in the T4R RHO model. Instead, disruption in rods of disc and plasma membranes within minutes after light exposure followed by increase in calpain activity and caspase-12 expression suggests a different mechanism of degeneration. PMID:25695253

  18. PERK regulated miR-424(322)-503 cluster fine-tunes activation of IRE1 and ATF6 during Unfolded Protein Response

    PubMed Central

    Gupta, Ananya; Hossain, Muhammad Mosaraf; Read, Danielle E.; Hetz, Claudio; Samali, Afshin; Gupta, Sanjeev

    2015-01-01

    The endoplasmic reticulum (ER) responds to changes in intracellular homeostasis through activation of the unfolded protein response (UPR). UPR can facilitate the restoration of cellular homeostasis, via the concerted activation of three ER stress sensors, namely IRE1, PERK and ATF6. Global approaches in several cellular contexts have revealed that UPR regulates the expression of many miRNAs that play an important role in the regulation of life and death decisions during UPR. Here we show that expression of miR-424(322)-503 cluster is downregulated during UPR. IRE1 inhibitor (4 μ8C) and deficiency of XBP1 had no effect on downregulation of miR-424(322)-503 during UPR. Treatment of cells with CCT030312, a selective activator of EIF2AK3/PERK signalling, leads to the downregulation of miR-424(322)-503 expression. The repression of miR-424(322)-503 cluster during conditions of ER stress is compromised in PERK-deficient MEFs. miR-424 regulates the expression of ATF6 via a miR-424 binding site in its 3′ UTR and attenuates the ATF6 transcriptional activity during UPR. Further miR-424 had no effect on IRE1-XBP1 axis but enhanced the regulated IRE1-dependent decay (RIDD). Our results suggest that miR-424 constitutes an obligatory fine-tuning mechanism where PERK-mediated downregulation of miR-424(322)-503 cluster regulates optimal activation of IRE1 and ATF6 during conditions of ER stress. PMID:26674075

  19. Sequential protein unfolding through a carbon nanotube pore.

    PubMed

    Xu, Zhonghe; Zhang, Shuang; Weber, Jeffrey K; Luan, Binquan; Zhou, Ruhong; Li, Jingyuan

    2016-06-16

    An assortment of biological processes, like protein degradation and the transport of proteins across membranes, depend on protein unfolding events mediated by nanopore interfaces. In this work, we exploit fully atomistic simulations of an artificial, CNT-based nanopore to investigate the nature of ubiquitin unfolding. With one end of the protein subjected to an external force, we observe non-canonical unfolding behaviour as ubiquitin is pulled through the pore opening. Secondary structural elements are sequentially detached from the protein and threaded into the nanotube, interestingly, the remaining part maintains native-like characteristics. The constraints of the nanopore interface thus facilitate the formation of stable "unfoldon" motifs above the nanotube aperture that can exist in the absence of specific native contacts with the other secondary structure. Destruction of these unfoldons gives rise to distinct force peaks in our simulations, providing us with a sensitive probe for studying the kinetics of serial unfolding events. Our detailed analysis of nanopore-mediated protein unfolding events not only provides insight into how related processes might proceed in the cell, but also serves to deepen our understanding of structural arrangements which form the basis for protein conformational stability. PMID:26899409

  20. Analysis and Interpretation of Single Molecule Protein Unfolding Kinetics

    NASA Astrophysics Data System (ADS)

    Lannon, Herbert; Brujic, Jasna

    2012-02-01

    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

  1. Effect of antimicrobial preservatives on partial protein unfolding and aggregation†

    PubMed Central

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

    2014-01-01

    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

  2. Forced unfolding of protein domains determines cytoskeletal rheology

    NASA Astrophysics Data System (ADS)

    Crocker, John

    2005-03-01

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

  3. Developing a Novel, Interdisciplinary Approach to Study Protein Unfolding

    NASA Astrophysics Data System (ADS)

    Bentley, Ian; Link, Justin

    2013-03-01

    The ability of a protein to function is a direct result of its ability to properly obtain its native, folded structure. In order to determine the structural stability of proteins and to gain knowledge of their folding mechanism, we must develop protocols that allow us to monitor the controlled unfolding of proteins. Here, we investigate the stability of cytochrome c, a well-studied, model protein, under denaturing conditions using circular dichroism (CD) and fluorescence. Using either a chemical denaturant (Guanidine HCl) or heat, we can cause a protein to gradually unfold. The changes in the fluorescence and CD spectra can provide insight into the stability of proteins by providing us with thermodynamic parameters such as the Gibbs free energy, melting temperature and enthalpy. Research in this lab has been explored with mutant proteins and change in CD signal, however further work must still be done to observe their unfolding monitored by fluorescence. This technique will allow us to determine which regions of native cytochrome c have the greatest impact on the protein folding process. The objective of this session is to present recent work in developing a protocol to observe the unfolding of wild type and mutant proteins with fluorescence. The Borcer Fund, The John A. Hauck Foundation, and Xavier University

  4. Rosiglitazone induces the unfolded protein response, but has no significant effect on cell viability, in monocytic and vascular smooth muscle cells

    SciTech Connect

    Caddy, J.; Isa, S.; Mainwaring, L.S.; Adam, E.; Roberts, A.; Lang, D.; Morris, R.H.K.; Thomas, A.W.; Webb, R.

    2010-10-01

    Research highlights: {yields} Rosiglitazone rapidly (30 min) inhibited microsomal Ca{sup 2+}ATPase activity (IC{sub 50} {approx}2 {mu}M). {yields} After 4 h rosiglitazone exposure, the UPR transcription factor XBP-1 was activated. {yields} Within 24-72 h, UPR target genes were upregulated, enhancing ER Ca{sup 2+} sequestration. {yields} Replenishment of ER Ca{sup 2+} stores appeared to restore normal cell physiology. {yields} Monocyte/VSMC viability was not decreased during 2 weeks' rosiglitazone treatment. -- Abstract: Given the safety concerns expressed over negative cardiovascular outcomes resulting from the clinical use of rosiglitazone, and the view that rosiglitazone exerts PPAR{gamma}-independent effects alongside its insulin-sensitising PPAR{gamma}-dependent effects, we hypothesised that rosiglitazone may trigger Unfolded Protein Responses (UPRs) due to disruptions in [Ca{sup 2+}]{sub i} homeostasis within two cardiovascular cell types: monocytic (MM6) and vascular smooth muscle (A7r5) cells. In microsomal samples derived from both cell types, pre-incubation with rosiglitazone rapidly (30 min) brought about concentration-dependent PPAR{gamma}-independent inhibition of Ca{sup 2+}ATPase activity (IC{sub 50} {approx}2 {mu}M). Fluo-3 fluorimetric data demonstrated in intact cells that 1 h treatment with 1 or 10 {mu}M rosiglitazone caused Ca{sup 2+} ions to leak into the cytoplasm. Gene expression analysis showed that within 4 h of rosiglitazone exposure, the UPR transcription factor XBP-1 was activated (likely due to corresponding ER Ca{sup 2+} depletion), and the UPR target genes BiP and SERCA2b were subsequently upregulated within 24-72 h. After 72 h 1 or 10 {mu}M rosiglitazone treatment, microsomal Ca{sup 2+}ATPase activity increased to >2-fold of that seen in control microsomes, while [Ca{sup 2+}]{sub i} returned to basal, indicating that UPR-triggered SERCA2b upregulation was responsible for enhanced enzymatic Ca{sup 2+} sequestration within the ER. This

  5. Connecting thermal and mechanical protein (un)folding landscapes.

    PubMed

    Sun, Li; Noel, Jeffrey K; Sulkowska, Joanna I; Levine, Herbert; Onuchic, José N

    2014-12-16

    Molecular dynamics simulations supplement single-molecule pulling experiments by providing the possibility of examining the full free energy landscape using many coordinates. Here, we use an all-atom structure-based model to study the force and temperature dependence of the unfolding of the protein filamin by applying force at both termini. The unfolding time-force relation τ(F) indicates that the force-induced unfolding behavior of filamin can be characterized into three regimes: barrier-limited low- and intermediate-force regimes, and a barrierless high-force regime. Slope changes of τ(F) separate the three regimes. We show that the behavior of τ(F) can be understood from a two-dimensional free energy landscape projected onto the extension X and the fraction of native contacts Q. In the low-force regime, the unfolding rate is roughly force-independent due to the small (even negative) separation in X between the native ensemble and transition state ensemble (TSE). In the intermediate-force regime, force sufficiently separates the TSE from the native ensemble such that τ(F) roughly follows an exponential relation. This regime is typically explored by pulling experiments. While X may fail to resolve the TSE due to overlap with the unfolded ensemble just below the folding temperature, the overlap is minimal at lower temperatures where experiments are likely to be conducted. The TSE becomes increasingly structured with force, whereas the average order of structural events during unfolding remains roughly unchanged. The high-force regime is characterized by barrierless unfolding, and the unfolding time approaches a limit of ∼10 μs for the highest forces we studied. Finally, a combination of X and Q is shown to be a good reaction coordinate for almost the entire force range. PMID:25517160

  6. Protein unfolding by biological unfoldases: insights from modeling.

    PubMed

    Wojciechowski, Michał; Szymczak, Piotr; Carrión-Vázquez, Mariano; Cieplak, Marek

    2014-10-01

    The molecular determinants of the high efficiency of biological machines like unfoldases (e.g., the proteasome) are not well understood. We propose a model to study protein translocation into the chamber of biological unfoldases represented as a funnel. It is argued that translocation is a much faster way of unfolding a protein than end-to-end stretching, especially in a low-force regime, because it allows for a conformational freedom while concentrating local tension on consecutive regions of a protein chain and preventing refolding. This results in a serial unfolding of the protein structures dominated by unzipping. Thus, pulling against the unfoldase pore is an efficient catalyst of the unfolding reaction. We also show that the presence of the funnel makes the tension along the backbone of the substrate protein nonuniform even when the protein gets unfolded. Hence, the stalling force measured by single-molecule force spectroscopy techniques may be smaller than the traction force of the unfoldase motor. PMID:25296319

  7. Protein Unfolding by Biological Unfoldases: Insights from Modeling

    PubMed Central

    Wojciechowski, Michał; Szymczak, Piotr; Carrión-Vázquez, Mariano; Cieplak, Marek

    2014-01-01

    The molecular determinants of the high efficiency of biological machines like unfoldases (e.g., the proteasome) are not well understood. We propose a model to study protein translocation into the chamber of biological unfoldases represented as a funnel. It is argued that translocation is a much faster way of unfolding a protein than end-to-end stretching, especially in a low-force regime, because it allows for a conformational freedom while concentrating local tension on consecutive regions of a protein chain and preventing refolding. This results in a serial unfolding of the protein structures dominated by unzipping. Thus, pulling against the unfoldase pore is an efficient catalyst of the unfolding reaction. We also show that the presence of the funnel makes the tension along the backbone of the substrate protein nonuniform even when the protein gets unfolded. Hence, the stalling force measured by single-molecule force spectroscopy techniques may be smaller than the traction force of the unfoldase motor. PMID:25296319

  8. Protein unfolding and subsequent refolding: a spectroscopic investigation.

    PubMed

    Anand, Uttam; Jash, Chandrima; Mukherjee, Saptarshi

    2011-12-01

    The mechanism by which the protein Bovine Serum Albumin (BSA) undergoes unfolding induced by Guanidine Hydrochloride (GdHCl) and then the subsequent refolding brought in by many-fold dilution was studied by steady-state fluorescence, anisotropy, time resolved measurements and Circular Dichroism (CD) spectroscopy. CD data reveal that the protein attains a degree of extra rigidity at low concentrations of the denaturant, GdHCl, and this observation was correlated with other techniques used in this present work. The unfolding and refolding of BSA appear to proceed through intermediates and both the processes are sequential in nature. The intrinsic fluorescence from the tryptophan amino acid residue of BSA and another external fluorophore Nile Red was made use of in order to investigate the mechanisms of unfolding and refolding and we have conclusively proved that both these processes follow a reversible mechanism. PMID:21993230

  9. Influence of hydrodynamic interactions on mechanical unfolding of proteins

    NASA Astrophysics Data System (ADS)

    Szymczak, P.; Cieplak, Marek

    2007-07-01

    We incorporate hydrodynamic interactions in a structure-based model of ubiquitin and demonstrate that the hydrodynamic coupling may reduce the peak force when stretching the protein at constant speed, especially at larger speeds. Hydrodynamic interactions are also shown to facilitate unfolding at constant force and inhibit stretching by fluid flows.

  10. Connecting thermal and mechanical protein (un)folding landscapes

    NASA Astrophysics Data System (ADS)

    Sun, Li; Noel, Jeffrey; Sulkowska, Joanna; Levine, Herbert; Onuchic, José

    2015-03-01

    Molecular dynamics simulations supplement single-molecule pulling experiments by providing the possibility of examining the full free energy landscape using many coordinates. Here, we use an all-atom structure-based model to study the force and temperature dependence of the unfolding of the protein filamin by applying force at both termini. The unfolding time-force relation τ(F) indicates that the unfolding behavior can be characterized into three regimes: barrier-limited low- and intermediate-force regimes, and a barrierless high-force regime. Slope changes of τ(F) separate the three regimes. We show that the behavior of τ(F) can be understood from a two-dimensional free energy landscape projected onto the extension X and the fraction of native contacts Q. In the low-force regime, the unfolding rate is roughly force-independent due to the small (even negative) separation in X between the native ensemble and transition state ensemble (TSE). In the intermediate-force regime, force sufficiently separates the TSE from the native ensemble such that τ(F) roughly follows an exponential relation. The TSE becomes increasingly structured with force. The high-force regime is characterized by barrierless unfolding, approaching a time limit of around 10 μs.

  11. COARSE-GRAINED MODELING OF PROTEIN UNFOLDING DYNAMICS*

    PubMed Central

    DENG, MINGGE

    2014-01-01

    We present a new dynamic elastic network model (DENM) that describes the unfolding process of a force-loaded protein. The protein interaction network and its potentials are constructed based on information of its native-state structure obtained from the Protein Data Bank, with network nodes positioned at the Cα coordinates of the protein backbone. Specifically, to mimic the unfolding process, i.e., to simulate the process of overcoming the local energy barrier on the free energy landscape with force loading, the noncovalent protein network bonds (i.e., hydrogen bonds, salt bridges, hydrophobic contacts, etc.) are broken one-by-one with a certain probability, while the strong covalent bonds along the backbone (i.e., peptide bonds, disulfide bonds, etc.) are kept intact. The jumping event from local energy minima (bonds breaking rate) are chosen according to Kramer’s theory and the Bell model. Moreover, we exploit the self-similar structure of proteins at different scales to design an effective coarse-graining procedure for DENM with optimal parameter selection. The robustness of DENM is validated by coarse-grained molecular dynamics (MD) simulation against atomistic MD simulation of force-extension processes of the Fibrinogen and Titin Immunoglobulin proteins. We observe that the native structure of the proteins determines the total unfolding dynamics (including large deviations) and not just the fluctuations around the native state. PMID:25400515

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

    PubMed

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

    2014-01-31

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

  13. Amyloid protein unfolding and insertion kinetics on neuronal membrane mimics

    NASA Astrophysics Data System (ADS)

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

    2010-03-01

    Atomistic details of beta-amyloid (Aβ ) 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 Aβ 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.

  14. [Unfolding chaperone as a prion protein relating molecule].

    PubMed

    Hachiya, Naomi S; Sakasegawa, Yuji; Kaneko, Kiyotoshi

    2003-11-01

    Prion protein exists in two different isoforms, a normal cellular isoform (PrPc) and an abnormal infectious isoform (PrPSc), the latter is a causative agent of prion disease such as mad cow disease and Creutzfeldt-Jakob disease. Amino acid sequences of PrPc and PrPSc are identical, but their conformations are rather different; PrPc rich in non beta-sheet vs. PrPSc rich in beta-sheet isoform. Since the two isoforms have quite different conformation, this host factor might be a molecular chaperone, which enables to override an energy barrier between PrPc and PrPSc. To examine the protein unfolding activities against collectively folded structure exist or not, we constructed an assay system and purified a novel molecular chaperone. Unfolding, from S. cerevisiae. Unfolding consists of oligomeric ring-like structure with the central cavity and has an ATP-dependent protein Unfoldingg activity with broad specificity in vitro, of which targets included PrP in beta-sheet form, alpha-synuclein, and A beta protein. We have also found that mouse neuroblastoma N2a cells contained the activity. Treatment of this factor with an ATP-hydrolyzing enzyme, apyrase, caused the decrease in its protein Unfoldingg activity. It was suggested that the purified protein probably formed homo-oligomer consisting of 4-5 subunits and its activity was ATP-dependent. PMID:15152473

  15. Protein under tension and mechanical unfolding

    NASA Astrophysics Data System (ADS)

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

    2003-03-01

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

  16. Prediction of change in protein unfolding rates upon point mutations in two state proteins.

    PubMed

    Chaudhary, Priyashree; Naganathan, Athi N; Gromiha, M Michael

    2016-09-01

    Studies on protein unfolding rates are limited and challenging due to the complexity of unfolding mechanism and the larger dynamic range of the experimental data. Though attempts have been made to predict unfolding rates using protein sequence-structure information there is no available method for predicting the unfolding rates of proteins upon specific point mutations. In this work, we have systematically analyzed a set of 790 single mutants and developed a robust method for predicting protein unfolding rates upon mutations (Δlnku) in two-state proteins by combining amino acid properties and knowledge-based classification of mutants with multiple linear regression technique. We obtain a mean absolute error (MAE) of 0.79/s and a Pearson correlation coefficient (PCC) of 0.71 between predicted unfolding rates and experimental observations using jack-knife test. We have developed a web server for predicting protein unfolding rates upon mutation and it is freely available at https://www.iitm.ac.in/bioinfo/proteinunfolding/unfoldingrace.html. Prominent features that determine unfolding kinetics as well as plausible reasons for the observed outliers are also discussed. PMID:27264959

  17. Ising Model Reprogramming of a Repeat Protein's Equilibrium Unfolding Pathway.

    PubMed

    Millership, C; Phillips, J J; Main, E R G

    2016-05-01

    Repeat proteins are formed from units of 20-40 aa that stack together into quasi one-dimensional non-globular structures. This modular repetitive construction means that, unlike globular proteins, a repeat protein's equilibrium folding and thus thermodynamic stability can be analysed using linear Ising models. Typically, homozipper Ising models have been used. These treat the repeat protein as a series of identical interacting subunits (the repeated motifs) that couple together to form the folded protein. However, they cannot describe subunits of differing stabilities. Here we show that a more sophisticated heteropolymer Ising model can be constructed and fitted to two new helix deletion series of consensus tetratricopeptide repeat proteins (CTPRs). This analysis, showing an asymmetric spread of stability between helices within CTPR ensembles, coupled with the Ising model's predictive qualities was then used to guide reprogramming of the unfolding pathway of a variant CTPR protein. The designed behaviour was engineered by introducing destabilising mutations that increased the thermodynamic asymmetry within a CTPR ensemble. The asymmetry caused the terminal α-helix to thermodynamically uncouple from the rest of the protein and preferentially unfold. This produced a specific, highly populated stable intermediate with a putative dimerisation interface. As such it is the first step in designing repeat proteins with function regulated by a conformational switch. PMID:26947150

  18. Reversible thermal unfolding of a yfdX protein with chaperone-like activity

    PubMed Central

    Saha, Paramita; Manna, Camelia; Chakrabarti, Jaydeb; Ghosh, Mahua

    2016-01-01

    yfdX proteins are ubiquitously present in a large number of virulent bacteria. A member of this family of protein in E. coli is known to be up-regulated by the multidrug response regulator. Their abundance in such bacteria suggests some important yet unidentified functional role of this protein. Here, we study the thermal response and stability of yfdX protein STY3178 from Salmonella Typhi using circular dichroism, steady state fluorescence, dynamic light scattering and nuclear magnetic resonance experiments. We observe the protein to be stable up to a temperature of 45 °C. It folds back to the native conformation from unfolded state at temperature as high as 80 °C. The kinetic measurements of unfolding and refolding show Arrhenius behavior where the refolding involves less activation energy barrier than that of unfolding. We propose a homology model to understand the stability of the protein. Our molecular dynamic simulation studies on this model structure at high temperature show that the structure of this protein is quite stable. Finally, we report a possible functional role of this protein as a chaperone, capable of preventing DTT induced aggregation of insulin. Our studies will have broader implication in understanding the role of yfdX proteins in bacterial function and virulence. PMID:27404435

  19. Reversible thermal unfolding of a yfdX protein with chaperone-like activity.

    PubMed

    Saha, Paramita; Manna, Camelia; Chakrabarti, Jaydeb; Ghosh, Mahua

    2016-01-01

    yfdX proteins are ubiquitously present in a large number of virulent bacteria. A member of this family of protein in E. coli is known to be up-regulated by the multidrug response regulator. Their abundance in such bacteria suggests some important yet unidentified functional role of this protein. Here, we study the thermal response and stability of yfdX protein STY3178 from Salmonella Typhi using circular dichroism, steady state fluorescence, dynamic light scattering and nuclear magnetic resonance experiments. We observe the protein to be stable up to a temperature of 45 °C. It folds back to the native conformation from unfolded state at temperature as high as 80 °C. The kinetic measurements of unfolding and refolding show Arrhenius behavior where the refolding involves less activation energy barrier than that of unfolding. We propose a homology model to understand the stability of the protein. Our molecular dynamic simulation studies on this model structure at high temperature show that the structure of this protein is quite stable. Finally, we report a possible functional role of this protein as a chaperone, capable of preventing DTT induced aggregation of insulin. Our studies will have broader implication in understanding the role of yfdX proteins in bacterial function and virulence. PMID:27404435

  20. Amino acid substitutions in the non-structural proteins 4A or 4B modulate the induction of autophagy in West Nile virus infected cells independently of the activation of the unfolded protein response

    PubMed Central

    Blázquez, Ana-Belén; Martín-Acebes, Miguel A.; Saiz, Juan-Carlos

    2015-01-01

    West Nile virus (WNV) is a neurotropic mosquito-borne flavivirus responsible for outbreaks of meningitis and encephalitis. Whereas the activation of autophagy in cells infected with other flaviviruses is well known, the interaction of WNV with the autophagic pathway still remains unclear and there are reports describing opposite findings obtained even analyzing the same viral strain. To clarify this controversy, we first analyzed the induction of autophagic features in cells infected with a panel of WNV strains. WNV was determined to induce autophagy in a strain dependent manner. We observed that all WNV strains or isolates analyzed, except for the WNV NY99 used, upregulated the autophagic pathway in infected cells. Interestingly, a variant derived from this WNV NY99 isolated from a persistently infected mouse increased LC3 modification and aggregation. Genome sequencing of this variant revealed only two non-synonymous nucleotide substitutions when compared to parental NY99 strain. These nucleotide substitutions introduced one amino acid replacement in NS4A and other in NS4B. Using genetically engineered viruses we showed that introduction of only one of these replacements was sufficient to upregulate the autophagic pathway. Thus, in this work we have shown that naturally occurring point mutations in the viral non-structural proteins NS4A and NS4B confer WNV with the ability to induce the hallmarks of autophagy such as LC3 modification and aggregation. Even more, the differences on the induction of an autophagic response observed among WNV variants in infected cells did not correlate with alterations on the activation of the unfolded protein response (UPR), suggesting an uncoupling of UPR and autophagy during flavivirus infection. The findings here reported could help to improve the knowledge of the cellular processes involved on flavivirus–host cell interactions and contribute to the design of effective strategies to combat these pathogens. PMID:25642225

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

    ERIC Educational Resources Information Center

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

    2005-01-01

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

  2. Activation of autophagy by unfolded proteins during endoplasmic reticulum stress.

    PubMed

    Yang, Xiaochen; Srivastava, Renu; Howell, Stephen H; Bassham, Diane C

    2016-01-01

    Endoplasmic reticulum stress is defined as the accumulation of unfolded proteins in the endoplasmic reticulum, and is caused by conditions such as heat or agents that cause endoplasmic reticulum stress, including tunicamycin and dithiothreitol. Autophagy, a major pathway for degradation of macromolecules in the vacuole, is activated by these stress agents in a manner dependent on inositol-requiring enzyme 1b (IRE1b), and delivers endoplasmic reticulum fragments to the vacuole for degradation. In this study, we examined the mechanism for activation of autophagy during endoplasmic reticulum stress in Arabidopsis thaliana. The chemical chaperones sodium 4-phenylbutyrate and tauroursodeoxycholic acid were found to reduce tunicamycin- or dithiothreitol-induced autophagy, but not autophagy caused by unrelated stresses. Similarly, over-expression of BINDING IMMUNOGLOBULIN PROTEIN (BIP), encoding a heat shock protein 70 (HSP70) molecular chaperone, reduced autophagy. Autophagy activated by heat stress was also found to be partially dependent on IRE1b and to be inhibited by sodium 4-phenylbutyrate, suggesting that heat-induced autophagy is due to accumulation of unfolded proteins in the endoplasmic reticulum. Expression in Arabidopsis of the misfolded protein mimics zeolin or a mutated form of carboxypeptidase Y (CPY*) also induced autophagy in an IRE1b-dependent manner. Moreover, zeolin and CPY* partially co-localized with the autophagic body marker GFP-ATG8e, indicating delivery to the vacuole by autophagy. We conclude that accumulation of unfolded proteins in the endoplasmic reticulum is a trigger for autophagy under conditions that cause endoplasmic reticulum stress. PMID:26616142

  3. 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

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

    2013-01-01

    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

  4. Tannin-assisted aggregation of natively unfolded proteins

    NASA Astrophysics Data System (ADS)

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

    2008-06-01

    Tannin-protein interactions are essentially physical: hydrophobic and hydrogen-bond-mediated. We explored the tannin-assisted protein aggregation on the case of β-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.

  5. Protein co-translocational unfolding depends on the direction of pulling

    NASA Astrophysics Data System (ADS)

    Rodriguez-Larrea, David; Bayley, Hagan

    2014-09-01

    Protein unfolding and translocation through pores occurs during trafficking between organelles, protein degradation and bacterial toxin delivery. In vivo, co-translocational unfolding can be affected by the end of the polypeptide that is threaded into the pore first. Recently, we have shown that co-translocational unfolding can be followed in a model system at the single-molecule level, thereby unravelling molecular steps and their kinetics. Here, we show that the unfolding kinetics of the model substrate thioredoxin, when pulled through an α-haemolysin pore, differ markedly depending on whether the process is initiated from the C terminus or the N terminus. Further, when thioredoxin is pulled from the N terminus, the unfolding pathway bifurcates: some molecules finish unfolding quickly, while others finish ~100 times slower. Our findings have important implications for the understanding of biological unfolding mechanisms and in the application of nanopore technology for the detection of proteins and their modifications.

  6. The mechanochemistry of copper reports on the directionality of unfolding in model cupredoxin proteins

    NASA Astrophysics Data System (ADS)

    Beedle, Amy E. M.; Lezamiz, Ainhoa; Stirnemann, Guillaume; Garcia-Manyes, Sergi

    2015-08-01

    Understanding the directionality and sequence of protein unfolding is crucial to elucidate the underlying folding free energy landscape. An extra layer of complexity is added in metalloproteins, where a metal cofactor participates in the correct, functional fold of the protein. However, the precise mechanisms by which organometallic interactions are dynamically broken and reformed on (un)folding are largely unknown. Here we use single molecule force spectroscopy AFM combined with protein engineering and MD simulations to study the individual unfolding pathways of the blue-copper proteins azurin and plastocyanin. Using the nanomechanical properties of the native copper centre as a structurally embedded molecular reporter, we demonstrate that both proteins unfold via two independent, competing pathways. Our results provide experimental evidence of a novel kinetic partitioning scenario whereby the protein can stochastically unfold through two distinct main transition states placed at the N and C termini that dictate the direction in which unfolding occurs.

  7. The mechanochemistry of copper reports on the directionality of unfolding in model cupredoxin proteins

    PubMed Central

    Beedle, Amy E. M.; Lezamiz, Ainhoa; Stirnemann, Guillaume; Garcia-Manyes, Sergi

    2015-01-01

    Understanding the directionality and sequence of protein unfolding is crucial to elucidate the underlying folding free energy landscape. An extra layer of complexity is added in metalloproteins, where a metal cofactor participates in the correct, functional fold of the protein. However, the precise mechanisms by which organometallic interactions are dynamically broken and reformed on (un)folding are largely unknown. Here we use single molecule force spectroscopy AFM combined with protein engineering and MD simulations to study the individual unfolding pathways of the blue-copper proteins azurin and plastocyanin. Using the nanomechanical properties of the native copper centre as a structurally embedded molecular reporter, we demonstrate that both proteins unfold via two independent, competing pathways. Our results provide experimental evidence of a novel kinetic partitioning scenario whereby the protein can stochastically unfold through two distinct main transition states placed at the N and C termini that dictate the direction in which unfolding occurs. PMID:26235284

  8. Mifepristone increases mRNA translation rate, triggers the unfolded protein response, increases autophagic flux, and kills ovarian cancer cells in combination with proteasome or lysosome inhibitors.

    PubMed

    Zhang, Lei; Hapon, Maria B; Goyeneche, Alicia A; Srinivasan, Rekha; Gamarra-Luques, Carlos D; Callegari, Eduardo A; Drappeau, Donis D; Terpstra, Erin J; Pan, Bo; Knapp, Jennifer R; Chien, Jeremy; Wang, Xuejun; Eyster, Kathleen M; Telleria, Carlos M

    2016-08-01

    The synthetic steroid mifepristone blocks the growth of ovarian cancer cells, yet the mechanism driving such effect is not entirely understood. Unbiased genomic and proteomic screenings using ovarian cancer cell lines of different genetic backgrounds and sensitivities to platinum led to the identification of two key genes upregulated by mifepristone and involved in the unfolded protein response (UPR): the master chaperone of the endoplasmic reticulum (ER), glucose regulated protein (GRP) of 78 kDa, and the CCAAT/enhancer binding protein homologous transcription factor (CHOP). GRP78 and CHOP were upregulated by mifepristone in ovarian cancer cells regardless of p53 status and platinum sensitivity. Further studies revealed that the three UPR-associated pathways, PERK, IRE1α, and ATF6, were activated by mifepristone. Also, the synthetic steroid acutely increased mRNA translation rate, which, if prevented, abrogated the splicing of XBP1 mRNA, a non-translatable readout of IRE1α activation. Moreover, mifepristone increased LC3-II levels due to increased autophagic flux. When the autophagic-lysosomal pathway was inhibited with chloroquine, mifepristone was lethal to the cells. Lastly, doses of proteasome inhibitors that are inadequate to block the activity of the proteasomes, caused cell death when combined with mifepristone; this phenotype was accompanied by accumulation of poly-ubiquitinated proteins denoting proteasome inhibition. The stimulation by mifepristone of ER stress and autophagic flux offers a therapeutic opportunity for utilizing this compound to sensitize ovarian cancer cells to proteasome or lysosome inhibitors. PMID:27233943

  9. The Unfolded Protein Response and the Phosphorylations of Activating Transcription Factor 2 in the trans-Activation of il23a Promoter Produced by β-Glucans*

    PubMed Central

    Rodríguez, Mario; Domingo, Esther; Alonso, Sara; Frade, Javier García; Eiros, José; Crespo, Mariano Sánchez; Fernández, Nieves

    2014-01-01

    Current views on the control of IL-23 production focus on the regulation of il23a, the gene encoding IL-23 p19, by NF-κB in combination with other transcription factors. C/EBP homologous protein (CHOP), X2-Box-binding protein 1 (XBP1), activator protein 1 (AP1), SMAD, CCAAT/enhancer-binding protein (C/EBPβ), and cAMP-response element-binding protein (CREB) have been involved in response to LPS, but no data are available regarding the mechanism triggered by the fungal mimic and β-glucan-containing stimulus zymosan, which produces IL-23 and to a low extent the related cytokine IL-12 p70. Zymosan induced the mobilization of CHOP from the nuclear fractions to phagocytic vesicles. Hypha-forming Candida also induced the nuclear disappearance of CHOP. Assay of transcription factor binding to the il23a promoter showed an increase of Thr(P)-71–Thr(P)-69-activating transcription factor 2 (ATF2) binding in response to zymosan. PKC and PKA/mitogen- and stress-activated kinase inhibitors down-regulated Thr(P)-71–ATF2 binding to the il23a promoter and il23a mRNA expression. Consistent with the current concept of complementary phosphorylations on N-terminal Thr-71 and Thr-69 of ATF2 by ERK and p38 MAPK, MEK, and p38 MAPK inhibitors blunted Thr(P)-69–ATF2 binding. Knockdown of atf2 mRNA with siRNA correlated with inhibition of il23a mRNA, but it did not affect the expression of il12/23b and il10 mRNA. These data indicate the following: (i) zymosan decreases nuclear proapoptotic CHOP, most likely by promoting its accumulation in phagocytic vesicles; (ii) zymosan-induced il23a mRNA expression is best explained through coordinated κB- and ATF2-dependent transcription; and (iii) il23a expression relies on complementary phosphorylation of ATF2 on Thr-69 and Thr-71 dependent on PKC and MAPK activities. PMID:24982422

  10. Preferential binding of an unfolded protein to DsbA.

    PubMed Central

    Frech, C; Wunderlich, M; Glockshuber, R; Schmid, F X

    1996-01-01

    The oxidoreductase DsbA from the periplasm of escherichia coli introduces disulfide bonds into proteins at an extremely high rate. During oxidation, a mixed disulfide is formed between DsbA and the folding protein chain, and this covalent intermediate reacts very rapidly either to form the oxidized protein or to revert back to oxidized DsbA. To investigate its properties, a stable form of the intermediate was produced by reacting the C33A variant of DsbA with a variant of RNase T1. We find that in this stable mixed disulfide the conformational stability of the substrate protein is decreased by 5 kJ/mol, whereas the conformational stability of DsbA is increased by 5 kJ/mol. This reciprocal effect suggests strongly that DsbA interacts with the unfolded substrate protein not only by the covalent disulfide bond, but also by preferential non-covalent interactions. The existence of a polypeptide binding site explains why DsbA oxidizes protein substrates much more rapidly than small thiol compounds. Such a very fast reaction is probably important for protein folding in the periplasm, because the accessibility of the thiol groups for DsbA can decrease rapidly when newly exported polypeptide chains begin to fold. PMID:8617214

  11. Sequence- and Temperature-Dependent Properties of Unfolded and Disordered Proteins from Atomistic Simulations.

    PubMed

    Zerze, Gül H; Best, Robert B; Mittal, Jeetain

    2015-11-19

    We use all-atom molecular simulation with explicit solvent to study the properties of selected intrinsically disordered proteins and unfolded states of foldable proteins, which include chain dimensions and shape, secondary structure propensity, solvent accessible surface area, and contact formation. We find that the qualitative scaling behavior of the chains matches expectations from theory under ambient conditions. In particular, unfolded globular proteins tend to be more collapsed under the same conditions than charged disordered sequences of the same length. However, inclusion of explicit solvent in addition naturally captures temperature-dependent solvation effects, which results in an initial collapse of the chains as temperature is increased, in qualitative agreement with experiment. There is a universal origin to the collapse, revealed in the change of hydration of individual residues as a function of temperature: namely, that the initial collapse is driven by unfavorable solvation free energy of individual residues, which in turn has a strong temperature dependence. We also observe that in unfolded globular proteins, increased temperature also initially favors formation of native-like (rather than non-native-like) structure. Our results help to establish how sequence encodes the degree of intrinsic disorder or order as well as its response to changes in environmental conditions. PMID:26498157

  12. SANS and DLS Studies of Protein Unfolding in Presence of Urea and Surfactant

    SciTech Connect

    Aswal, V. K.; Chodankar, S. N.; Wagh, A. G.; Kohlbrecher, J.; Vavrin, R.

    2008-03-17

    Small-angle neutron scattering (SANS) and dynamic light scattering (DLS) have been used to study conformational changes in protein bovine serum albumin (BSA) during its unfolding in presence of protein denaturating agents urea and surfactant. On addition of urea, the BSA protein unfolds for urea concentrations greater than 4 M and acquires a random coil configuration with its radius of gyration increasing with urea concentration. The addition of surfactant unfolds the protein by the formation of micelle-like aggregates of surfactants along the unfolded polypeptide chains of the protein. The fractal dimension of such a protein-surfactant complex decreases and the overall size of the complex increases on increasing the surfactant concentration. The conformation of the unfolded protein in the complex has been determined directly using contrast variation SANS measurements by contrast matching the surfactant to the medium. Results of DLS measurements are found to be in good agreement with those obtained using SANS.

  13. Unfolded protein response induced by Brefeldin A increases collagen type I levels in hepatic stellate cells through an IRE1α, p38 MAPK and Smad-dependent pathway.

    PubMed

    de Galarreta, Marina Ruiz; Navarro, Amaia; Ansorena, Eduardo; Garzón, Antonia García; Mòdol, Teresa; López-Zabalza, María J; Martínez-Irujo, Juan J; Iraburu, María J

    2016-08-01

    Unfolded protein response (UPR) triggered as a consequence of ER stress has been shown to be involved in the development of different pathologies, including fibrotic disorders. In the present paper we explore the role played by UPR on a key fibrogenic parameter in the liver: collagen type I levels in activated hepatic stellate cells (HSC). Using Brefeldin A (BFA) as an ER stress inducer we found that UPR correlated with enhanced mRNA and protein levels of collagen type I in a cell line of immortalized non-tumoral rat HSC. Analysis of the three branches of UPR revealed the activation of IRE1α, PERK and ATF6 in response to BFA, although PERK activation was shown not to be involved in the fibrogenic action of BFA. BFA also activated p38 MAPK in an IRE1α-dependent way and the p38 MAPK inhibitor SB203580 prevented the increase in collagen type I mRNA and protein levels caused by BFA, suggesting the involvement of this kinase on this effect. Analysis of Smad activation showed that phosphorylated nuclear levels of Smad2 and 3 were increased in response to BFA treatment. Inhibition of Smad3 phosphorylation by SIS3 prevented the enhancement of collagen type I levels caused by BFA. Pretreatment with IRE1α and p38 MAPK inhibitors also prevented the increased p-Smad3 accumulation in the nucleus, suggesting an IRE1α-p38 MAPK-Smad pathway to be responsible for the fibrogenic action of BFA on HSC. PMID:27155082

  14. Hsp70 chaperones accelerate protein translocation and the unfolding of stable protein aggregates by entropic pulling.

    PubMed

    De Los Rios, Paolo; Ben-Zvi, Anat; Slutsky, Olga; Azem, Abdussalam; Goloubinoff, Pierre

    2006-04-18

    Hsp70s are highly conserved ATPase molecular chaperones mediating the correct folding of de novo synthesized proteins, the translocation of proteins across membranes, the disassembly of some native protein oligomers, and the active unfolding and disassembly of stress-induced protein aggregates. Here, we bring thermodynamic arguments and biochemical evidences for a unifying mechanism named entropic pulling, based on entropy loss due to excluded-volume effects, by which Hsp70 molecules can convert the energy of ATP hydrolysis into a force capable of accelerating the local unfolding of various protein substrates and, thus, perform disparate cellular functions. By means of entropic pulling, individual Hsp70 molecules can accelerate unfolding and pulling of translocating polypeptides into mitochondria in the absence of a molecular fulcrum, thus settling former contradictions between the power-stroke and the Brownian ratchet models for Hsp70-mediated protein translocation across membranes. Moreover, in a very different context devoid of membrane and components of the import pore, the same physical principles apply to the forceful unfolding, solubilization, and assisted native refolding of stable protein aggregates by individual Hsp70 molecules, thus providing a mechanism for Hsp70-mediated protein disaggregation. PMID:16606842

  15. Hsp70 chaperones accelerate protein translocation and the unfolding of stable protein aggregates by entropic pulling

    PubMed Central

    De Los Rios, Paolo; Ben-Zvi, Anat; Slutsky, Olga; Azem, Abdussalam; Goloubinoff, Pierre

    2006-01-01

    Hsp70s are highly conserved ATPase molecular chaperones mediating the correct folding of de novo synthesized proteins, the translocation of proteins across membranes, the disassembly of some native protein oligomers, and the active unfolding and disassembly of stress-induced protein aggregates. Here, we bring thermodynamic arguments and biochemical evidences for a unifying mechanism named entropic pulling, based on entropy loss due to excluded-volume effects, by which Hsp70 molecules can convert the energy of ATP hydrolysis into a force capable of accelerating the local unfolding of various protein substrates and, thus, perform disparate cellular functions. By means of entropic pulling, individual Hsp70 molecules can accelerate unfolding and pulling of translocating polypeptides into mitochondria in the absence of a molecular fulcrum, thus settling former contradictions between the power-stroke and the Brownian ratchet models for Hsp70-mediated protein translocation across membranes. Moreover, in a very different context devoid of membrane and components of the import pore, the same physical principles apply to the forceful unfolding, solubilization, and assisted native refolding of stable protein aggregates by individual Hsp70 molecules, thus providing a mechanism for Hsp70-mediated protein disaggregation. PMID:16606842

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

    ERIC Educational Resources Information Center

    Johnson, Matthew S.; Junker, Brian W.

    2003-01-01

    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…

  17. A human coronavirus OC43 variant harboring persistence-associated mutations in the S glycoprotein differentially induces the unfolded protein response in human neurons as compared to wild-type virus

    SciTech Connect

    Favreau, Dominique J.; Desforges, Marc; St-Jean, Julien R.; Talbot, Pierre J.

    2009-12-20

    We have reported that human respiratory coronavirus OC43 (HCoV-OC43) is neurotropic and neuroinvasive in humans and mice, and that neurons are the primary target of infection in mice, leading to neurodegenerative disabilities. We now report that an HCoV-OC43 mutant harboring two persistence-associated S glycoprotein point mutations (H183R and Y241H), induced a stronger unfolded protein response (UPR) and translation attenuation in infected human neurons. There was a major contribution of the IRE1/XBP1 pathway, followed by caspase-3 activation and nuclear fragmentation, with no significant role of the ATF6 and eIF2-alpha/ATF4 pathways. Our results show the importance of discrete molecular viral S determinants in virus-neuronal cell interactions that lead to increased production of viral proteins and infectious particles, enhanced UPR activation, and increased cytotoxicity and cell death. As this mutant virus is more neurovirulent in mice, our results also suggest that two mutations in the S glycoprotein could eventually modulate viral neuropathogenesis.

  18. ATF4-dependent transcription is a key mechanism in VEGF up-regulation by oxidized phospholipids: critical role of oxidized sn-2 residues in activation of unfolded protein response

    PubMed Central

    Oskolkova, Olga V.; Afonyushkin, Taras; Leitner, Alexander; von Schlieffen, Elena; Gargalovic, Peter S.; Lusis, Aldons J.; Binder, Bernd R.

    2008-01-01

    We have shown previously that oxidized phospholipids (OxPLs), known to accumulate in atherosclerotic vessels, stimulate angiogenesis via induction of autocrine mediators, such as vascular endothelial growth factor (VEGF). We now address the pathways mediating up-regulation of VEGF in human endothelial cells treated with OxPLs. Analysis of structure-function relationship using individual species of OxPLs demonstrated a close relation between induction of VEGF and activation of the unfolded protein response (UPR). Inducers of UPR up-regulated VEGF, whereas inhibition of UPR by chemical chaperones or knock-down of cochaperone HTJ-1 inhibited elevation of VEGF mRNA induced by OxPLs. OxPLs induced protein expression of activating transcription factor-4 (ATF4), an important effector of UPR. Expression levels of VEGF in OxPL-treated cells strongly correlated with induction of the ATF4 target genes ATF3 and TRB3. Knocking down ATF4 was paralleled by loss of VEGF induction by OxPLs. Chromatin immunoprecipitation demonstrated that OxPLs stimulated binding of ATF4 to a regulatory site in the VEGFA gene. Taken together, these data characterize UPR and more specifically its ATF4 branch as an important mechanism mediating up-regulation of VEGF by OxPLs, and allow hypothesizing that the UPR cascade might play a role in pathologic angiogenesis in atherosclerotic plaques. PMID:18451308

  19. Implementation of Multiple Spectroscopic Techniques to Simultaneously Observe Native and Mutated Protein Unfolding

    NASA Astrophysics Data System (ADS)

    Cull, Brennan; Ben, Kelty; Link, Justin

    A protein's natural, correctly folded structure can determine the protein's ability to carry out its function. If the unfolding process of proteins can be observed, then the relative stability can be better understood between native and mutated proteins. A global picture of the unfolding process may be completed through the studies of strategically mutated proteins using tryptophan as a probe. Horse heart cytochrome c, a thoroughly studied, model protein was used in our investigation to explore this idea. Various spectroscopic techniques such as circular dichroism (CD), absorbance, and fluorescence were simultaneously applied while slowly unfolding our protein by increasing the concentration of a chemical denaturant, guanidine hydrochloride. This provided us information about the thermodynamic properties of the protein and several mutants which can then be interpreted to gain relative stability information among mutations. Efforts to utilize these techniques on native and mutated proteins in comparison to current scientific unfolding theories will be presented in this session.

  20. Protein unfolding as a switch from self-recognition to high-affinity client binding.

    PubMed

    Groitl, Bastian; Horowitz, Scott; Makepeace, Karl A T; Petrotchenko, Evgeniy V; Borchers, Christoph H; Reichmann, Dana; Bardwell, James C A; Jakob, Ursula

    2016-01-01

    Stress-specific activation of the chaperone Hsp33 requires the unfolding of a central linker region. This activation mechanism suggests an intriguing functional relationship between the chaperone's own partial unfolding and its ability to bind other partially folded client proteins. However, identifying where Hsp33 binds its clients has remained a major gap in our understanding of Hsp33's working mechanism. By using site-specific Fluorine-19 nuclear magnetic resonance experiments guided by in vivo crosslinking studies, we now reveal that the partial unfolding of Hsp33's linker region facilitates client binding to an amphipathic docking surface on Hsp33. Furthermore, our results provide experimental evidence for the direct involvement of conditionally disordered regions in unfolded protein binding. The observed structural similarities between Hsp33's own metastable linker region and client proteins present a possible model for how Hsp33 uses protein unfolding as a switch from self-recognition to high-affinity client binding. PMID:26787517

  1. Protein unfolding as a switch from self-recognition to high-affinity client binding

    PubMed Central

    Groitl, Bastian; Horowitz, Scott; Makepeace, Karl A. T.; Petrotchenko, Evgeniy V.; Borchers, Christoph H.; Reichmann, Dana; Bardwell, James C. A.; Jakob, Ursula

    2016-01-01

    Stress-specific activation of the chaperone Hsp33 requires the unfolding of a central linker region. This activation mechanism suggests an intriguing functional relationship between the chaperone's own partial unfolding and its ability to bind other partially folded client proteins. However, identifying where Hsp33 binds its clients has remained a major gap in our understanding of Hsp33's working mechanism. By using site-specific Fluorine-19 nuclear magnetic resonance experiments guided by in vivo crosslinking studies, we now reveal that the partial unfolding of Hsp33's linker region facilitates client binding to an amphipathic docking surface on Hsp33. Furthermore, our results provide experimental evidence for the direct involvement of conditionally disordered regions in unfolded protein binding. The observed structural similarities between Hsp33's own metastable linker region and client proteins present a possible model for how Hsp33 uses protein unfolding as a switch from self-recognition to high-affinity client binding. PMID:26787517

  2. Elucidation of GB1 Protein Unfolding Mechanism via a Long-timescale Molecular Dynamics Simulation

    NASA Astrophysics Data System (ADS)

    Sumaryada, T.; Hati, J.; Wahyudi, S. T.; Malau, N. D.; Sawitri, K. N.

    2016-01-01

    This study investigates the unfolding mechanism of 1GB1 protein at various simulation temperatures using a long-timescale molecular dynamics simulation. Analysis of structural parameters of molecular dynamics simulation have indicated that the unfolding process of GB1 protein has started at 95 ns for 475 K simulation, and at 745 ps for 500 K simulation. The unfolding process in this simulation exhibit the feature of hydrophobic core collapse model, in which the beta-hairpin destruction precedes the a-helix to coil transition. The unfolding was started with the increasing flexibility of the beta-sheets and hydrophobic core region, continued with beta-hairpins destruction, and ended with a-helix to coil and turn transition. The final structures of GB1 protein after unfolding, suggest an unfinished denaturation of protein as seen from the small remains of α-helix structure.

  3. Intermediate states of globular proteins during temperature-induced folding and unfolding studied using small angle x-ray scattering

    NASA Astrophysics Data System (ADS)

    Banuelos, Jose; Urquidi, Jacob

    2009-03-01

    The ability of proteins to change their conformation in response to changes in their environment has consequences in biological processes like metabolism, chemical regulation in cells, and is believed to play a role in the onset of several neurodegenerative diseases. Factors such as concentration, degree of crowding from other entities, and solvent medium affect how a protein folds. As a protein unfolds, the ratio of nonpolar to polar groups exposed to water changes, affecting a protein's thermodynamic properties. Using small angle x-ray scattering (SAXS), we are currently studying the intermediate protein conformations that arise during the folding/unfolding process as a function of temperature for a series of globular proteins. The temporal stability of these ensembles is also under investigation. Trends in the scattering profiles, along with correlations with protein thermodynamics, may help elucidate shared characteristics between all proteins in their folding behavior.

  4. Shifting transition states in the unfolding of a large ankyrin repeat protein

    PubMed Central

    Werbeck, Nicolas D.; Rowling, Pamela J. E.; Chellamuthu, Vasuki R.; Itzhaki, Laura S.

    2008-01-01

    The 33-amino-acid ankyrin motif comprises a β-turn followed by two anti-parallel α-helices and a loop and tandem arrays of the motif pack in a linear fashion to produce elongated structures characterized by short-range interactions. In this article we use site-directed mutagenesis to investigate the kinetic unfolding mechanism of D34, a 426-residue, 12-ankyrin repeat fragment of the protein ankyrinR. The data are consistent with a model in which the N-terminal half of the protein unfolds first by unraveling progressively from the start of the polypeptide chain to form an intermediate; in the next step, the C-terminal half of the protein unfolds via two pathways whose transition states have either the early or the late C-terminal ankyrin repeats folded. We conclude that the two halves of the protein unfold by different mechanisms because the N-terminal moiety folds and unfolds in the context of a folded C-terminal moiety, which therefore acts as a “seed” and confers a unique directionality on the process, whereas the C-terminal moiety folds and unfolds in the context of an unfolded N-terminal moiety and therefore behaves like a single-domain ankyrin repeat protein, having a high degree of symmetry and consequently more than one unfolding pathway accessible to it. PMID:18632570

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

    NASA Astrophysics Data System (ADS)

    Discher, Dennis; Law, Richard; Klein, Mike

    2004-03-01

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

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

    PubMed Central

    Vitarelli, Michael J.; Talaga, David S.

    2013-01-01

    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

  7. Fibroblast growth factor 21 is regulated by the IRE1α-XBP1 branch of the unfolded protein response and counteracts endoplasmic reticulum stress-induced hepatic steatosis.

    PubMed

    Jiang, Shan; Yan, Cheng; Fang, Qi-chen; Shao, Meng-le; Zhang, Yong-liang; Liu, Yang; Deng, Yi-ping; Shan, Bo; Liu, Jing-qi; Li, Hua-ting; Yang, Liu; Zhou, Jian; Dai, Zhi; Liu, Yong; Jia, Wei-ping

    2014-10-24

    Endoplasmic reticulum (ER) stress activates the adaptive unfolded protein response (UPR) and represents a critical mechanism that underlies metabolic dysfunctions. Fibroblast growth factor 21 (FGF21), a hormone that is predominantly secreted by the liver, exerts a broad range of effects upon the metabolism of carbohydrates and lipids. Although increased circulating levels of FGF21 have been documented in animal models and human subjects with obesity and nonalcoholic fatty liver disease, the functional interconnections between metabolic ER stress and FGF21 are incompletely understood. Here, we report that increased ER stress along with the simultaneous elevation of FGF21 expression were associated with the occurrence of nonalcoholic fatty liver disease both in diet-induced obese mice and human patients. Intraperitoneal administration of the ER stressor tunicamycin in mice resulted in hepatic steatosis, accompanied by activation of the three canonical UPR branches and increased the expression of FGF21. Furthermore, the IRE1α-XBP1 pathway of the UPR could directly activate the transcriptional expression of Fgf21. Administration of recombinant FGF21 in mice alleviated tunicamycin-induced liver steatosis, in parallel with reduced eIF2α-ATF4-CHOP signaling. Taken together, these results suggest that FGF21 is an integral physiological component of the cellular UPR program, which exerts beneficial feedback effects upon lipid metabolism through counteracting ER stress. PMID:25170079

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

    PubMed Central

    Bernadó, Pau; Blackledge, Martin; Sancho, Javier

    2006-01-01

    Protein stability cannot be understood without the correct description of the unfolded state. We present here an efficient method for accurate calculation of atomic solvent exposures for denatured protein ensembles. The method used to generate the ensembles has been shown to reproduce diverse biophysical experimental data corresponding to natively and chemically unfolded proteins. Using a data set of 19 nonhomologous proteins containing from 98 to 579 residues, we report average accessibilities for all residue types. These averaged accessibilities are considerably lower than those previously reported for tripeptides and close to the lower limit reported by Creamer and co-workers. Of importance, we observe remarkable sequence dependence for the exposure to solvent of all residue types, which indicates that average residue solvent exposures can be inappropriate to interpret mutational studies. In addition, we observe smaller influences of both protein size and protein amino acid composition in the averaged residue solvent exposures for individual proteins. Calculating residue-specific solvent accessibilities within the context of real sequences is thus necessary and feasible. The approach presented here may allow a more precise parameterization of protein energetics as a function of polar- and apolar-area burial and opens new ways to investigate the energetics of the unfolded state of proteins. PMID:17012314

  9. Valosin-containing protein (VCP/p97) is capable of unfolding polyubiquitinated proteins through its ATPase domains.

    PubMed

    Song, Changcheng; Wang, Qing; Song, Changzheng; Rogers, Thomas J

    2015-07-31

    Valosin-containing protein (VCP or p97) is required for the proteasomal degradation of polyubiquitinated proteins. However, the molecular mechanism for VCP to process the polyubiquitinated proteins remains unclear. Here, we show that VCP can unfold polyubiquitinated proteins. It preferably unfolds the pentaubiquitin-over monoubiquin-conjugated dihydrofolate reductase (Ub5-DHFR or Ub-DHFR) in a dose dependent manner. In addition, the unfolding activity of VCP does not depend on its ATPase activity, on the contrary, ATP and its non-hydrolysable analogs suppress the unfolding of Ub5-DHFR. The structural and functional analysis showed that either D1 or D2 domain of VCP is sufficient to carry out this unfolding activity. The structure of the substrates also affects its unfolding by VCP. VCP is unable to unfold Ub5-DHFR in a tight structure when it binds with methotrexate, a folate analog with high affinity to DHFR. Thus, these results support that VCP is capable of unfolding polyubiquitinated proteins and suggest that VCP may facilitate the proteasomal degradation of polyubiquitinated proteins through its unfolding activity. PMID:26043696

  10. Mimicking Ribosomal Unfolding of RNA Pseudoknot in a Protein Channel.

    PubMed

    Zhang, Xinyue; Xu, Xiaojun; Yang, Zhiyu; Burcke, Andrew J; Gates, Kent S; Chen, Shi-Jie; Gu, Li-Qun

    2015-12-23

    Pseudoknots are a fundamental RNA tertiary structure with important roles in regulation of mRNA translation. Molecular force spectroscopic approaches such as optical tweezers can track the pseudoknot's unfolding intermediate states by pulling the RNA chain from both ends, but the kinetic unfolding pathway induced by this method may be different from that in vivo, which occurs during translation and proceeds from the 5' to 3' end. Here we developed a ribosome-mimicking, nanopore pulling assay for dissecting the vectorial unfolding mechanism of pseudoknots. The pseudoknot unfolding pathway in the nanopore, either from the 5' to 3' end or in the reverse direction, can be controlled by a DNA leader that is attached to the pseudoknot at the 5' or 3' ends. The different nanopore conductance between DNA and RNA translocation serves as a marker for the position and structure of the unfolding RNA in the pore. With this design, we provided evidence that the pseudoknot unfolding is a two-step, multistate, metal ion-regulated process depending on the pulling direction. Most notably, unfolding in both directions is rate-limited by the unzipping of the first helix domain (first step), which is Helix-1 in the 5' → 3' direction and Helix-2 in the 3' → 5' direction, suggesting that the initial unfolding step in either pulling direction needs to overcome an energy barrier contributed by the noncanonical triplex base-pairs and coaxial stacking interactions for the tertiary structure stabilization. These findings provide new insights into RNA vectorial unfolding mechanisms, which play an important role in biological functions including frameshifting. PMID:26595106

  11. Fully Atomistic Simulations of Protein Unfolding in Low Speed Atomic Force Microscope and Force Clamp Experiments with the Help of Boxed Molecular Dynamics.

    PubMed

    Booth, Jonathan J; Shalashilin, Dmitrii V

    2016-02-01

    The results of boxed dynamics (BXD) fully atomistic simulations of protein unfolding by atomic force microscopy (AFM) in both force clamp (FC) and velocity clamp (VC) modes are reported. In AFM experiments the unfolding occurs on a time scale which is too long for standard atomistic molecular dynamics (MD) simulations, which are usually performed with the addition of forces which exceed those of experiment by many orders of magnitude. BXD can reach the time scale of slow unfolding and sample the very high free energy unfolding pathway, reproducing the experimental dependence of pulling force against extension and extension against time. Calculations show the presence of the pulling force "humps" previously observed in the VC AFM experiments and allow the identification of intermediate protein conformations responsible for them. Fully atomistic BXD simulations can estimate the rate of unfolding in the FC experiments up to the time scale of seconds. PMID:26760898

  12. Development and Application of a High Throughput Protein Unfolding Kinetic Assay

    PubMed Central

    Wang, Qiang; Waterhouse, Nicklas; Feyijinmi, Olusegun; Dominguez, Matthew J.; Martinez, Lisa M.; Sharp, Zoey; Service, Rachel; Bothe, Jameson R.; Stollar, Elliott J.

    2016-01-01

    The kinetics of folding and unfolding underlie protein stability and quantification of these rates provides important insights into the folding process. Here, we present a simple high throughput protein unfolding kinetic assay using a plate reader that is applicable to the studies of the majority of 2-state folding proteins. We validate the assay by measuring kinetic unfolding data for the SH3 (Src Homology 3) domain from Actin Binding Protein 1 (AbpSH3) and its stabilized mutants. The results of our approach are in excellent agreement with published values. We further combine our kinetic assay with a plate reader equilibrium assay, to obtain indirect estimates of folding rates and use these approaches to characterize an AbpSH3-peptide hybrid. Our high throughput protein unfolding kinetic assays allow accurate screening of libraries of mutants by providing both kinetic and equilibrium measurements and provide a means for in-depth ϕ-value analyses. PMID:26745729

  13. ATP-dependent Proteases Differ Substantially in Their Ability to Unfold Globular Proteins*

    PubMed Central

    Koodathingal, Prakash; Jaffe, Neil E.; Kraut, Daniel A.; Prakash, Sumit; Fishbain, Susan; Herman, Christophe; Matouschek, Andreas

    2009-01-01

    ATP-dependent proteases control the concentrations of hundreds of regulatory proteins and remove damaged or misfolded proteins from cells. They select their substrates primarily by recognizing sequence motifs or covalent modifications. Once a substrate is bound to the protease, it has to be unfolded and translocated into the proteolytic chamber to be degraded. Some proteases appear to be promiscuous, degrading substrates with poorly defined targeting signals, which suggests that selectivity may be controlled at additional levels. Here we compare the abilities of representatives from all classes of ATP-dependent proteases to unfold a model substrate protein and find that the unfolding abilities range over more than 2 orders of magnitude. We propose that these differences in unfolding abilities contribute to the fates of substrate proteins and may act as a further layer of selectivity during protein destruction. PMID:19383601

  14. High-Pressure SAXS Study of Folded and Unfolded Ensembles of Proteins

    PubMed Central

    Schroer, Martin A.; Paulus, Michael; Jeworrek, Christoph; Krywka, Christina; Schmacke, Saskia; Zhai, Yong; Wieland, D. C. Florian; Sahle, Christoph J.; Chimenti, Michael; Royer, Catherine A.; Garcia-Moreno, Bertrand; Tolan, Metin; Winter, Roland

    2010-01-01

    A structural interpretation of the thermodynamic stability of proteins requires an understanding of the structural properties of the unfolded state. High-pressure small-angle x-ray scattering was used to measure the effects of temperature, pressure, denaturants, and stabilizing osmolytes on the radii of gyration of folded and unfolded state ensembles of staphylococcal nuclease. A set of variants with the internal Val-66 replaced with Ala, Tyr, or Arg was used to examine how changes in the volume and polarity of an internal microcavity affect the dimensions of the native state and the pressure sensitivity of the ensemble. The unfolded state ensembles achieved for these proteins with high pressure were more compact than those achieved at high temperature, and were all very sensitive to the presence of urea and glycerol. Substitutions at the hydrophobic core detectably altered the conformation of the protein, even in the folded state. The introduction of a charged residue, such as Arg, inside the hydrophobic interior of a protein could dramatically alter the structural properties, even those of the unfolded state. The data suggest that a charge at an internal position can interfere with the formation of transient hydrophobic clusters in the unfolded state, and ensure that the pressure-unfolded form of a protein occupies the maximum volume possible. Only at high temperatures does the radius of gyration of the unfolded state ensemble approach the value for a statistical random coil. PMID:21081092

  15. High-pressure SAXS study of folded and unfolded ensembles of proteins.

    PubMed

    Schroer, Martin A; Paulus, Michael; Jeworrek, Christoph; Krywka, Christina; Schmacke, Saskia; Zhai, Yong; Wieland, D C Florian; Sahle, Christoph J; Chimenti, Michael; Royer, Catherine A; Garcia-Moreno, Bertrand; Tolan, Metin; Winter, Roland

    2010-11-17

    A structural interpretation of the thermodynamic stability of proteins requires an understanding of the structural properties of the unfolded state. High-pressure small-angle x-ray scattering was used to measure the effects of temperature, pressure, denaturants, and stabilizing osmolytes on the radii of gyration of folded and unfolded state ensembles of staphylococcal nuclease. A set of variants with the internal Val-66 replaced with Ala, Tyr, or Arg was used to examine how changes in the volume and polarity of an internal microcavity affect the dimensions of the native state and the pressure sensitivity of the ensemble. The unfolded state ensembles achieved for these proteins with high pressure were more compact than those achieved at high temperature, and were all very sensitive to the presence of urea and glycerol. Substitutions at the hydrophobic core detectably altered the conformation of the protein, even in the folded state. The introduction of a charged residue, such as Arg, inside the hydrophobic interior of a protein could dramatically alter the structural properties, even those of the unfolded state. The data suggest that a charge at an internal position can interfere with the formation of transient hydrophobic clusters in the unfolded state, and ensure that the pressure-unfolded form of a protein occupies the maximum volume possible. Only at high temperatures does the radius of gyration of the unfolded state ensemble approach the value for a statistical random coil. PMID:21081092

  16. Local Unfolding of Fatty Acid Binding Protein to Allow Ligand Entry for Binding.

    PubMed

    Xiao, Tianshu; Fan, Jing-Song; Zhou, Hu; Lin, Qingsong; Yang, Daiwen

    2016-06-01

    Fatty acid binding proteins are responsible for the transportation of fatty acids in biology. Despite intensive studies, the molecular mechanism of fatty acid entry to and exit from the protein cavity is still unclear. Here a cap-closed variant of human intestinal fatty acid binding protein was generated by mutagenesis, in which the helical cap is locked to the β-barrel by a disulfide linkage. Structure determination shows that this variant adopts a closed conformation, but still uptakes fatty acids. Stopped-flow experiments indicate that a rate-limiting step exists before the ligand association and this step corresponds to the conversion of the closed form to the open one. NMR relaxation dispersion and H-D exchange data demonstrate the presence of two excited states: one is native-like, but the other adopts a locally unfolded structure. Local unfolding of helix 2 generates an opening for ligands to enter the protein cavity, and thus controls the ligand association rate. PMID:27105780

  17. Unfolding and inactivation of proteins by counterions in protein-nanoparticles interaction.

    PubMed

    Ghosh, Goutam; Gaikwad, Pallavi S; Panicker, Lata; Nath, Bimalendu B; Mukhopadhyaya, Rita

    2016-09-01

    In this work, the structure and activity of proteins; such as, hen egg lysozyme (HEWL) and calf intestine alkaline phosphatase (CIAP); have been investigated after incubation with surface coated iron oxide nanoparticles (IONPs) in water. IONPs were coated with counterions bound charge-ligands and were named as the charge-ligand counterions iron oxide nanoparticles (CLC-IONPs). The coating was done with tri-lithium citrate (TLC) and tri-potassium citrate (TKC) to have negative surface charge of CLC-IONPs and Li(+) and K(+), respectively, as counterions. To have positive surface charge, IONPs were coated with cetylpyridinium chloride (CPC) and cetylpyridinium iodide (CPI) having Cl(-) and I(-), respectively, as counterions. The secondary structure of proteins was measured using far ultraviolet circular dichroism (CD) spectroscopy which showed that both proteins were irreversibly unfolded after incubation with CLC-IONPs. The unfolded proteins were seen to be functionally inactive, as confirmed through their activity assays, i.e., HEWL with Escherichia coli (E. coli) and CIAP with para-nitrophenyl phosphate (pNPP). Additionally, we have observed that monomeric hemoglobin (Hb) from radio-resistant insect Chironomus ramosus (ChHb) was also partially unfolded upon interaction with CLC-IONPs. This work clearly shows the role of counterions in protein inactivation via protein-nanoparticles interaction and, therefore, CLC-IONPs could be used for therapeutic purpose. PMID:27182654

  18. Assaying the kinetics of protein denaturation catalyzed by AAA+ unfolding machines and proteases.

    PubMed

    Baytshtok, Vladimir; Baker, Tania A; Sauer, Robert T

    2015-04-28

    ATP-dependent molecular machines of the AAA+ superfamily unfold or remodel proteins in all cells. For example, AAA+ ClpX and ClpA hexamers collaborate with the self-compartmentalized ClpP peptidase to unfold and degrade specific proteins in bacteria and some eukaryotic organelles. Although degradation assays are straightforward, robust methods to assay the kinetics of enzyme-catalyzed protein unfolding in the absence of proteolysis have been lacking. Here, we describe a FRET-based assay in which enzymatic unfolding converts a mixture of donor-labeled and acceptor-labeled homodimers into heterodimers. In this assay, ClpX is a more efficient protein-unfolding machine than ClpA both kinetically and in terms of ATP consumed. However, ClpP enhances the mechanical activities of ClpA substantially, and ClpAP degrades the dimeric substrate faster than ClpXP. When ClpXP or ClpAP engage the dimeric subunit, one subunit is actively unfolded and degraded, whereas the other subunit is passively unfolded by loss of its partner and released. This assay should be broadly applicable for studying the mechanisms of AAA+ proteases and remodeling chaperones. PMID:25870262

  19. Investigation of folding unfolding process of a new variant of dihydrofolate reductase protein from Zebrafish.

    PubMed

    Thapliyal, Charu; Jain, Neha; Chaudhuri Chattopadhyay, Pratima

    2016-10-01

    The folding and unfolding mechanisms of a small monomeric protein, Dihydrofolate reductase (1.5.1.3.) from a new variant, Zebrafish (zDHFR) has been studied through GdnHCl denaturation, followed by its refolding through dilution of the denaturant. Intrinsic and extrinsic fluorescence, far-UV CD and enzyme activity were employed to monitor structural and functional changes due to chemical denaturation. The unfolding transitions monitored by intrinsic fluorescence showed that GdnHCl based denaturation of zDHFR is reversible. At low concentration of the denaturant, zDHFR forms intermediate species as reflected by increased fluorescence intensity compared to the native and fully unfolded form. Equilibrium unfolding transition study of zDHFR induced by GdnHCl exhibited three- state process. The non- coincidence of fluorescence and far-UVCD based transitions curves support the establishment of three state model of zDHFR protein which involves native, intermediate and unfolded forms. Analysis of the equilibrium unfolding transition suggests the presence of non- native intermediate species. A comparative study of various species of DHFR shows that zDHFR has comparable thermodynamic stability with human counterpart and thus proved to be a good in vitro model system for structure- function relationship studies. Understanding various conformational states during the folding unfolding process of the zDHFR protein may provide important clues towards designing inhibitors against this important protein involved in cell cycle regulation. PMID:27287769

  20. Point Mutations in Membrane Proteins Reshape Energy Landscape and Populate Different Unfolding Pathways

    PubMed Central

    Sapra, K. Tanuj; Balasubramanian, G. Prakash; Labudde, Dirk; Bowie, James U.; Muller, Daniel J.

    2009-01-01

    Using single-molecule force spectroscopy, we investigated the effect of single point mutations on the energy landscape and unfolding pathways of the transmembrane protein bacteriorhodopsin. We show that the unfolding energy barriers in the energy landscape of the membrane protein followed a simple two-state behavior and represent a manifestation of many converging unfolding pathways. Although the unfolding pathways of wild-type and mutant bacteriorhodopsin did not change, indicating the presence of same ensemble of structural unfolding intermediates, the free energies of the rate-limiting transition states of the bacteriorhodopsin mutants decreased as the distance of those transition states to the folded intermediate states decreased. Thus, all mutants exhibited Hammond behavior and a change in the free energies of the intermediates along the unfolding reaction coordinate and, consequently, their relative occupancies. This is the first experimental proof showing that point mutations can reshape the free energy landscape of a membrane protein and force single proteins to populate certain unfolding pathways over others. PMID:18191146

  1. The Paracrine Effect of Skeletal Myoblasts Is Cardioprotective Against Oxidative Stress and Involves EGFR-ErbB4 Signaling, Cystathionase, and the Unfolded Protein Response.

    PubMed

    Siltanen, Antti; Nuutila, Kristo; Imanishi, Yukiko; Uenaka, Hisazumi; Mäkelä, Johanna; Pätilä, Tommi; Vento, Antti; Miyagawa, Shigeru; Sawa, Yoshiki; Harjula, Ari; Kankuri, Esko

    2016-01-01

    Therapeutic effects of skeletal myoblast transplantation into the myocardium are mediated via paracrine factors. We investigated the ability of myoblast-derived soluble mediators to protect cardiomyocytes from oxidative stress. Fetal rat cardiac cells were treated with conditioned medium from cultures of myoblasts or cardiac fibroblasts, and oxidative stress was induced with H2O2. Myoblast-derived factors effectively prevented oxidative stress-induced cardiac cell death and loss of mitochondrial membrane potential. This protective effect was mediated via epidermal growth factor (EGF) receptor and c-Met signaling, and mimicked by neuregulin 1 but not EGF. Microarray analysis of cardiac cells treated with myoblast versus cardiac fibroblast-derived mediators revealed differential regulation of genes associated with antioxidative effects: cystathionine-γ-lyase (cst), xanthine oxidase, and thioredoxin-interacting protein as well as tribbles homolog 3 (trib3). Cardiac cell pretreatment with tunicamycin, an inducer of trib3, also protected them against H2O2-induced cell death. Epicardial transplantation of myoblast sheets in a rat model of acute myocardial infarction was used to evaluate the expression of CST and trib3 as markers of myoblasts' paracrine effect in vivo. Myoblast sheets induced expression of the CST as well as trib3 in infarcted myocardium. CST localized around blood vessels, suggesting smooth muscle cell localization. Our results provide a deeper molecular insight into the therapeutic mechanisms of myoblast-derived paracrine signaling in cardiac cells and suggest that myoblast transplantation therapy may prevent oxidative stress-induced cardiac deterioration and progression of heart failure. PMID:26021843

  2. Correction: Reversibility and two state behaviour in the thermal unfolding of oligomeric TIM barrel proteins.

    PubMed

    Romero-Romero, Sergio; Costas, Miguel; Rodríguez-Romero, Adela; Fernández-Velasco, D Alejandro

    2016-04-21

    Correction for 'Reversibility and two state behaviour in the thermal unfolding of oligomeric TIM barrel proteins' by Sergio Romero-Romero et al., Phys. Chem. Chem. Phys., 2015, 17, 20699-20714. PMID:27010946

  3. Frequency of COL4A3/COL4A4 Mutations amongst Families Segregating Glomerular Microscopic Hematuria and Evidence for Activation of the Unfolded Protein Response. Focal and Segmental Glomerulosclerosis Is a Frequent Development during Ageing

    PubMed Central

    Papazachariou, Louiza; Demosthenous, Panayiota; Pieri, Myrtani; Papagregoriou, Gregory; Savva, Isavella; Stavrou, Christoforos; Zavros, Michael; Athanasiou, Yiannis; Ioannou, Kyriakos; Patsias, Charalambos; Panagides, Alexia; Potamitis, Costas; Demetriou, Kyproula; Prikis, Marios; Hadjigavriel, Michael; Kkolou, Maria; Loukaidou, Panayiota; Pastelli, Androulla; Michael, Aristos; Lazarou, Akis; Arsali, Maria; Damianou, Loukas; Goutziamani, Ioanna; Soloukides, Andreas; Yioukas, Lakis; Elia, Avraam; Zouvani, Ioanna; Polycarpou, Polycarpos; Pierides, Alkis; Voskarides, Konstantinos; Deltas, Constantinos

    2014-01-01

    Familial glomerular hematuria(s) comprise a genetically heterogeneous group of conditions which include Alport Syndrome (AS) and thin basement membrane nephropathy (TBMN). Here we investigated 57 Greek-Cypriot families presenting glomerular microscopic hematuria (GMH), with or without proteinuria or chronic kidney function decline, but excluded classical AS. We specifically searched the COL4A3/A4 genes and identified 8 heterozygous mutations in 16 families (28,1%). Eight non-related families featured the founder mutation COL4A3-p.(G1334E). Renal biopsies from 8 patients showed TBMN and focal segmental glomerulosclerosis (FSGS). Ten patients (11.5%) reached end-stage kidney disease (ESKD) at ages ranging from 37-69-yo (mean 50,1-yo). Next generation sequencing of the patients who progressed to ESKD failed to reveal a second mutation in any of the COL4A3/A4/A5 genes, supporting that true heterozygosity for COL4A3/A4 mutations predisposes to CRF/ESKD. Although this could be viewed as a milder and late-onset form of autosomal dominant AS, we had no evidence of ultrastructural features or extrarenal manifestations that would justify this diagnosis. Functional studies in cultured podocytes transfected with wild type or mutant COL4A3 chains showed retention of mutant collagens and differential activation of the unfolded protein response (UPR) cascade. This signifies the potential role of the UPR cascade in modulating the final phenotype in patients with collagen IV nephropathies. PMID:25514610

  4. Frequency of COL4A3/COL4A4 mutations amongst families segregating glomerular microscopic hematuria and evidence for activation of the unfolded protein response. Focal and segmental glomerulosclerosis is a frequent development during ageing.

    PubMed

    Papazachariou, Louiza; Demosthenous, Panayiota; Pieri, Myrtani; Papagregoriou, Gregory; Savva, Isavella; Stavrou, Christoforos; Zavros, Michael; Athanasiou, Yiannis; Ioannou, Kyriakos; Patsias, Charalambos; Panagides, Alexia; Potamitis, Costas; Demetriou, Kyproula; Prikis, Marios; Hadjigavriel, Michael; Kkolou, Maria; Loukaidou, Panayiota; Pastelli, Androulla; Michael, Aristos; Lazarou, Akis; Arsali, Maria; Damianou, Loukas; Goutziamani, Ioanna; Soloukides, Andreas; Yioukas, Lakis; Elia, Avraam; Zouvani, Ioanna; Polycarpou, Polycarpos; Pierides, Alkis; Voskarides, Konstantinos; Deltas, Constantinos

    2014-01-01

    Familial glomerular hematuria(s) comprise a genetically heterogeneous group of conditions which include Alport Syndrome (AS) and thin basement membrane nephropathy (TBMN). Here we investigated 57 Greek-Cypriot families presenting glomerular microscopic hematuria (GMH), with or without proteinuria or chronic kidney function decline, but excluded classical AS. We specifically searched the COL4A3/A4 genes and identified 8 heterozygous mutations in 16 families (28,1%). Eight non-related families featured the founder mutation COL4A3-p.(G1334E). Renal biopsies from 8 patients showed TBMN and focal segmental glomerulosclerosis (FSGS). Ten patients (11.5%) reached end-stage kidney disease (ESKD) at ages ranging from 37-69-yo (mean 50,1-yo). Next generation sequencing of the patients who progressed to ESKD failed to reveal a second mutation in any of the COL4A3/A4/A5 genes, supporting that true heterozygosity for COL4A3/A4 mutations predisposes to CRF/ESKD. Although this could be viewed as a milder and late-onset form of autosomal dominant AS, we had no evidence of ultrastructural features or extrarenal manifestations that would justify this diagnosis. Functional studies in cultured podocytes transfected with wild type or mutant COL4A3 chains showed retention of mutant collagens and differential activation of the unfolded protein response (UPR) cascade. This signifies the potential role of the UPR cascade in modulating the final phenotype in patients with collagen IV nephropathies. PMID:25514610

  5. Adsorption and Unfolding of a Single Protein Triggers Nanoparticle Aggregation

    PubMed Central

    2016-01-01

    The response of living systems to nanoparticles is thought to depend on the protein corona, which forms shortly after exposure to physiological fluids and which is linked to a wide array of pathophysiologies. A mechanistic understanding of the dynamic interaction between proteins and nanoparticles and thus the biological fate of nanoparticles and associated proteins is, however, often missing mainly due to the inadequacies in current ensemble experimental approaches. Through the application of a variety of single molecule and single particle spectroscopic techniques in combination with ensemble level characterization tools, we identified different interaction pathways between gold nanorods and bovine serum albumin depending on the protein concentration. Overall, we found that local changes in protein concentration influence everything from cancer cell uptake to nanoparticle stability and even protein secondary structure. We envision that our findings and methods will lead to strategies to control the associated pathophysiology of nanoparticle exposure in vivo. PMID:26751094

  6. Adsorption and Unfolding of a Single Protein Triggers Nanoparticle Aggregation.

    PubMed

    Dominguez-Medina, Sergio; Kisley, Lydia; Tauzin, Lawrence J; Hoggard, Anneli; Shuang, Bo; D S Indrasekara, A Swarnapali; Chen, Sishan; Wang, Lin-Yung; Derry, Paul J; Liopo, Anton; Zubarev, Eugene R; Landes, Christy F; Link, Stephan

    2016-02-23

    The response of living systems to nanoparticles is thought to depend on the protein corona, which forms shortly after exposure to physiological fluids and which is linked to a wide array of pathophysiologies. A mechanistic understanding of the dynamic interaction between proteins and nanoparticles and thus the biological fate of nanoparticles and associated proteins is, however, often missing mainly due to the inadequacies in current ensemble experimental approaches. Through the application of a variety of single molecule and single particle spectroscopic techniques in combination with ensemble level characterization tools, we identified different interaction pathways between gold nanorods and bovine serum albumin depending on the protein concentration. Overall, we found that local changes in protein concentration influence everything from cancer cell uptake to nanoparticle stability and even protein secondary structure. We envision that our findings and methods will lead to strategies to control the associated pathophysiology of nanoparticle exposure in vivo. PMID:26751094

  7. Native topology determines force-induced unfolding pathways in globular proteins

    NASA Astrophysics Data System (ADS)

    Klimov, D. K.; Thirumalai, D.

    2000-06-01

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

  8. Computer simulations of the translocation and unfolding of a protein pulled mechanically through a pore

    NASA Astrophysics Data System (ADS)

    Huang, Lei; Kirmizialtin, Serdal; Makarov, Dmitrii E.

    2005-09-01

    Protein degradation by ATP-dependent proteases and protein import into the mitochondrial matrix involve the unfolding of proteins upon their passing through narrow constrictions. It has been hypothesized that the cellular machinery accomplishes protein unfolding by pulling mechanically at one end of the polypeptide chain. Here, we use Langevin dynamics simulations of a minimalist off-lattice model to examine this hypothesis and to study the unfolding of a protein domain pulled mechanically through a long narrow pore. We compute the potential of mean force (PMF) experienced by the domain as a function of its displacement along the pore and identify the unfolding intermediates corresponding to the local minima of the PMF. The observed unfolding mechanism is different from that found when the two termini are pulled apart, as in single-molecule mechanical unfolding experiments. It depends on the pore diameter, the magnitude of the pulling force, and on whether the force is applied at the N- or the C-terminus of the chain. Consequently, the translocation time exhibits a pulling force dependence that is more complex than a simple exponential function expected on the basis of simple phenomenological models of translocation.

  9. Dynamics and Energy Contributions for Transport of Unfolded Pertactin through a Protein Nanopore.

    PubMed

    Cressiot, Benjamin; Braselmann, Esther; Oukhaled, Abdelghani; Elcock, Adrian H; Pelta, Juan; Clark, Patricia L

    2015-09-22

    To evaluate the physical parameters governing translocation of an unfolded protein across a lipid bilayer, we studied protein transport through aerolysin, a passive protein channel, at the single-molecule level. The protein model used was the passenger domain of pertactin, an autotransporter virulence protein. Transport of pertactin through the aerolysin nanopore was detected as transient partial current blockades as the unfolded protein partially occluded the aerolysin channel. We compared the dynamics of entry and transport for unfolded pertactin and a covalent end-to-end dimer of the same protein. For both the monomer and the dimer, the event frequency of current blockades increased exponentially with the applied voltage, while the duration of each event decreased exponentially as a function of the electrical potential. The blockade time was twice as long for the dimer as for the monomer. The calculated activation free energy includes a main enthalpic component that we attribute to electrostatic interactions between pertactin and the aerolysin nanopore (despite the low Debye length), plus an entropic component due to confinement of the unfolded chain within the narrow pore. Comparing our experimental results to previous studies and theory suggests that unfolded proteins cross the membrane by passing through the nanopore in a somewhat compact conformation according to the "blob" model of Daoud and de Gennes. PMID:26302243

  10. Dynamics and Energy Contributions for Transport of Unfolded Pertactin through a Protein Nanopore

    PubMed Central

    Cressiot, Benjamin; Braselmann, Esther; Oukhaled, Abdelghani; Elcock, Adrian H.; Pelta, Juan; Clark, Patricia L.

    2016-01-01

    To evaluate the physical parameters governing translocation of an unfolded protein across a lipid bilayer, we studied protein transport through aerolysin, a passive protein channel, at the single molecule level. The protein model used was the passenger domain of pertactin, an autotransporter virulence protein. Transport of pertactin through the aerolysin nanopore was detected as transient partial current blockades as the unfolded protein partially occluded the aerolysin channel. We compared the dynamics of entry and transport for unfolded pertactin and a covalent end-to-end dimer of the same protein. For both the monomer and the dimer, the event frequency of current blockades increased exponentially with the applied voltage, while the duration of each event decreased exponentially as a function of the electrical potential. The blockade time was twice as long for the dimer as for the monomer. The calculated activation free energy includes a main enthalpic component that we attribute to electrostatic interactions between pertactin and the aerolysin nanopore (despite the low Debye length), plus an entropic component due to confinement of the unfolded chain within the narrow pore. Comparing our experimental results to previous studies and theory suggests that unfolded proteins cross the membrane by passing through the nanopore in a somewhat compact conformation according to the “blob” model of Daoud and de Gennes. PMID:26302243

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

    ERIC Educational Resources Information Center

    Sgammato, Adrienne N.

    2009-01-01

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

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

    SciTech Connect

    Millett, I.S.; Doniach, S.; Plaxco, K.W.

    2005-02-15

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

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

    PubMed Central

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

    2013-01-01

    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

  14. Mechanical Folding and Unfolding of Protein Barnase at the Single-Molecule Level.

    PubMed

    Alemany, Anna; Rey-Serra, Blanca; Frutos, Silvia; Cecconi, Ciro; Ritort, Felix

    2016-01-01

    The unfolding and folding of protein barnase has been extensively investigated in bulk conditions under the effect of denaturant and temperature. These experiments provided information about structural and kinetic features of both the native and the unfolded states of the protein, and debates about the possible existence of an intermediate state in the folding pathway have arisen. Here, we investigate the folding/unfolding reaction of protein barnase under the action of mechanical force at the single-molecule level using optical tweezers. We measure unfolding and folding force-dependent kinetic rates from pulling and passive experiments, respectively, and using Kramers-based theories (e.g., Bell-Evans and Dudko-Hummer-Szabo models), we extract the position of the transition state and the height of the kinetic barrier mediating unfolding and folding transitions, finding good agreement with previous bulk measurements. Measurements of the force-dependent kinetic barrier using the continuous effective barrier analysis show that protein barnase verifies the Leffler-Hammond postulate under applied force and allow us to extract its free energy of folding, ΔG0. The estimated value of ΔG0 is in agreement with our predictions obtained using fluctuation relations and previous bulk studies. To address the possible existence of an intermediate state on the folding pathway, we measure the power spectrum of force fluctuations at high temporal resolution (50 kHz) when the protein is either folded or unfolded and, additionally, we study the folding transition-path time at different forces. The finite bandwidth of our experimental setup sets the lifetime of potential intermediate states upon barnase folding/unfolding in the submillisecond timescale. PMID:26745410

  15. Physical modeling of the conformation of the unfolded proteins of the Nuclear Pore Complex

    NASA Astrophysics Data System (ADS)

    Zilman, Anton; Opferman, Michael; Coalson, Rob; Jasnow, David

    2013-03-01

    Nuclear Pore Complex (NPC) is a biological ``nano-machine'' that controls the macromolecular transport between the cell nucleus and the cytoplasm. NPC functions without direct input of metabolic energy and without transitions of the gate from a ``closed'' to an ``open'' state during transport. The key and unique aspect of transport is the interaction of the transported molecules with the unfolded, natively unstructured proteins that cover the lumen of the NPC. Recently, the NPC inspired creation of artificial bio-mimetic for nano-technology applications. Although several models have been proposed, it is still not clear how the passage of the transport factors is coupled to the conformational dynamics of the unfolded proteins within the NPC. Morphology changes in assemblies of the unfolded proteins induced by the transport factors have been investigated experimentally in vitro. I will present a coarse-grained theoretical and simulation framework that mimics the interactions of unfolded proteins with nano-sized transport factors. The simple physical model predicts morphology changes that explain the recent puzzling experimental results and suggests possible new modes of transport through the NPC. It also provides insights into the physics of the behavior of unfolded proteins.

  16. Unfolding of Proteins and Long Transient Conformations Detected by Single Nanopore Recording

    NASA Astrophysics Data System (ADS)

    Oukhaled, G.; Mathé, J.; Biance, A.-L.; Bacri, L.; Betton, J.-M.; Lairez, D.; Pelta, J.; Auvray, L.

    2007-04-01

    We study the electrophoretic blockades due to entries of partially unfolded proteins into a nanopore as a function of the concentration of the denaturing agent. Short and long pore blockades are observed by electrical detection. Short blockades are due to the passage of completely unfolded proteins, their frequency increases as the concentration of the denaturing agent increases, following a sigmoidal denaturation curve. Long blockades reveal partially folded conformations. Their duration increases as the proteins are more folded. The observation of a Vogel-Fulcher law suggests a glassy behavior.

  17. Computational modeling of acrylodan-labeled cAMP dependent protein kinase catalytic subunit unfolding.

    PubMed

    Kuznetsov, Aleksei; Kivi, Rait; Järv, Jaak

    2016-04-01

    Structure of the cAMP-dependent protein kinase catalytic subunit, where the asparagine residue 326 was replaced with acrylodan-cystein conjugate to implement this fluorescence reporter group into the enzyme, was modeled by molecular dynamics (MD) method and the positioning of the dye molecule in protein structure was characterized at temperatures 300K, 500K and 700K. It was found that the acrylodan moiety, which fluorescence is very sensitive to solvating properties of its microenvironment, was located on the surface of the native protein at 300K that enabled its partial solvation with water. At high temperatures the protein structure significantly changed, as the secondary and tertiary structure elements were unfolded and these changes were sensitively reflected in positioning of the dye molecule. At 700K complete unfolding of the protein occurred and the reporter group was entirely expelled into water. However, at 500K an intermediate of the protein unfolding process was formed, where the fluorescence reporter group was directed towards the protein interior and buried in the core of the formed molten globule state. This different positioning of the reporter group was in agreement with the two different shifts of emission spectrum of the covalently bound acrylodan, observed in the unfolding process of the protein. PMID:26896699

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

    SciTech Connect

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

    2008-01-01

    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, β-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, β-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 β-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

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

    PubMed Central

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

    2012-01-01

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

  20. ClpXP, an ATP-powered unfolding and protein-degradation machine

    PubMed Central

    Baker, Tania A.; Sauer, Robert T.

    2011-01-01

    ClpXP is a AAA+ protease that uses the energy of ATP binding and hydrolysis to perform mechanical work during targeted protein degradation within cells. ClpXP consists of hexamers of a AAA+ ATPase (ClpX) and a tetradecameric peptidase (ClpP). Asymmetric ClpX hexamers bind unstructured peptide tags in protein substrates, unfold stable tertiary structure in the substrate, and then translocate the unfolded polypeptide chain into an internal proteolytic compartment in ClpP. Here, we review our present understanding of ClpXP structure and function, as revealed by two decades of biochemical and biophysical studies. PMID:21736903

  1. Reversibility and two state behaviour in the thermal unfolding of oligomeric TIM barrel proteins.

    PubMed

    Romero-Romero, Sergio; Costas, Miguel; Rodríguez-Romero, Adela; Alejandro Fernández-Velasco, D

    2015-08-28

    Temperature is one of the main variables that modulate protein function and stability. Thermodynamic studies of oligomeric proteins, the dominant protein natural form, have been often hampered because irreversible aggregation and/or slow reactions are common. There are no reports on the reversible equilibrium thermal unfolding of proteins composed of (β/α)8 barrel subunits, albeit this "TIM barrel" topology is one of the most abundant and versatile in nature. We studied the eponymous TIM barrel, triosephosphate isomerase (TIM), belonging to five species of different bacterial taxa. All of them were found to be catalytically efficient dimers. The three-dimensional structure of four enzymes was solved at high/medium resolution. Irreversibility and kinetic control were observed in the thermal unfolding of two TIMs, while for the other three the thermal unfolding was found to follow a two-state equilibrium reversible process. Shifts in the global stability curves of these three proteins are related to the organismal temperature range of optimal growth and modulated by variations in maximum stability temperature and in the enthalpy change at that temperature. Reversibility appears to correlate with the low isoelectric point, the absence of a residual structure in the unfolded state, small cavity volume in the native state, low conformational stability and a low melting temperature. Furthermore, the strong coupling between dimer dissociation and monomer unfolding may reduce aggregation and favour reversibility. It is therefore very thought-provoking to find that a common topological ensemble, such as the TIM barrel, can unfold/refold in the Anfinsen way, i.e. without the help of the cellular machinery. PMID:26206330

  2. Interactions of urea with native and unfolded proteins: a volumetric study.

    PubMed

    Son, Ikbae; Shek, Yuen Lai; Tikhomirova, Anna; Baltasar, Eduardo Hidalgo; Chalikian, Tigran V

    2014-11-26

    We describe a statistical thermodynamic approach to analyzing urea-dependent volumetric properties of proteins. We use this approach to analyze our urea-dependent data on the partial molar volume and adiabatic compressibility of lysozyme, apocytochrome c, ribonuclease A, and α-chymotrypsinogen A. The analysis produces the thermodynamic properties of elementary urea-protein association reactions while also yielding estimates of the effective solvent-accessible surface areas of the native and unfolded protein states. Lysozyme and apocytochrome c do not undergo urea-induced transitions. The former remains folded, while the latter is unfolded between 0 and 8 M urea. In contrast, ribonuclease A and α-chymotrypsinogen A exhibit urea-induced unfolding transitions. Thus, our data permit us to characterize urea-protein interactions in both the native and unfolded states. We interpreted the urea-dependent volumetric properties of the proteins in terms of the equilibrium constant, k, and changes in volume, ΔV0, and compressibility, ΔKT0, for a reaction in which urea binds to a protein with a concomitant release of two waters of hydration to the bulk. Comparison of the values of k, ΔV0, and ΔKT0 with the similar data obtained on small molecules mimicking protein groups reveals lack of cooperative effects involved in urea-protein interactions. In general, the volumetric approach, while providing a unique characterization of cosolvent-protein interactions, offers a practical way for evaluating the effective solvent accessible surface area of biologically significant fully or partially unfolded polypeptides. PMID:25365737

  3. Application of principal component analysis in protein unfolding: An all-atom molecular dynamics simulation study

    NASA Astrophysics Data System (ADS)

    Das, Atanu; Mukhopadhyay, Chaitali

    2007-10-01

    We have performed molecular dynamics (MD) simulation of the thermal denaturation of one protein and one peptide—ubiquitin and melittin. To identify the correlation in dynamics among various secondary structural fragments and also the individual contribution of different residues towards thermal unfolding, principal component analysis method was applied in order to give a new insight to protein dynamics by analyzing the contribution of coefficients of principal components. The cross-correlation matrix obtained from MD simulation trajectory provided important information regarding the anisotropy of backbone dynamics that leads to unfolding. Unfolding of ubiquitin was found to be a three-state process, while that of melittin, though smaller and mostly helical, is more complicated.

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

    NASA Astrophysics Data System (ADS)

    Arteca, Gustavo A.; Tapia, O.

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

  5. Protein unfolding is an essential requirement for transport across the parasitophorous vacuolar membrane of Plasmodium falciparum.

    PubMed

    Gehde, Nina; Hinrichs, Corinna; Montilla, Irine; Charpian, Stefan; Lingelbach, Klaus; Przyborski, Jude M

    2009-02-01

    Plasmodium falciparum traffics a large number of proteins to its host cell, the mature human erythrocyte. How exactly these proteins gain access to the red blood cell is poorly understood. Here we have investigated the effect of protein folding on the transport of model substrate proteins to the host cell. We find that proteins must pass into the erythrocyte cytoplasm in an unfolded state. Our data strongly support the presence of a protein-conducting channel in the parasitophorous vacuolar membrane, and additionally imply an important role for molecular chaperones in keeping parasite proteins in a 'translocation competent' state prior to membrane passage. PMID:19040635

  6. Unfolding proteins in an external field: Can we always observe the intermediate states?

    NASA Astrophysics Data System (ADS)

    Lemak, Alexander S.; Lepock, James R.; Chen, Jeff Z.

    2003-03-01

    A protein molecule under the stress of an external denaturing force acting on a terminal end or on the entire molecule is expected to unfold, possibly through a few intermediate stages depending on the magnitude of the denaturing force. We have investigated two protein minimal models under various types of denaturing force fields using the collision molecular-dynamics simulation, in order to critically examine the relationship between the folding pathways observed in different protein denaturing experiments.

  7. Secondary Structural Change Can Occur Diffusely and Not Modularly during Protein Folding and Unfolding Reactions.

    PubMed

    Malhotra, Pooja; Udgaonkar, Jayant B

    2016-05-11

    A major goal of protein folding studies is to understand the structural basis of the coupling between stabilizing interactions, which leads to cooperative conformational change. The goal is challenging because of the difficulty in simultaneously measuring global cooperativity by determining population distributions of the conformations present, and the structures of these conformations. Here, hydrogen exchange (HX) into the small protein monellin was carried out under conditions where structure-opening is rate limiting for most backbone amide sites. Detection by mass spectrometry allowed characterization of not only segment-specific structure-opening rates but also the cooperativity of unfolding of the different secondary structural segments of the protein. The segment-specific pattern of HX reveals that the backbone hydrogen-bonding network disassembles in a structurally diffuse, asynchronous manner. A comparison of the site-specific transient opening rates of secondary and tertiary structure in the protein provides a structural rationale for the observation that unfolding is hierarchical and describable by exponential kinetics, despite being diffuse. Since unfolding was studied in native conditions, the sequence of events during folding in the same conditions will be the reverse of the sequence of events observed during unfolding. Hence, the formation of secondary structural units during folding would also occur in a non-cooperative, diffuse, and asynchronous manner. PMID:27093885

  8. The study of unfoldable self-avoiding walks - Application to protein structure prediction software.

    PubMed

    Guyeux, Christophe; Nicod, Jean-Marc; Philippe, Laurent; Bahi, Jacques M

    2015-08-01

    Self-avoiding walks (SAWs) are the source of very difficult problems in probability and enumerative combinatorics. They are of great interest as, for example, they are the basis of protein structure prediction (PSP) in bioinformatics. The authors of this paper have previously shown that, depending on the prediction algorithm, the sets of obtained walk conformations differ: For example, all the SAWs can be generated using stretching-based algorithms whereas only the unfoldable SAWs can be obtained with methods that iteratively fold the straight line. A deeper study of (non-)unfoldable SAWs is presented in this paper. The contribution is first a survey of what is currently known about these sets. In particular, we provide clear definitions of various subsets of SAWs related to pivot moves (unfoldable and non-unfoldable SAWs, etc.) and the first results that we have obtained, theoretically or computationally, on these sets. Then a new theorem on the number of non-unfoldable SAWs is demonstrated. Finally, a list of open questions is provided and the consequences on the PSP problem is proposed. PMID:25669327

  9. Force-dependent switch in protein unfolding pathways and transition-state movements.

    PubMed

    Zhuravlev, Pavel I; Hinczewski, Michael; Chakrabarti, Shaon; Marqusee, Susan; Thirumalai, D

    2016-02-01

    Although it is known that single-domain proteins fold and unfold by parallel pathways, demonstration of this expectation has been difficult to establish in experiments. Unfolding rate, [Formula: see text], as a function of force f, obtained in single-molecule pulling experiments on src SH3 domain, exhibits upward curvature on a [Formula: see text] plot. Similar observations were reported for other proteins for the unfolding rate [Formula: see text]. These findings imply unfolding in these single-domain proteins involves a switch in the pathway as f or [Formula: see text] is increased from a low to a high value. We provide a unified theory demonstrating that if [Formula: see text] as a function of a perturbation (f or [Formula: see text]) exhibits upward curvature then the underlying energy landscape must be strongly multidimensional. Using molecular simulations we provide a structural basis for the switch in the pathways and dramatic shifts in the transition-state ensemble (TSE) in src SH3 domain as f is increased. We show that a single-point mutation shifts the upward curvature in [Formula: see text] to a lower force, thus establishing the malleability of the underlying folding landscape. Our theory, applicable to any perturbation that affects the free energy of the protein linearly, readily explains movement in the TSE in a β-sandwich (I27) protein and single-chain monellin as the denaturant concentration is varied. We predict that in the force range accessible in laser optical tweezer experiments there should be a switch in the unfolding pathways in I27 or its mutants. PMID:26818842

  10. Structure and stability of recombinant bovine odorant-binding protein: II. Unfolding of the monomeric forms.

    PubMed

    Stepanenko, Olga V; Roginskii, Denis O; Stepanenko, Olesya V; Kuznetsova, Irina M; Uversky, Vladimir N; Turoverov, Konstantin K

    2016-01-01

    In a family of monomeric odorant-binding proteins (OBPs), bovine OBP (bOBP), that lacks conserved disulfide bond found in other OBPs, occupies unique niche because of its ability to form domain-swapped dimers. In this study, we analyzed conformational stabilities of the recombinant bOBP and its monomeric variants, the bOBP-Gly121+ mutant containing an additional glycine residue after the residue 121 of the bOBP, and the GCC-bOBP mutant obtained from the bOBP-Gly121+ form by introduction of the Trp64Cys/His155Cys double mutation to restore the canonical disulfide bond. We also analyzed the effect of the natural ligand binding on the conformational stabilities of these bOBP variants. Our data are consistent with the conclusion that the unfolding-refolding pathways of the recombinant bOBP and its mutant monomeric forms bOBP-Gly121+ and GCC-bOBP are similar and do not depend on the oligomeric status of the protein. This clearly shows that the information on the unfolding-refolding mechanism is encoded in the structure of the bOBP monomers. However, the process of the bOBP unfolding is significantly complicated by the formation of the domain-swapped dimer, and the rates of the unfolding-refolding reactions essentially depend on the conditions in which the protein is located. PMID:27114857

  11. Single-molecule spectroscopy of the temperature-induced collapse of unfolded proteins.

    PubMed

    Nettels, Daniel; Müller-Späth, Sonja; Küster, Frank; Hofmann, Hagen; Haenni, Dominik; Rüegger, Stefan; Reymond, Luc; Hoffmann, Armin; Kubelka, Jan; Heinz, Benjamin; Gast, Klaus; Best, Robert B; Schuler, Benjamin

    2009-12-01

    We used single-molecule FRET in combination with other biophysical methods and molecular simulations to investigate the effect of temperature on the dimensions of unfolded proteins. With single-molecule FRET, this question can be addressed even under near-native conditions, where most molecules are folded, allowing us to probe a wide range of denaturant concentrations and temperatures. We find a compaction of the unfolded state of a small cold shock protein with increasing temperature in both the presence and the absence of denaturant, with good agreement between the results from single-molecule FRET and dynamic light scattering. Although dissociation of denaturant from the polypeptide chain with increasing temperature accounts for part of the compaction, the results indicate an important role for additional temperature-dependent interactions within the unfolded chain. The observation of a collapse of a similar extent in the extremely hydrophilic, intrinsically disordered protein prothymosin alpha suggests that the hydrophobic effect is not the sole source of the underlying interactions. Circular dichroism spectroscopy and replica exchange molecular dynamics simulations in explicit water show changes in secondary structure content with increasing temperature and suggest a contribution of intramolecular hydrogen bonding to unfolded state collapse. PMID:19933333

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

    PubMed Central

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

    2011-01-01

    All cells employ ATP-powered proteases for protein-quality control and regulation. In the ClpXP protease, ClpX is a AAA+ machine that recognizes specific protein substrates, unfolds these molecules, and then translocates the denatured polypeptide through a central pore and into ClpP for degradation. Here, we use optical-trapping nanometry to probe the mechanics of enzymatic unfolding and translocation of single molecules of a multidomain substrate. Our experiments demonstrate the capacity of ClpXP and ClpX to perform mechanical work under load, reveal very fast and highly cooperative unfolding of individual substrate domains, suggest a translocation step size of 5–8 amino acids, and support a power-stroke model of denaturation in which successful enzyme-mediated unfolding of stable domains requires coincidence between mechanical pulling by the enzyme and a transient stochastic reduction in protein stability. We anticipate that single-molecule studies of the mechanical properties of other AAA+ proteolytic machines will reveal many shared features with ClpXP. PMID:21496645

  13. Structure and stability of recombinant bovine odorant-binding protein: II. Unfolding of the monomeric forms

    PubMed Central

    Stepanenko, Olga V.; Roginskii, Denis O.; Stepanenko, Olesya V.; Kuznetsova, Irina M.

    2016-01-01

    In a family of monomeric odorant-binding proteins (OBPs), bovine OBP (bOBP), that lacks conserved disulfide bond found in other OBPs, occupies unique niche because of its ability to form domain-swapped dimers. In this study, we analyzed conformational stabilities of the recombinant bOBP and its monomeric variants, the bOBP-Gly121+ mutant containing an additional glycine residue after the residue 121 of the bOBP, and the GCC-bOBP mutant obtained from the bOBP-Gly121+ form by introduction of the Trp64Cys/His155Cys double mutation to restore the canonical disulfide bond. We also analyzed the effect of the natural ligand binding on the conformational stabilities of these bOBP variants. Our data are consistent with the conclusion that the unfolding-refolding pathways of the recombinant bOBP and its mutant monomeric forms bOBP-Gly121+ and GCC-bOBP are similar and do not depend on the oligomeric status of the protein. This clearly shows that the information on the unfolding-refolding mechanism is encoded in the structure of the bOBP monomers. However, the process of the bOBP unfolding is significantly complicated by the formation of the domain-swapped dimer, and the rates of the unfolding-refolding reactions essentially depend on the conditions in which the protein is located. PMID:27114857

  14. Studying the unfolding kinetics of proteins under pressure using long molecular dynamic simulation runs.

    PubMed

    Chara, Osvaldo; Grigera, José Raúl; McCarthy, Andrés N

    2007-12-01

    The usefulness of computational methods such as molecular dynamics simulation has been extensively established for studying systems in equilibrium. Nevertheless, its application to complex non-equilibrium biological processes such as protein unfolding has been generally regarded as producing results which cannot be interpreted straightforwardly. In the present study, we present results for the kinetics of unfolding of apomyoglobin, based on the analysis of long simulation runs of this protein in solution at 3 kbar (1 atm = 1.01325, bar = 101,325 Pa). We hereby demonstrate that the analysis of the data collected within a simulated time span of 0.18 mus suffices for producing results, which coincide remarkably with the available unfolding kinetics experimental data. This not only validates molecular dynamics simulation as a valuable alternative for studying non-equilibrium processes, but also enables a detailed analysis of the actual structural mechanism which underlies the unfolding process of proteins under elusive denaturing conditions such as high pressure. PMID:19669536

  15. Polymer scaling laws of unfolded and intrinsically disordered proteins quantified with single-molecule spectroscopy

    PubMed Central

    Hofmann, Hagen; Soranno, Andrea; Borgia, Alessandro; Gast, Klaus; Nettels, Daniel; Schuler, Benjamin

    2012-01-01

    The dimensions of unfolded and intrinsically disordered proteins are highly dependent on their amino acid composition and solution conditions, especially salt and denaturant concentration. However, the quantitative implications of this behavior have remained unclear, largely because the effective theta-state, the central reference point for the underlying polymer collapse transition, has eluded experimental determination. Here, we used single-molecule fluorescence spectroscopy and two-focus correlation spectroscopy to determine the theta points for six different proteins. While the scaling exponents of all proteins converge to 0.62 ± 0.03 at high denaturant concentrations, as expected for a polymer in good solvent, the scaling regime in water strongly depends on sequence composition. The resulting average scaling exponent of 0.46 ± 0.05 for the four foldable protein sequences in our study suggests that the aqueous cellular milieu is close to effective theta conditions for unfolded proteins. In contrast, two intrinsically disordered proteins do not reach the Θ-point under any of our solvent conditions, which may reflect the optimization of their expanded state for the interactions with cellular partners. Sequence analyses based on our results imply that foldable sequences with more compact unfolded states are a more recent result of protein evolution. PMID:22984159

  16. Mechanical Unfolding of Cardiac Myosin Binding Protein-C by Atomic Force Microscopy

    PubMed Central

    Karsai, Árpád; Kellermayer, Miklós S.Z.; Harris, Samantha P.

    2011-01-01

    Cardiac myosin-binding protein-C (cMyBP-C) is a thick-filament-associated protein that performs regulatory and structural roles within cardiac sarcomeres. It is a member of the immunoglobulin (Ig) superfamily of proteins consisting of eight Ig- and three fibronectin (FNIII)-like domains, along with a unique regulatory sequence referred to as the M-domain, whose structure is unknown. Domains near the C-terminus of cMyBP-C bind tightly to myosin and mediate the association of cMyBP-C with thick (myosin-containing) filaments, whereas N-terminal domains, including the regulatory M-domain, bind reversibly to myosin S2 and/or actin. The ability of MyBP-C to bind to both myosin and actin raises the possibility that cMyBP-C cross-links myosin molecules within the thick filament and/or cross-links myosin and thin (actin-containing) filaments together. In either scenario, cMyBP-C could be under mechanical strain. However, the physical properties of cMyBP-C and its behavior under load are completely unknown. Here, we investigated the mechanical properties of recombinant baculovirus-expressed cMyBP-C using atomic force microscopy to assess the stability of individual cMyBP-C molecules in response to stretch. Force-extension curves showed the presence of long extensible segment(s) that became stretched before the unfolding of individual Ig and FNIII domains, which were evident as sawtooth peaks in force spectra. The forces required to unfold the Ig/FNIII domains at a stretch rate of 500 nm/s increased monotonically from ∼30 to ∼150 pN, suggesting a mechanical hierarchy among the different Ig/FNIII domains. Additional experiments using smaller recombinant proteins showed that the regulatory M-domain lacks significant secondary or tertiary structure and is likely an intrinsically disordered region of cMyBP-C. Together, these data indicate that cMyBP-C exhibits complex mechanical behavior under load and contains multiple domains with distinct mechanical properties. PMID

  17. Insight into Early-Stage Unfolding of GPI-Anchored Human Prion Protein.

    PubMed

    Wu, Emilia L; Qi, Yifei; Park, Soohyung; Mallajosyula, Sairam S; MacKerell, Alexander D; Klauda, Jeffery B; Im, Wonpil

    2015-11-17

    Prion diseases are fatal neurodegenerative disorders, which are characterized by the accumulation of misfolded prion protein (PrPSc) converted from a normal host cellular prion protein (PrPC). Experimental studies suggest that PrPC is enriched with α-helical structure, whereas PrPSc contains a high proportion of β-sheet. In this study, we report the impact of N-glycosylation and the membrane on the secondary structure stability utilizing extensive microsecond molecular dynamics simulations. Our results reveal that the HB (residues 173 to 194) C-terminal fragment undergoes conformational changes and helix unfolding in the absence of membrane environments because of the competition between protein backbone intramolecular and protein-water intermolecular hydrogen bonds as well as its intrinsic instability originated from the amino acid sequence. This initiation of the unfolding process of PrPC leads to a subsequent increase in the length of the HB-HC loop (residues 195 to 199) that may trigger larger rigid body motions or further unfolding around this region. Continuous interactions between prion protein and the membrane not only constrain the protein conformation but also decrease the solvent accessibility of the backbone atoms, thereby stabilizing the secondary structure, which is enhanced by N-glycosylation via additional interactions between the N-glycans and the membrane surface. PMID:26588568

  18. Electron transfer dissociation provides higher-order structural information of native and partially unfolded protein complexes.

    PubMed

    Lermyte, Frederik; Sobott, Frank

    2015-08-01

    Top-down sequencing approaches are becoming ever more popular for protein characterization, due to the ability to distinguish and characterize different protein isoforms. Under non-denaturing conditions, electron transfer dissociation (ETD) can furthermore provide important information on the exposed surface of proteins or complexes, thereby contributing to the characterization of their higher-order structure. Here, we investigate this approach using top-down ETD of tetrameric hemoglobin, concanavalin A, and alcohol dehydrogenase combined with ion mobility (IM) on a commercially available quadrupole/ion mobility/time-of-flight instrument (Waters Synapt G2). By applying supplemental activation in the transfer cell (post-IM), we release ETD fragments and attain good sequence coverage in the exposed terminal regions of the protein. We investigate the correlation between observed sites of fragmentation with regions of solvent accessibility, as derived from the crystal structure. Ion acceleration prior to ETD is also used to cause collision-induced unfolding (CIU) of the complexes without monomer ejection, as evidenced by the IM profiles. These partially unfolded tetramers show efficient fragmentation in some regions which are not sequenced under more gentle MS conditions. We show that by increasing CIU in small increments and monitoring the changes in the fragmentation pattern, it is possible to follow the initial steps of gas-phase protein unfolding. Fragments from partially unfolded protein complexes are released immediately after electron transfer, prior to IM (they do not share the drift time of their precursor), and observed without the need for supplemental activation. This is further evidence that the higher-order structure in these protein regions has been disrupted. PMID:26081219

  19. Code System for NE-213 Unfolding of Neutron Spectra up to 100 MeV with Response Function Error Propagation.

    Energy Science and Technology Software Center (ESTSC)

    1987-09-30

    Version 00 The REFERDOU system can be used to calculate the response function of a NE-213 scintillation detector for energies up to 100 MeV, to interpolate and spread (Gaussian) the response function, and unfold the measured spectrum of neutrons while propagating errors from the response functions to the unfolded spectrum.

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

    NASA Astrophysics Data System (ADS)

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

    2013-02-01

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

  1. Conformational equilibration time of unfolded protein chains and the folding speed limit†

    PubMed Central

    Abel, Christina J.; Goldbeck, Robert A.; Latypov, Ramil F.; Roder, Heinrich; Kliger, David S.

    2015-01-01

    The speed with which the conformers of unfolded protein chains interconvert is a fundamental question in the study of protein folding. Kinetic evidence is presented here for the time constant for interconversion of disparate unfolded chain conformations of a small globular protein, cytochrome c, in the presence of guanidine HCl denaturant. The axial binding reactions of histidine and methionine residues with the Fe(II) heme cofactor were monitored with time-resolved magnetic circular dichroism spectroscopy after photodissociation of the CO complexes of unfolded protein obtained from horse and tuna, and from several histidine mutants of the horse protein. A kinetic model fitting both the reaction rate constants and spectra of the intermediates was used to obtain a quantitative estimate of the conformational diffusion time. The latter parameter was approximated as a first-order time constant for exchange between conformational subensembles presenting either a methionine or a histidine residue to the heme iron for facile binding. The mean diffusional time constant of the wild type and variants was 3 ± 2 μs, close to the folding "speed limit". The implications of the relatively rapid conformational equilibration time observed are discussed in terms of the energy landscape and classical pathway time regimes of folding, for which the conformational diffusion time can be considered a pivot point. PMID:17352458

  2. Effects of gene dosage, promoters, and substrates on unfolded protein stress of recombinant Pichia pastoris.

    PubMed

    Hohenblum, Hubertus; Gasser, Brigitte; Maurer, Michael; Borth, Nicole; Mattanovich, Diethard

    2004-02-20

    The expression of heterologous proteins may exert severe stress on the host cells at different levels. Depending on the specific features of the product, different steps may be rate-limiting. For the secretion of recombinant proteins from yeast cells, folding and disulfide bond formation were identified as rate-limiting in several cases and the induction of the chaperone BiP (binding protein) is described. During the development of Pichia pastoris strains secreting human trypsinogen, a severe limitation of the amount of secreted product was identified. Strains using either the AOX1 or the GAP promoter were compared at different gene copy numbers. With the constitutive GAP promoter, no effect on the expression level was observed, whereas with the inducible AOX1 promoter an increase of the copy number above two resulted in a decrease of expression. To identify whether part of the product remained in the cells, lysates were fractionated and significant amounts of the product were identified in the insoluble fraction containing the endoplasmic reticulum, while the soluble cytosolic fraction contained product only in clones using the GAP promoter. An increase of BiP was observed upon induction of expression, indicating that the intracellular product fraction exerts an unfolded protein response in the host cells. A strain using the GAP promoter was grown both on glucose and methanol and trypsinogen was identified in the insoluble fractions of both cultures, but only in the soluble fraction of the glucose grown cultures, indicating that the amounts and distribution of intracellularly retained product depends on the culture conditions, especially the carbon source. PMID:14755554

  3. Effect of Interdomain Linker Length on an Antagonistic Folding-Unfolding Equilibrium between Two Protein Domains

    PubMed Central

    Cutler, Thomas A.; Mills, Brandon M.; Lubin, David J.; Chong, Lillian T.; Loh, Stewart N.

    2009-01-01

    Fusion of one protein domain with another is a common event in both evolution and protein engineering experiments. When insertion is at an internal site (e.g., a surface loop or turn), as opposed to one of the termini, conformational strain can be introduced into both domains. Strain is manifested by an antagonistic folding-unfolding equilibrium between the two domains, which we previously showed can be parameterized by a coupling free-energy term (ΔGX). The extent of strain is predicted to depend primarily on the ratio of the N-to-C distance of the guest protein to the distance between ends of the surface loop in the host protein. Here, we test that hypothesis by inserting ubiquitin (Ub) into the bacterial ribonuclease barnase (Bn), using peptide linkers from zero to 10 amino acids each. ΔGX values are determined by measuring the extent to which Co2+ binding to an engineered site on the Ub domain destabilizes the Bn domain. All-atom, unforced Langevin dynamics simulations are employed to gain structural insight into the mechanism of mechanically induced unfolding. Experimental and computational results find that the two domains are structurally and energetically uncoupled when linkers are long and that ΔGX increases with decreasing linker length. When the linkers are fewer than two amino acids, strain is so great that one domain unfolds the other. However, the protein is able to refold as dimers and higher-order oligomers. The likely mechanism is a three-dimensional domain swap of the Bn domain, which relieves conformational strain. The simulations suggest that an effective route to mechanical unfolding begins with disruption of the hydrophobic core of Bn near the Ub insertion site. PMID:19038264

  4. A protein's conformational stability is an immunologically dominant factor: evidence that free-energy barriers for protein unfolding limit the immunogenicity of foreign proteins.

    PubMed

    Ohkuri, Takatoshi; Nagatomo, Satoko; Oda, Kenji; So, Takanori; Imoto, Taiji; Ueda, Tadashi

    2010-10-01

    Foreign protein Ags are incorporated into APCs and then degraded by endosomal proteases. The peptides are then mounted on MHC II molecules on the surfaces of APCs. The T cell-triggering response and, therefore, the immune response, were suggested to be governed by the degree of conformational stability of the foreign protein Ags. However, there is little evidence that a protein's conformational stability is an immunologically dominant factor. In this study, we show that a protein has a threshold of conformational stability to prevent the immunogenicity of foreign proteins. Inverse and linear correlations were found between the amount of IgG production against lysozymes and the free-energy change for the unfolding of lysozymes, based on the correlation between the free-energy changes of the protein unfolding and the amount of IgG production against lysozymes with different stabilities in mice using hen egg white lysozyme derivatives and mutant mouse lysozymes, in which the sequence between 107 and 116 is replaced with that of hen egg white lysozyme, which can produce autoantibodies in mice. Interestingly, the thresholds of free-energy changes for both lysozymes to prevent their immunogenicity were almost identical (21-23 kcal/mol). To confirm the results, we also showed that the cross-linking of Phl p 7, in which intact Phl p 7 has stability greater than ∼20 kcal/mol under physiological conditions, induced minimal IgG production in mice, whereas intact Phl p 7 was antigenic. From the above results, we suggest that protein conformational stability was an immunologically dominant factor. PMID:20817878

  5. Thermal stability and unfolding pathways of hyperthermophilic and mesophilic periplasmic binding proteins studied by molecular dynamics simulation.

    PubMed

    Chen, Lin; Li, Xue; Wang, Ruige; Fang, Fengqin; Yang, Wanli; Kan, Wei

    2016-07-01

    The ribose binding protein (RBP), a sugar-binding periplasmic protein, is involved in the transport and signaling processes in both prokaryotes and eukaryotes. Although several cellular and structural studies have been reported, a description of the thermostability of RBP at the molecular level remains elusive. Focused on the hyperthermophilic Thermoytoga maritima RBP (tmRBP) and mesophilic Escherichia coli homolog (ecRBP), we applied molecular dynamics simulations at four different temperatures (300, 380, 450, and 500 K) to obtain a deeper insight into the structural features responsible for the reduced thermostability of the ecRBP. The simulations results indicate that there are distinct structural differences in the unfolding pathway between the two homologs and the ecRBP unfolds faster than the hyperthermophilic homologs at certain temperatures in accordance with the lower thermal stability found experimentally. Essential dynamics analysis uncovers that the essential subspaces of ecRBP and tmRBP are non-overlapping and these two proteins show different directions of motion within the simulations trajectories. Such an understanding is required for designing efficient proteins with characteristics for a particular application. PMID:26292713

  6. Unfolding Thermodynamics of Cysteine-Rich Proteins and Molecular Thermal-Adaptation of Marine Ciliates

    PubMed Central

    Cazzolli, Giorgia; Škrbić, Tatjana; Guella, Graziano; Faccioli, Pietro

    2013-01-01

    Euplotes nobilii and Euplotes raikovi are phylogenetically closely allied species of marine ciliates, living in polar and temperate waters, respectively. Their evolutional relation and the sharply different temperatures of their natural environments make them ideal organisms to investigate thermal-adaptation. We perform a comparative study of the thermal unfolding of disulfide-rich protein pheromones produced by these ciliates. Recent circular dichroism (CD) measurements have shown that the two psychrophilic (E. nobilii) and mesophilic (E. raikovi) protein families are characterized by very different melting temperatures, despite their close structural homology. The enhanced thermal stability of the E. raikovi pheromones is realized notwithstanding the fact that these proteins form, as a rule, a smaller number of disulfide bonds. We perform Monte Carlo (MC) simulations in a structure-based coarse-grained (CG) model to show that the higher stability of the E. raikovi pheromones is due to the lower locality of the disulfide bonds, which yields a lower entropy increase in the unfolding process. Our study suggests that the higher stability of the mesophilic E. raikovi phermones is not mainly due to the presence of a strongly hydrophobic core, as it was proposed in the literature. In addition, we argue that the molecular adaptation of these ciliates may have occurred from cold to warm, and not from warm to cold. To provide a testable prediction, we identify a point-mutation of an E. nobilii pheromone that should lead to an unfolding temperature typical of that of E. raikovi pheromones. PMID:24970199

  7. Overexpression of smooth muscle myosin heavy chain leads to activation of the unfolded protein response and autophagic turnover of thick filament-associated proteins in vascular smooth muscle cells.

    PubMed

    Kwartler, Callie S; Chen, Jiyuan; Thakur, Dhananjay; Li, Shumin; Baskin, Kedryn; Wang, Shanzhi; Wang, Zhao V; Walker, Lori; Hill, Joseph A; Epstein, Henry F; Taegtmeyer, Heinrich; Milewicz, Dianna M

    2014-05-16

    Duplications spanning nine genes at the genomic locus 16p13.1 predispose individuals to acute aortic dissections. The most likely candidate gene in this region leading to the predisposition for dissection is MYH11, which encodes smooth muscle myosin heavy chain (SM-MHC). The effects of increased expression of MYH11 on smooth muscle cell (SMC) phenotypes were explored using mouse aortic SMCs with transgenic overexpression of one isoform of SM-MHC. We found that these cells show increased expression of Myh11 and myosin filament-associated contractile genes at the message level when compared with control SMCs, but not at the protein level due to increased protein degradation. Increased expression of Myh11 resulted in endoplasmic reticulum (ER) stress in SMCs, which led to a paradoxical decrease of protein levels through increased autophagic degradation. An additional consequence of ER stress in SMCs was increased intracellular calcium ion concentration, resulting in increased contractile signaling and contraction. The increased signals for contraction further promote transcription of contractile genes, leading to a feedback loop of metabolic abnormalities in these SMCs. We suggest that overexpression of MYH11 can lead to increased ER stress and autophagy, findings that may be globally implicated in disease processes associated with genomic duplications. PMID:24711452

  8. Deconvoluting Protein (Un)folding Structural Ensembles Using X-Ray Scattering, Nuclear Magnetic Resonance Spectroscopy and Molecular Dynamics Simulation

    PubMed Central

    Nasedkin, Alexandr; Marcellini, Moreno; Religa, Tomasz L.; Freund, Stefan M.; Menzel, Andreas; Fersht, Alan R.; Jemth, Per; van der Spoel, David; Davidsson, Jan

    2015-01-01

    The folding and unfolding of protein domains is an apparently cooperative process, but transient intermediates have been detected in some cases. Such (un)folding intermediates are challenging to investigate structurally as they are typically not long-lived and their role in the (un)folding reaction has often been questioned. One of the most well studied (un)folding pathways is that of Drosophila melanogaster Engrailed homeodomain (EnHD): this 61-residue protein forms a three helix bundle in the native state and folds via a helical intermediate. Here we used molecular dynamics simulations to derive sample conformations of EnHD in the native, intermediate, and unfolded states and selected the relevant structural clusters by comparing to small/wide angle X-ray scattering data at four different temperatures. The results are corroborated using residual dipolar couplings determined by NMR spectroscopy. Our results agree well with the previously proposed (un)folding pathway. However, they also suggest that the fully unfolded state is present at a low fraction throughout the investigated temperature interval, and that the (un)folding intermediate is highly populated at the thermal midpoint in line with the view that this intermediate can be regarded to be the denatured state under physiological conditions. Further, the combination of ensemble structural techniques with MD allows for determination of structures and populations of multiple interconverting structures in solution. PMID:25946337

  9. Does deamidation cause protein unfolding? A top-down tandem mass spectrometry study

    PubMed Central

    Soulby, Andrew J; Heal, Jack W; Barrow, Mark P; Roemer, Rudolf A; O'Connor, Peter B

    2015-01-01

    Deamidation is a nonenzymatic post-translational modification of asparagine to aspartic acid or glutamine to glutamic acid, converting an uncharged amino acid to a negatively charged residue. It is plausible that deamidation of asparagine and glutamine residues would result in disruption of a proteins' hydrogen bonding network and thus lead to protein unfolding. To test this hypothesis Calmodulin and B2M were deamidated and analyzed using tandem mass spectrometry on a Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS). The gas phase hydrogen bonding networks of deamidated and nondeamidated protein isoforms were probed by varying the infra-red multi-photon dissociation laser power in a linear fashion and plotting the resulting electron capture dissociation fragment intensities as a melting curve at each amino acid residue. Analysis of the unfolding maps highlighted increased fragmentation at lower laser powers localized around heavily deamidated regions of the proteins. In addition fragment intensities were decreased across the rest of the proteins which we propose is because of the formation of salt-bridges strengthening the intramolecular interactions of the central regions. These results were supported by a computational flexibility analysis of the mutant and unmodified proteins, which would suggest that deamidation can affect the global structure of a protein via modification of the hydrogen bonding network near the deamidation site and that top down FTICR-MS is an appropriate technique for studying protein folding. PMID:25653127

  10. Protein unfolding accounts for the unusual mechanical behavior of fibrin networks

    PubMed Central

    Purohit, Prashant K.; Litvinov, Rustem I.; Brown, Andre E. X.; Discher, Dennis E.; Weisel, John W.

    2011-01-01

    We describe the mechanical behavior of isotropic fibrin networks at the macroscopic scale in terms of the nanoscale force response of fibrin molecules that are its basic building blocks. We show that the remarkable extensibility and compressibility of fibrin networks have their origins in the unfolding of fibrin molecules. The force-stretch behavior of a single fibrin fiber is described using a two-state model in which the fiber has a linear force-stretch relation in the folded phase and behaves like a worm-like-chain in the unfolded phase. The nanoscale force-stretch response is connected to the macro-scale stress-stretch response by means of the eight-chain model. This model is able to capture the macroscopic response of a fibrin network in uniaxial tension and appears remarkably simple given the molecular complexity. We use the eight-chain model to explain why fibrin networks have negative compressibility and Poisson’s ratio greater than one due to unfolding of fibrin molecules. PMID:21342665

  11. Unusual Reversible Oligomerization of Unfolded Dengue Envelope Protein Domain 3 at High Temperatures and Its Abolition by a Point Mutation.

    PubMed

    Saotome, Tomonori; Nakamura, Shigeyoshi; Islam, Mohammad M; Nakazawa, Akiko; Dellarole, Mariano; Arisaka, Fumio; Kidokoro, Shun-Ichi; Kuroda, Yutaka

    2016-08-16

    We report differential scanning calorimetry (DSC) experiments between 10 and 120 °C of Dengue 4 envelope protein domain 3 (DEN4 ED3), a small 107-residue monomeric globular protein domain. The thermal unfolding of DEN4 ED3 was fully reversible and exhibited two peculiar endothermic peaks. AUC (analytical ultracentrifugation) experiments at 25 °C indicated that DEN4 ED3 was monomeric. Detailed thermodynamic analysis indicated that the two endothermic peaks separated with an increasing protein concentration, and global fitting of the DSC curves strongly suggested the presence of unfolded tetramers at temperatures around 80-90 °C, which dissociated to unfolded monomers at even higher temperatures. To further characterize this rare thermal unfolding process, we designed and constructed a DEN4 ED3 variant that would unfold according to a two-state model, typical of globular proteins. We thus substituted Val 380, the most buried residue at the dimeric interface in the protein crystal, with less hydrophobic amino acids (Ala, Ser, Thr, Asn, and Lys). All variants showed a single heat absorption peak, typical of small globular proteins. In particular, the DSC thermogram of DEN4 V380K indicated a two-state reversible thermal unfolding independent of protein concentration, indicating that the high-temperature oligomeric state was successfully abolished by a single mutation. These observations confirmed the standard view that small monomeric globular proteins undergo a two-state unfolding. However, the reversible formation of unfolded oligomers at high temperatures is a truly new phenomenon, which was fully inhibited by an accurately designed single mutation. PMID:27433922

  12. Stochastic but highly coordinated protein unfolding and translocation by the ClpXP proteolytic machine.

    PubMed

    Cordova, Juan Carlos; Olivares, Adrian O; Shin, Yongdae; Stinson, Benjamin M; Calmat, Stephane; Schmitz, Karl R; Aubin-Tam, Marie-Eve; Baker, Tania A; Lang, Matthew J; Sauer, Robert T

    2014-07-31

    ClpXP and other AAA+ proteases recognize, mechanically unfold, and translocate target proteins into a chamber for proteolysis. It is not known whether these remarkable molecular machines operate by a stochastic or sequential mechanism or how power strokes relate to the ATP-hydrolysis cycle. Single-molecule optical trapping allows ClpXP unfolding to be directly visualized and reveals translocation steps of ∼1-4 nm in length, but how these activities relate to solution degradation and the physical properties of substrate proteins remains unclear. By studying single-molecule degradation using different multidomain substrates and ClpXP variants, we answer many of these questions and provide evidence for stochastic unfolding and translocation. We also present a mechanochemical model that accounts for single-molecule, biochemical, and structural results for our observation of enzymatic memory in translocation stepping, for the kinetics of translocation steps of different sizes, and for probabilistic but highly coordinated subunit activity within the ClpX ring. PMID:25083874

  13. β-sheet-like formation during the mechanical unfolding of prion protein

    NASA Astrophysics Data System (ADS)

    Tao, Weiwei; Yoon, Gwonchan; Cao, Penghui; Eom, Kilho; Park, Harold S.

    2015-09-01

    Single molecule experiments and simulations have been widely used to characterize the unfolding and folding pathways of different proteins. However, with few exceptions, these tools have not been applied to study prion protein, PrPC, whose misfolded form PrPSc can induce a group of fatal neurodegenerative diseases. Here, we apply novel atomistic modeling based on potential energy surface exploration to study the constant force unfolding of human PrP at time scales inaccessible with standard molecular dynamics. We demonstrate for forces around 100 pN, prion forms a stable, three-stranded β-sheet-like intermediate configuration containing residues 155-214 with a lifetime exceeding hundreds of nanoseconds. A mutant without the disulfide bridge shows lower stability during the unfolding process but still forms the three-stranded structure. The simulations thus not only show the atomistic details of the mechanically induced structural conversion from the native α-helical structure to the β-rich-like form but also lend support to the structural theory that there is a core of the recombinant PrP amyloid, a misfolded form reported to induce transmissible disease, mapping to C-terminal residues ≈160-220.

  14. Mechanisms of triggering H1 helix in prion proteins unfolding revealed by molecular dynamic simulation

    NASA Astrophysics Data System (ADS)

    Tseng, Chih-Yuan; Lee, H. C.

    2006-03-01

    In template-assistance model, normal Prion protein (PrP^C), the pathogen to cause several prion diseases such as Creutzfeldt-Jakob (CJD) in human, Bovine Spongiform Encephalopathy (BSE) in cow, and scrapie in sheep, converts to infectious prion (PrP^Sc) through a transient interaction with PrP^Sc. Furthermore, conventional studies showed S1-H1-S2 region in PrP^C to be the template of S1-S2 β-sheet in PrP^Sc, and Prion protein's conformational conversion may involve an unfolding of H1 and refolding into β-sheet. Here we prepare several mouse prion peptides that contain S1-H1-S2 region with specific different structures, which are corresponding to specific interactions, to investigate possible mechanisms to trigger H1 α-helix unfolding process via molecular dynamic simulation. Three properties, conformational transition, salt-bridge in H1, and hydrophobic solvent accessible surface (SAS) are analyzed. From these studies, we found the interaction that triggers H1 unfolding to be the one that causes dihedral angle at residue Asn^143 changes. Whereas interactions that cause S1 segment's conformational changes play a minor in this process. These studies offers an additional evidence for template-assistance model.

  15. β-sheet-like formation during the mechanical unfolding of prion protein.

    PubMed

    Tao, Weiwei; Yoon, Gwonchan; Cao, Penghui; Eom, Kilho; Park, Harold S

    2015-09-28

    Single molecule experiments and simulations have been widely used to characterize the unfolding and folding pathways of different proteins. However, with few exceptions, these tools have not been applied to study prion protein, PrP(C), whose misfolded form PrP(Sc) can induce a group of fatal neurodegenerative diseases. Here, we apply novel atomistic modeling based on potential energy surface exploration to study the constant force unfolding of human PrP at time scales inaccessible with standard molecular dynamics. We demonstrate for forces around 100 pN, prion forms a stable, three-stranded β-sheet-like intermediate configuration containing residues 155-214 with a lifetime exceeding hundreds of nanoseconds. A mutant without the disulfide bridge shows lower stability during the unfolding process but still forms the three-stranded structure. The simulations thus not only show the atomistic details of the mechanically induced structural conversion from the native α-helical structure to the β-rich-like form but also lend support to the structural theory that there is a core of the recombinant PrP amyloid, a misfolded form reported to induce transmissible disease, mapping to C-terminal residues ≈160-220. PMID:26429042

  16. β-sheet-like formation during the mechanical unfolding of prion protein

    SciTech Connect

    Tao, Weiwei; Cao, Penghui; Park, Harold S.; Yoon, Gwonchan; Eom, Kilho

    2015-09-28

    Single molecule experiments and simulations have been widely used to characterize the unfolding and folding pathways of different proteins. However, with few exceptions, these tools have not been applied to study prion protein, PrP{sup C}, whose misfolded form PrP{sup Sc} can induce a group of fatal neurodegenerative diseases. Here, we apply novel atomistic modeling based on potential energy surface exploration to study the constant force unfolding of human PrP at time scales inaccessible with standard molecular dynamics. We demonstrate for forces around 100 pN, prion forms a stable, three-stranded β-sheet-like intermediate configuration containing residues 155-214 with a lifetime exceeding hundreds of nanoseconds. A mutant without the disulfide bridge shows lower stability during the unfolding process but still forms the three-stranded structure. The simulations thus not only show the atomistic details of the mechanically induced structural conversion from the native α-helical structure to the β-rich-like form but also lend support to the structural theory that there is a core of the recombinant PrP amyloid, a misfolded form reported to induce transmissible disease, mapping to C-terminal residues ≈160-220.

  17. An Item Response Unfolding Model for Graphic Rating Scales

    ERIC Educational Resources Information Center

    Liu, Ying

    2009-01-01

    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…

  18. Accelerated simulation of unfolding and refolding of a large single chain globular protein

    PubMed Central

    Seddon, Gavin M.; Bywater, Robert P.

    2012-01-01

    We have developed novel strategies for contracting simulation times in protein dynamics that enable us to study a complex protein with molecular weight in excess of 34 kDa. Starting from a crystal structure, we produce unfolded and then refolded states for the protein. We then compare these quantitatively using both established and new metrics for protein structure and quality checking. These include use of the programs Concoord and Darvols. Simulation of protein-folded structure well beyond the molten globule state and then recovery back to the folded state is itself new, and our results throw new light on the protein-folding process. We accomplish this using a novel cooling protocol developed for this work. PMID:22870389

  19. Accelerated simulation of unfolding and refolding of a large single chain globular protein.

    PubMed

    Seddon, Gavin M; Bywater, Robert P

    2012-07-01

    We have developed novel strategies for contracting simulation times in protein dynamics that enable us to study a complex protein with molecular weight in excess of 34 kDa. Starting from a crystal structure, we produce unfolded and then refolded states for the protein. We then compare these quantitatively using both established and new metrics for protein structure and quality checking. These include use of the programs Concoord and Darvols. Simulation of protein-folded structure well beyond the molten globule state and then recovery back to the folded state is itself new, and our results throw new light on the protein-folding process. We accomplish this using a novel cooling protocol developed for this work. PMID:22870389

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

    NASA Astrophysics Data System (ADS)

    Banci, Lucia

    1998-12-01

    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.

  1. Direct observation of multimer stabilization in the mechanical unfolding pathway of a protein undergoing oligomerization.

    PubMed

    Scholl, Zackary N; Yang, Weitao; Marszalek, Piotr E

    2015-02-24

    Understanding how protein oligomerization affects the stability of monomers in self-assembled structures is crucial to the development of new protein-based nanomaterials and protein cages for drug delivery. Here, we use single-molecule force spectroscopy (AFM-SMFS), protein engineering, and computer simulations to evaluate how dimerization and tetramerization affects the stability of the monomer of Streptavidin, a model homotetrameric protein. The unfolding force directly relates to the folding stability, and we find that monomer of Streptavidin is mechanically stabilized by 40% upon dimerization, and that it is stabilized an additional 24% upon tetramerization. We also find that biotin binding increases stability by another 50% as compared to the apo-tetrameric form. We used the distribution of unfolding forces to extract properties of the underlying energy landscape and found that the distance to the transition state is decreased and the barrier height is increased upon multimerization. Finally, we investigated the origin of the strengthening by ligand binding. We found that, rather than being strengthened through intramolecular contacts, it is strengthened due to the contacts provided by the biotin-binding loop that crosses the interface between the dimers. PMID:25639698

  2. Pressure-induced protein unfolding in the ternary system AOT-octane-water is different from that in bulk water.

    PubMed

    Meersman, Filip; Dirix, Carolien; Shipovskov, Stepan; Klyachko, Natalia L; Heremans, Karel

    2005-04-12

    In a cellular environment, the presence of macromolecular cosolutes and membrane interfaces can influence the folding-unfolding behavior of proteins. Here we report on the pressure stability of alpha-chymotrypsin in the ternary system bis(2-ethylhexyl)sodium sulfosuccinate-octane-water using FTIR spectroscopy. The ternary system forms anionic reverse micelles which mimic cellular conditions. We find that inclusion of a single protein molecule in a reverse micelle does not alter its conformation. When pressurized in bulk water, alpha-chymotrypsin unfolds at 750 MPa into a partially unfolded structure. In contrast, in the ternary system, the same pressure increase induces a random coil-like unfolded state, which collapses into an amorphous aggregate during the decompression phase. It is suggested that the unfolding pathway is different in a cell-mimicking environment due to the combined effect of multiple factors, including confinement. A phase transition of the reverse micellar to the lamellar phase is thought to be essential to provide the conditions required for unfolding and aggregation, though the unfolding is not a direct result of the phase transition. Our observations therefore suggest that membranes may cause the formation of alternative conformations that are more susceptible to aggregation. PMID:15807607

  3. Local unfolding is required for the site-specific protein modification by transglutaminase.

    PubMed

    Spolaore, Barbara; Raboni, Samanta; Ramos Molina, Amparo; Satwekar, Abhijeet; Damiano, Nunzio; Fontana, Angelo

    2012-10-30

    The transglutaminase (TGase) from Streptomyces mobaraensis catalyzes transamidation reactions in a protein substrate leading to the modification of the side chains of Gln and Lys residues according to the A-CONH(2) + H(2)N-B → A-CONH-B + NH(3) reaction, where both A and B can be a protein or a ligand. A noteworthy property of TGase is its susbstrate specificity, so that often only a few specific Gln or Lys residues can be modified in a globular protein. The molecular features of a globular protein dictating the site-specific reactions mediated by TGase are yet poorly understood. Here, we have analyzed the reactivity toward TGase of apomyoglobin (apoMb), α-lactalbumin (α-LA), and fragment 205-316 of thermolysin. These proteins are models of protein structure and folding that have been studied previously using the limited proteolysis technique to unravel regions of local unfolding in their amino acid sequences. The three proteins were modified by TGase at the level of Gln or Lys residues with dansylcadaverine or carbobenzoxy-l-glutaminylglycine, respectively. Despite these model proteins containing several Gln and Lys residues, the sites of TGase derivatization occur over restricted chain regions of the protein substrates. In particular, the TGase-mediated modifications occur in the "helix F" region in apoMb, in the β-domain in apo-α-LA in its molten globule state, and in the N-terminal region in fragment 205-316 of thermolysin. Interestingly, the sites of limited proteolysis are located in the same chain regions of these proteins, thus providing a clear-cut demonstration that chain flexibility or local unfolding overwhelmingly dictates the site-specific modification by both TGase and a protease. PMID:23083324

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

    PubMed Central

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

    2012-01-01

    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 γD Crystallin (HγD-Crys), a structural protein of the eye lens, and two cataract-related mutants, L5S HγD-Crys and I90F HγD-Crys. In equilibrium unfolding-refolding studies, the presence of 250 mM sodium citrate increased the transition midpoint of the N-td of WT HγD-Crys and L5S HγD-Crys by 0.3 M GuHCl, the C-td by 0.6M GuHCl, and the single transition of I90F HγD-Crys by 0.4M GuHCl. In kinetic unfolding reactions, sodium citrate demonstrates a measurable stabilization effect only for the mutant I90F HγD-Crys. In the presence of citrate, a kinetic unfolding intermediate of I90F HγD-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 HγD-Crys, but considerably slows the rate of aggregation of both L5S HγD-Crys and I90F HγD-Crys. The presence of sodium citrate dramatically slows refolding of WT HγD-Crys and I90F HγD-Crys, but has a significantly smaller effect on the refolding of L5S HγD-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. PMID:21600897

  5. Slow transition between two beta-strand registers is dictated by protein unfolding.

    PubMed

    Evans, Matthew R; Gardner, Kevin H

    2009-08-19

    The aryl hydrocarbon receptor nuclear translocator (ARNT) is a promiscuous, basic helix-loop-helix Period/ARNT/Single-minded protein that forms dimeric transcriptional regulator complexes with other bHLH-PAS proteins to regulate various biological pathways. Intriguingly, the introduction of a single point mutation into the C-terminal PAS-B domain resulted in a protein that can simultaneously exist in two distinct conformations. The difference between these two structures is a +3 slip and inversion of a central Ibeta-strand and an accompanying N448-P449 peptide bond isomerization in the preceding HI loop. Previous studies have indicated these two forms of Y456T interconvert on the approximate time scale of tens of minutes, allowing these two conformations to be separated by ion exchange chromatography. Here, we use time-resolved solution NMR spectroscopy to quantitatively characterize this rate and its temperature dependence, providing information into the transition state. When compared with fluorescence measurements of protein unfolding rates, we find data that suggest a linkage between interconversion and unfolding based on comparable temperature dependence and corresponding energetics of these processes. Notably, the N448-P449 peptide bond also plays a critical role for the interconversion between states, with a mutant unable to adopt a cis configuration at this bond (P449A/Y456T) being kinetically trapped under nondenaturing conditions. Taken together, these data provide information about a rare equilibrium model system for beta-strand slippage. PMID:19722642

  6. Decoding Structural Properties of a Partially Unfolded Protein Substrate: En Route to Chaperone Binding

    PubMed Central

    Nagpal, Suhani; Tiwari, Satyam; Mapa, Koyeli; Thukral, Lipi

    2015-01-01

    Many proteins comprising of complex topologies require molecular chaperones to achieve their unique three-dimensional folded structure. The E.coli chaperone, GroEL binds with a large number of unfolded and partially folded proteins, to facilitate proper folding and prevent misfolding and aggregation. Although the major structural components of GroEL are well defined, scaffolds of the non-native substrates that determine chaperone-mediated folding have been difficult to recognize. Here we performed all-atomistic and replica-exchange molecular dynamics simulations to dissect non-native ensemble of an obligate GroEL folder, DapA. Thermodynamics analyses of unfolding simulations revealed populated intermediates with distinct structural characteristics. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native β-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spectrometry (HDX-MS). Further, we constructed network graphs to elucidate long-range intra-protein connectivity of native and intermediate topologies, demonstrating regions that serve as central “hubs”. Overall, our results implicate that genomic variations (or mutations) in the distinct regions of protein structures might disrupt these topological signatures disabling chaperone-mediated folding, leading to formation of aggregates. PMID:26394388

  7. CIUSuite: A Quantitative Analysis Package for Collision Induced Unfolding Measurements of Gas-Phase Protein Ions.

    PubMed

    Eschweiler, Joseph D; Rabuck-Gibbons, Jessica N; Tian, Yuwei; Ruotolo, Brandon T

    2015-11-17

    Ion mobility-mass spectrometry (IM-MS) is a technology of growing importance for structural biology, providing complementary 3D structure information for biomolecules within samples that are difficult to analyze using conventional analytical tools through the near-simultaneous acquisition of ion collision cross sections (CCSs) and masses. Despite recent advances in IM-MS instrumentation, the resolution of closely related protein conformations remains challenging. Collision induced unfolding (CIU) has been demonstrated as a useful tool for resolving isocrossectional protein ions, as they often follow distinct unfolding pathways when subjected to collisional heating in the gas phase. CIU has been used for a variety of applications, from differentiating binding modes of activation state-selective kinase inhibitors to characterizing the domain structure of multidomain proteins. With the growing utilization of CIU as a tool for structural biology, significant challenges have emerged in data analysis and interpretation, specifically the normalization and comparison of CIU data sets. Here, we present CIUSuite, a suite of software modules designed for the rapid processing, analysis, comparison, and classification of CIU data. We demonstrate these tools as part of a series of workflows for applications in comparative structural biology, biotherapeutic analysis, and high throughput screening of kinase inhibitors. These examples illustrate both the potential for CIU in general protein analysis as well as a demonstration of best practices in the interpretation of CIU data. PMID:26489593

  8. Hofmeister effects in protein unfolding kinetics: estimation of changes in surface area upon formation of the transition state.

    PubMed

    López-Arenas, Leticia; Solís-Mendiola, Silvia; Padilla-Zúñiga, Jaqueline; Hernández-Arana, Andrés

    2006-07-01

    We studied the effect of three electrolytes (LiCl, Na(2)SO(4), GuHCl) on the unfolding reaction of chymopapain, a two-domain protein belonging in the papain family of cysteine proteinases. Due to methodological reasons, these studies were carried out at pH 1.5 where the protein unfolds following biphasic kinetics. We have observed the presence of two different effects of electrolyte concentration on the unfolding reactions. At low ionic strength, the ionic atmosphere brought about an increase in reaction rates, regardless of the type of ions being present; this effect is attributed to a general "electrostatic screening" of charge-charge interactions in the macromolecule. At high ionic strength, each electrolyte exerted a distinctively different effect: both rate constants were largely increased by GuHCl (a well-known protein denaturant), but only slightly by LiCl; in contrast, Na(2)SO(4) (a good precipitant) decreased the value of both unfolding rates. These ion-specific (Hofmeister) effects were further used to estimate changes in accessible surface area (DeltaASA) upon formation of the transition states (TS) for unfolding. Results obtained with LiCl and Na(2)SO(4), which we analyzed by means of a parameterization derived from published solubility data of amino acid derivatives, are consistent with DeltaASA increments (for each phase) of about 8.0% of the total theoretical DeltaASA for complete unfolding of the chymopapain molecule. Results in the presence of GuHCl, which were analyzed by using a previous parameterization of protein unfolding data, gave larger DeltaASAs of activation, equivalent to 13 and 16% of the total unfolding DeltaASA. PMID:16837256

  9. Thermal denaturation of Bungarus fasciatus acetylcholinesterase: Is aggregation a driving force in protein unfolding?

    PubMed

    Shin, I; Wachtel, E; Roth, E; Bon, C; Silman, I; Weiner, L

    2002-08-01

    A monomeric form of acetylcholinesterase from the venom of Bungarus fasciatus is converted to a partially unfolded molten globule species by thermal inactivation, and subsequently aggregates rapidly. To separate the kinetics of unfolding from those of aggregation, single molecules of the monomeric enzyme were encapsulated in reverse micelles of Brij 30 in 2,2,4-trimethylpentane, or in large unilamellar vesicles of egg lecithin/cholesterol at various protein/micelle (vesicle) ratios. The first-order rate constant for thermal inactivation at 45 degrees C, of single molecules entrapped within the reverse micelles (0.031 min(-1)), was higher than in aqueous solution (0.007 min(-1)) or in the presence of normal micelles (0.020 min(-1)). This clearly shows that aggregation does not provide the driving force for thermal inactivation of BfAChE. Within the large unilamellar vesicles, at average protein/vesicle ratios of 1:1 and 10:1, the first-order rate constants for thermal inactivation of the encapsulated monomeric acetylcholinesterase, at 53 degrees C, were 0.317 and 0.342 min(-1), respectively. A crosslinking technique, utilizing the photosensitive probe, hypericin, showed that thermal denaturation produces a distribution of species ranging from dimers through to large aggregates. Consequently, at a protein/vesicle ratio of 10:1, aggregation can occur upon thermal denaturation. Thus, these experiments also demonstrate that aggregation does not drive the thermal unfolding of Bungarus fasciatus acetylcholinesterase. Our experimental approach also permitted monitoring of recovery of enzymic activity after thermal denaturation in the absence of a competing aggregation process. Whereas no detectable recovery of enzymic activity could be observed in aqueous solution, up to 23% activity could be obtained for enzyme sequestered in the reverse micelles. PMID:12142456

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

    PubMed

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

    2009-01-01

    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

  11. Highly anomalous energetics of protein cold denaturation linked to folding-unfolding kinetics.

    PubMed

    Romero-Romero, M Luisa; Inglés-Prieto, Alvaro; Ibarra-Molero, Beatriz; Sanchez-Ruiz, Jose M

    2011-01-01

    Despite several careful experimental analyses, it is not yet clear whether protein cold-denaturation is just a "mirror image" of heat denaturation or whether it shows unique structural and energetic features. Here we report that, for a well-characterized small protein, heat denaturation and cold denaturation show dramatically different experimental energetic patterns. Specifically, while heat denaturation is endothermic, the cold transition (studied in the folding direction) occurs with negligible heat effect, in a manner seemingly akin to a gradual, second-order-like transition. We show that this highly anomalous energetics is actually an apparent effect associated to a large folding/unfolding free energy barrier and that it ultimately reflects kinetic stability, a naturally-selected trait in many protein systems. Kinetics thus emerges as an important factor linked to differential features of cold denaturation. We speculate that kinetic stabilization against cold denaturation may play a role in cold adaptation of psychrophilic organisms. Furthermore, we suggest that folding-unfolding kinetics should be taken into account when analyzing in vitro cold-denaturation experiments, in particular those carried out in the absence of destabilizing conditions. PMID:21829584

  12. Unfolding the response of a zero-degree magnetic spectrometer from measurements of the Δ resonance

    NASA Astrophysics Data System (ADS)

    Vargas, J.; Benlliure, J.; Caamaño, M.

    2013-04-01

    The magnetic spectrometer FRagment Separator at GSI has been used to investigate the in-medium Δ-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 Δ 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 Δ 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.

  13. Tracking Unfolding and Refolding Reactions of Single Proteins using Atomic Force Microscopy Methods

    PubMed Central

    Bujalowski, Paul J.; Oberhauser, Andres F.

    2013-01-01

    During the last two decades single-molecule manipulation techniques such as atomic force microscopy (AFM) has risen to prominence through their unique capacity to provide fundamental information on the structure and function of biomolecules. Here we describe the use of single-molecule AFM to track protein unfolding and refolding pathways, enzymatic catalysis and the effects of osmolytes and chaperones on protein stability and folding. We will outline the principles of operation for two different AFM pulling techniques: length clamp and force-clamp discuss prominent applications. We provide protocols for the construction of polyproteins which are amenable for AFM experiments, the preparation of different coverslips, choice and calibration of AFM cantilevers. We also discuss the selection criteria for AFM recordings, the calibration of AFM cantilevers, protein sample preparations and analysis of the obtained data. PMID:23523554

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

    PubMed Central

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

    2008-01-01

    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. PMID:19048998

  15. Effect of osmolytes on pressure-induced unfolding of proteins: a high-pressure SAXS study.

    PubMed

    Krywka, Christina; Sternemann, Christian; Paulus, Michael; Tolan, Metin; Royer, Catherine; Winter, Roland

    2008-12-22

    Herein, we explore the effect of different types of osmolytes on the high-pressure stability and tertiary structure of a well-characterized monomeric protein, staphylococcal nuclease (SNase). Changes in the denaturation pressure and the radius of gyration are obtained in the presence of different concentrations of trimethylamine N-oxide (TMAO), glycerol and urea. To reveal structural changes in the protein upon compression at various osmolyte conditions, small-angle X-ray scattering (SAXS) experiments were carried out. To this end, a new high-pressure cell suitable for high-precision SAXS studies at synchrotron sources was built, which allows one to carry out scattering experiments up to maximum pressures of about 7 kbar. Our data clearly indicate that the osmolytes that stabilize proteins against temperature-induced unfolding drastically increase their pressure stability and that the elliptically shaped curve of the pressure-temperature-stability diagram of proteins is shifted to higher temperatures and pressures with increasing osmolyte concentration. A drastic stabilization is observed for the osmolyte TMAO, which exhibits not only a significant stabilization against temperature-induced unfolding, but also a particularly strong stabilization of the protein against pressure. In fact, such findings are in accordance with in vivo studies (for example P. J. Yancey, J. Exp. Biol. 2005, 208, 2819-2830), where unusually high TMAO concentrations in some deep-sea animals were found. Conversely, chaotropic agents such as urea have a strong destabilizing effect on both the temperature and pressure stability of the protein. Our data also indicate that sufficiently high TMAO concentrations might be able to largely offset the destabilizing effect of urea. The different scenarios observed are discussed in the context of recent experimental and theoretical studies. PMID:18924198

  16. Unfolding knots by proteasome-like systems: simulations of the behaviour of folded and neurotoxic proteins.

    PubMed

    Wojciechowski, Michał; Gómez-Sicilia, Àngel; Carrión-Vázquez, Mariano; Cieplak, Marek

    2016-08-16

    Knots in proteins have been proposed to resist proteasomal degradation. Ample evidence associates proteasomal degradation with neurodegeneration. One interesting possibility is that indeed knotted conformers stall this machinery leading to toxicity. However, although the proteasome is known to unfold mechanically its substrates, at present there are no experimental methods to emulate this particular traction geometry. Here, we consider several dynamical models of the proteasome in which the complex is represented by an effective potential with an added pulling force. This force is meant to induce the translocation of a protein or a polypeptide into the catalytic chamber. The force is either constant or applied periodically. The translocated proteins are modelled in a coarse-grained fashion. We do comparative analysis of several knotted globular proteins and the transiently knotted polyglutamine tracts of length 60 alone and fused in exon 1 of the huntingtin protein. Huntingtin is associated with Huntington's disease, a well-known genetically determined neurodegenerative disease. We show that the presence of a knot hinders and sometimes even jams translocation. We demonstrate that the probability to do so depends on the protein, the model of the proteasome, the magnitude of the pulling force, and the choice of the pulled terminus. In any case, the net effect would be a hindrance in the proteasomal degradation process in the cell. This would then yield toxicity via two different mechanisms: one through toxic monomers compromising degradation and another by the formation of toxic oligomers. Our work paves the way for the mechanistic investigation of the mechanical unfolding of knotted structures by the proteasome and its relation to toxicity and disease. PMID:27425826

  17. Origin and Functional Evolution of the Cdc48/p97/VCP AAA+ Protein Unfolding and Remodeling Machine.

    PubMed

    Barthelme, Dominik; Sauer, Robert T

    2016-05-01

    The AAA+ Cdc48 ATPase (alias p97 or VCP) is a key player in multiple ubiquitin-dependent cell signaling, degradation, and quality control pathways. Central to these broad biological functions is the ability of Cdc48 to interact with a large number of adaptor proteins and to remodel macromolecular proteins and their complexes. Different models have been proposed to explain how Cdc48 might couple ATP hydrolysis to forcible unfolding, dissociation, or remodeling of cellular clients. In this review, we provide an overview of possible mechanisms for substrate unfolding/remodeling by this conserved and essential AAA+ protein machine and their adaption and possible biological function throughout evolution. PMID:26608813

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

    PubMed Central

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

    2009-01-01

    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

  19. Excluded volume entropic effects on protein unfolding times and intermediary stability

    NASA Astrophysics Data System (ADS)

    Chapagain, Prem P.; Gerstman, Bernard S.

    2004-02-01

    The dynamics of protein folding result from both enthalpic and entropic contributions to the free energy. In this paper we focus on entropic volume exclusion effects. We carry out computer simulations using a model that allows us to independently change the size or biochemical properties of amino acid residues. To determine the importance of excluded volume effects, we investigate the effects of changing the size of side chains on the unfolding dynamics of a model four-helix bundle protein. In addition, we also investigate the effects of changing the thickness of the chain's backbone. This has relevance to the behavior of synthetic polymers where the size of the constituent units can be varied. We find that entropic excluded volume effects are crucially important for stabilizing the organized native state relative to the molten globule.

  20. Excluded volume entropic effects on protein unfolding times and intermediary stability

    NASA Astrophysics Data System (ADS)

    Chapagain, Prem

    2005-03-01

    The dynamics of protein folding result from both enthalpic and entropic contributions to the free energy. In this paper we focus on entropic volume exclusion effects. We carry out computer simulations using a model that allows us to independently change the size or biochemical properties of amino acid residues. To determine the importance of excluded volume effects, we investigate the effects of changing the size of side chains on the unfolding dynamics of a model four-helix bundle protein. In addition, we also investigate the effects of changing the thickness of the chain's backbone. This has relevance to the behaviour of synthetic polymers where the size of the constituent units can be varied. We find that entropic excluded volume effects are crucially important for stabilizing the organized native state relative to the molten globule.

  1. Core-Shell Model of Folding-Unfolding Transitions (UFT) in Proteins

    NASA Astrophysics Data System (ADS)

    Aroutiounian, Svetlana

    2008-03-01

    There are ˜10^N conformations for a protein of length N to sort out randomly in search of lowest free energy state. Can protein folding be simple and fast? Core-shell model introduces principles, proposes mechanisms and scores residues of fast, reversible UFT in protein. According to it, during UFT the realm of intra-residual interactions leads the residue motion. The scaffold of hydrophilic residues forms external shell of unstructured, tube-like protein in unfolded state, just as the hydrophobic residues form internal scaffold -- core, of the protein in folded state. As UFT proceeds, residue slides into lowest-score position permitted by its structure. Model accounts for experimentally observed features of UFT. It is based on three principles: 1) During UFT protein is virtual - its features or structure are inferred only statistically and with limited precision; 2) Mechanism of UFT memory is not longitudinal, but transverse; 3) Native design overrides specific features of residues - the alphabet of amino acids assumes an intrinsic score-function. Per-residue mechanism of UFT is proposed and score-function is described. Difference graphs of transitional score-function and average genome-wide abundance index show that our score-function is the order parameter of UFT in protein and by virtue of being it, reveals transitional key residues. It echoes the multiple-tier and funnel concepts of FEL perspective. Monte Carlo simulations of UFT in myoglobin illustrate the idea.

  2. Quasiequilibrium unfolding thermodynamics of a small protein studied by molecular dynamics simulation with an explicit water model

    NASA Astrophysics Data System (ADS)

    Wang, Jihua; Zhang, Zhiyong; Liu, Haiyan; Shi, Yunyu

    2003-06-01

    The 124 independent molecular dynamics simulations are completed with total time of 196.8 ns. The calculated unfolding quasiequilibrium thermodynamics of G-IgG-binding domain B1 (GB1) shows the experimentally observed protein transitions: a coil to disordered globule transition, a disordered globule to molten globule transition, a molten globule to nativelike transition, and a nativelike to solidlike state transition. The first protein unfolding phase diagram has been constructed from molecular dynamics simulations with an explicit water model. The calculated melting temperature of GB1 agrees with early experiment. The results also agree with the recent experiment result in which GB1 has more than one intermediate.

  3. Probing the Folding-Unfolding Transition of a Thermophilic Protein, MTH1880

    PubMed Central

    Jung, Youngjin; Han, Jeongmin; Yun, Ji-Hye; Chang, Iksoo; Lee, Weontae

    2016-01-01

    The folding mechanism of typical proteins has been studied widely, while our understanding of the origin of the high stability of thermophilic proteins is still elusive. Of particular interest is how an atypical thermophilic protein with a novel fold maintains its structure and stability under extreme conditions. Folding-unfolding transitions of MTH1880, a thermophilic protein from Methanobacterium thermoautotrophicum, induced by heat, urea, and GdnHCl, were investigated using spectroscopic techniques including circular dichorism, fluorescence, NMR combined with molecular dynamics (MD) simulations. Our results suggest that MTH1880 undergoes a two-state N to D transition and it is extremely stable against temperature and denaturants. The reversibility of refolding was confirmed by spectroscopic methods and size exclusion chromatography. We found that the hyper-stability of the thermophilic MTH1880 protein originates from an extensive network of both electrostatic and hydrophobic interactions coordinated by the central β-sheet. Spectroscopic measurements, in combination with computational simulations, have helped to clarify the thermodynamic and structural basis for hyper-stability of the novel thermophilic protein MTH1880. PMID:26766214

  4. Cooperative hydration effect causes thermal unfolding of proteins and water activity plays a key role in protein stability in solutions.

    PubMed

    Miyawaki, Osato; Dozen, Michiko; Hirota, Kaede

    2016-08-01

    The protein unfolding process observed in a narrow temperature range was clearly explained by evaluating the small difference in the enthalpy of hydrogen-bonding between amino acid residues and the hydration of amino acid residue separately. In aqueous solutions, the effect of cosolute on the protein stability is primarily dependent on water activity, aw, the role of which has been long neglected in the literature. The effect of aw on protein stability works as a power law so that a small change in aw is amplified substantially through the cooperative hydration effect. In the present approach, the role of hydrophobic interaction stands behind. This affects protein stability indirectly through the change in solution structure caused by the existence of cosolute. PMID:26896315

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

    PubMed Central

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

    2013-01-01

    Biogenesis of β-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 β-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. PMID:22391420

  6. MARCKS is a natively unfolded protein with an inaccessible actin-binding site: evidence for long-range intramolecular interactions.

    PubMed

    Tapp, Hazel; Al-Naggar, Iman M; Yarmola, Elena G; Harrison, Alexis; Shaw, Gerry; Edison, Arthur S; Bubb, Michael R

    2005-03-18

    Myristoylated alanine-rich C kinase substrate (MARCKS) is an unfolded protein that contains well characterized actin-binding sites within the phosphorylation site domain (PSD), yet paradoxically, we now find that intact MARCKS does not bind to actin. Intact MARCKS also does not bind as well to calmodulin as does the PSD alone. Myristoylation at the N terminus alters how calmodulin binds to MARCKS, implying that, despite its unfolded state, the distant N terminus influences binding events at the PSD. We show that the free PSD binds with site specificity to MARCKS, suggesting that long-range intramolecular interactions within MARCKS are also possible. Because of the unusual primary sequence of MARCKS with an overall isoelectric point of 4.2 yet a very basic PSD (overall charge of +13), we speculated that ionic interactions between oppositely charged domains of MARCKS were responsible for long-range interactions within MARCKS that sterically influence binding events at the PSD and that explain the observed differences between properties of the PSD and MARCKS. Consistent with this hypothesis, chemical modifications of MARCKS that neutralize negatively charged residues outside of the PSD allow the PSD to bind to actin and increase the affinity of MARCKS for calmodulin. Similarly, both myristoylation of MARCKS and cleavage of MARCKS by calpain are shown to increase the availability of the PSD so as to activate its actin-binding activity. Because abundant evidence supports the conclusion that MARCKS is an important protein in regulating actin dynamics, our data imply that post-translational modifications of MARCKS are necessary and sufficient to regulate actin-binding activity. PMID:15640140

  7. The secondary structure and the thermal unfolding parameters of the S-layer protein from Lactobacillus salivarius.

    PubMed

    Lighezan, Liliana; Georgieva, Ralitsa; Neagu, Adrian

    2016-09-01

    Surface layer (S-layer) proteins have been identified in the cell envelope of many organisms, such as bacteria and archaea. They self-assemble, forming monomolecular crystalline arrays. Isolated S-layer proteins are able to recrystallize into regular lattices, which proved useful in biotechnology. Here we investigate the structure and thermal unfolding of the S-layer protein isolated from Lactobacillus salivarius 16 strain of human origin. Using circular dichroism (CD) spectroscopy, and the software CDSSTR from DICHROWEB, CONTINLL from CDPro, as well as CDNN, we assess the fractions of the protein's secondary structural elements at temperatures ranging between 10 and 90 °C, and predict the tertiary class of the protein. To study the thermal unfolding of the protein, we analyze the temperature dependence of the CD signal in the far- and near-UV domains. Fitting the experimental data by two- and three-state models of thermal unfolding, we infer the midpoint temperatures, the temperature dependence of the changes in Gibbs free energy, enthalpy, and entropy of the unfolding transitions in standard conditions, and the temperature dependence of the equilibrium constant. We also estimate the changes in heat capacity at constant pressure in standard conditions. The results indicate that the thermal unfolding of the S-layer protein from L. salivarius is highly cooperative, since changes in the secondary and tertiary structures occur simultaneously. The thermodynamic analysis predicts a "cold" transition, at about -3 °C, of both the secondary and tertiary structures. Our findings may be important for the use of S-layer proteins in biotechnology and in biomedical applications. PMID:26992716

  8. Mechanical Unfolding of an Autotransporter Passenger Protein Reveals the Secretion Starting Point and Processive Transport Intermediates.

    PubMed

    Baclayon, Marian; Ulsen, Peter van; Mouhib, Halima; Shabestari, Maryam Hashemi; Verzijden, Timo; Abeln, Sanne; Roos, Wouter H; Wuite, Gijs J L

    2016-06-28

    The backbone of secreted autotransporter passenger proteins generally attains a stable β-helical structure. The secretion of passengers across the outer membrane was proposed to be driven by sequential folding of this structure at the cell surface. This mechanism would require a relatively stable intermediate as starting point. Here, we investigated the mechanics of secreted truncated versions of the autotransporter hemoglobin protease (Hbp) of Escherichia coli using atomic force microscopy. The data obtained reveal a β-helical structure at the C terminus that is very stable. In addition, several other distinct metastable intermediates are found which are connected during unfolding by multiroute pathways. Computational analysis indicates that these intermediates correlate to the β-helical rungs in the Hbp structure which are clamped by stacked aromatic residues. Our results suggest a secretion mechanism that is initiated by a stable C-terminal structure and driven forward by several folding intermediates that build up the β-helical backbone. PMID:27219538

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

    PubMed Central

    Hagelueken, Gregor; Naismith, James H

    2013-01-01

    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 μg of material. The approach is robust for both soluble and detergent-solubilized membrane proteins. PMID:24091556

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

    PubMed Central

    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

    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

  11. Equilibrium properties of realistic random heteropolymers and their relevance for globular and naturally unfolded proteins

    NASA Astrophysics Data System (ADS)

    Tiana, G.; Sutto, L.

    2011-12-01

    Random heteropolymers do not display the typical equilibrium properties of globular proteins, but are the starting point to understand the physics of proteins and, in particular, to describe their non-native states. So far, they have been studied with mean-field models in the thermodynamic limit, or with computer simulations of very small chains on lattice. After describing a self-adjusting parallel-tempering technique to sample efficiently the low-energy states of frustrated systems without the need of tuning the system-dependent parameters of the algorithm, we apply it to random heteropolymers moving in continuous space. We show that if the mean interaction between monomers is negative, the usual description through the random-energy model is nearly correct, provided that it is extended to account for noncompact conformations. If the mean interaction is positive, such a simple description breaks out and the system behaves in a way more similar to Ising spin glasses. The former case is a model for the denatured state of globular proteins, the latter of naturally unfolded proteins, whose equilibrium properties thus result as qualitatively different.

  12. Hydration-responsive folding and unfolding in graphene oxide liquid crystal phases.

    PubMed

    Guo, Fei; Kim, Franklin; Han, Tae Hee; Shenoy, Vivek B; Huang, Jiaxing; Hurt, Robert H

    2011-10-25

    Graphene oxide is promising as a plate-like giant molecular building block for the assembly of new carbon materials. Its water dispersibility, liquid crystallinity, and ease of reduction offer advantages over other carbon precursors if its fundamental assembly rules can be identified. This article shows that graphene oxide sheets of known lateral dimension form nematic liquid crystal phases with transition points in agreement with the Onsager hard-plate theory. The liquid crystal phases can be systematically ordered into defined supramolecular patterns using surface anchoring, complex fluid flow, and microconfinement. Graphene oxide is seen to exhibit homeotropic surface anchoring at interfaces driven by excluded volume entropy and by adsorption enthalpy associated with its partially hydrophobic basal planes. Surprisingly, some of the surface-ordered graphene oxide phases dry into graphene oxide solids that undergo a dramatic anisotropic swelling upon rehydration to recover their initial size and shape. This behavior is shown to be a unique hydration-responsive folding and unfolding transition. During drying, surface tension forces acting parallel to the layer planes cause a buckling instability that stores elastic energy in accordion-folded structures in the dry solid. Subsequent water infiltration reduces interlayer frictional forces and triggers release of the stored elastic energy in the form of dramatic unidirectional expansion. We explain the folding/unfolding phenomena by quantitative nanomechanics and introduce the potential of liquid crystal-derived graphene oxide phases as new stimuli-response materials. PMID:21877716

  13. Hydration-Responsive Folding and Unfolding in Graphene Oxide Liquid Crystal Phases

    PubMed Central

    Guo, Fei; Kim, Franklin; Han, Tae Hee; Shenoy, Vivek B.; Huang, Jiaxing; Hurt, Robert H.

    2011-01-01

    Graphene oxide is promising as a plate-like giant molecular building block for the assembly of new carbon materials. Its water dispersibility, liquid crystallinity, and ease of reduction offer advantages over other carbon precursors if its fundamental assembly rules can be identified. This article shows that graphene oxide sheets of known lateral dimension form nematic liquid crystal phases with transition points in agreement with the Onsager hard-plate theory. The liquid crystal phases can be systematic ordered into defined supramolecular patterns using surface anchoring, complex fluid flow, and micro-confinement. Graphene oxide is seen to exhibit homeotropic surface anchoring at interfaces driven by excluded volume entropy and by adsorption enthalpy associated with its partially hydrophobic basal planes. Surprisingly, some of the surface-ordered graphene oxide phases dry into graphene oxide solids that undergo a dramatic anisotropic swelling upon rehydration to recover their initial size and shape. This behavior is shown to be a unique hydration-responsive folding and unfolding transition. During drying, surface tension forces acting parallel to the layer planes cause a buckling instability that stores elastic energy in accordion-folded structures in the dry solid. Subsequent water infiltration reduces interlayer frictional forces and triggers release of the stored elastic energy in the form of dramatic unidirectional expansion. We explain the folding/unfolding phenomena by quantitative nanomechanics, and introduce the potential of liquid crystal-derived graphene oxide phases as new stimuli-response materials. PMID:21877716

  14. Ligand-Induced Changes of the Apparent Transition-State Position in Mechanical Protein Unfolding

    PubMed Central

    Stigler, Johannes; Rief, Matthias

    2015-01-01

    Force-spectroscopic measurements of ligand-receptor systems and the unfolding/folding of nucleic acids or proteins reveal information on the underlying energy landscape along the pulling coordinate. The slope Δx‡ of the force-dependent unfolding/unbinding rates is interpreted as the distance from the folded/bound state to the transition state for unfolding/unbinding and, hence, often related to the mechanical compliance of the sample molecule. Here we show that in ligand-binding proteins, the experimentally inferred Δx‡ can depend on the ligand concentration, unrelated to changes in mechanical compliance. We describe the effect in single-molecule, force-spectroscopy experiments of the calcium-binding protein calmodulin and explain it in a simple model where mechanical unfolding and ligand binding occur on orthogonal reaction coordinates. This model predicts changes in the experimentally inferred Δx‡, depending on ligand concentration and the associated shift of the dominant barrier between the two reaction coordinates. We demonstrate quantitative agreement between experiments and simulations using a realistic six-state kinetic scheme using literature values for calcium-binding kinetics and affinities. Our results have important consequences for the interpretation of force-spectroscopic data of ligand-binding proteins. PMID:26200872

  15. Susceptibility of Nrf2-Null Mice to Steatohepatitis and Cirrhosis upon Consumption of a High-Fat Diet Is Associated with Oxidative Stress, Perturbation of the Unfolded Protein Response, and Disturbance in the Expression of Metabolic Enzymes but Not with Insulin Resistance

    PubMed Central

    Meakin, Paul J.; Chowdhry, Sudhir; Sharma, Ritu S.; Ashford, Fiona B.; Walsh, Shaun V.; McCrimmon, Rory J.; Dinkova-Kostova, Albena T.; Dillon, John F.

    2014-01-01

    Mice lacking the transcription factor NF-E2 p45-related factor 2 (Nrf2) develop more severe nonalcoholic steatohepatitis (NASH), with cirrhosis, than wild-type (Nrf2+/+) mice when fed a high-fat (HF) diet for 24 weeks. Although NASH is usually associated with insulin resistance, HF-fed Nrf2−/− mice exhibited better insulin sensitivity than HF-fed Nrf2+/+ mice. In livers of HF-fed mice, loss of Nrf2 resulted in greater induction of lipogenic genes, lower expression of β-oxidation genes, greater reduction in AMP-activated protein kinase (AMPK) levels, and diminished acetyl coenzyme A (CoA) carboxylase phosphorylation than in the wild-type livers, which is consistent with greater fatty acid (FA) synthesis in Nrf2−/− livers. Moreover, primary Nrf2−/− hepatocytes displayed lower glucose and FA oxidation than Nrf2+/+ hepatocytes, with FA oxidation partially rescued by treatment with AMPK activators. The unfolded protein response (UPR) was perturbed in control regular-chow (RC)-fed Nrf2−/− mouse livers, and this was associated with constitutive activation of NF-κB and JNK, along with upregulation of inflammatory genes. The HF diet elicited an antioxidant response in Nrf2+/+ livers, and as this was compromised in Nrf2−/− livers, they suffered oxidative stress. Therefore, Nrf2 protects against NASH by suppressing lipogenesis, supporting mitochondrial function, increasing the threshold for the UPR and inflammation, and enabling adaptation to HF-diet-induced oxidative stress. PMID:24958099

  16. Protein denaturation at a single-molecule level: the effect of nonpolar environments and its implications on the unfolding mechanism by proteases

    NASA Astrophysics Data System (ADS)

    Cheng, Bo; Wu, Shaogui; Liu, Shixin; Rodriguez-Aliaga, Piere; Yu, Jin; Cui, Shuxun

    2015-02-01

    Most proteins are typically folded into predetermined three-dimensional structures in the aqueous cellular environment. However, proteins can be exposed to a nonpolar environment under certain conditions, such as inside the central cavity of chaperones and unfoldases during protein degradation. It remains unclear how folded proteins behave when moved from an aqueous solvent to a nonpolar one. Here, we employed single-molecule atomic force microscopy and molecular dynamics (MD) simulations to investigate the structural and mechanical variations of a polyprotein, I278, during the change from a polar to a nonpolar environment. We found that the polyprotein was unfolded into an unstructured polypeptide spontaneously when pulled into nonpolar solvents. This finding was corroborated by MD simulations where I27 was dragged from water into a nonpolar solvent, revealing details of the unfolding process at the water/nonpolar solvent interface. These results highlight the importance of water in maintaining folding stability, and provide insights into the response of folded proteins to local hydrophobic environments.Most proteins are typically folded into predetermined three-dimensional structures in the aqueous cellular environment. However, proteins can be exposed to a nonpolar environment under certain conditions, such as inside the central cavity of chaperones and unfoldases during protein degradation. It remains unclear how folded proteins behave when moved from an aqueous solvent to a nonpolar one. Here, we employed single-molecule atomic force microscopy and molecular dynamics (MD) simulations to investigate the structural and mechanical variations of a polyprotein, I278, during the change from a polar to a nonpolar environment. We found that the polyprotein was unfolded into an unstructured polypeptide spontaneously when pulled into nonpolar solvents. This finding was corroborated by MD simulations where I27 was dragged from water into a nonpolar solvent, revealing

  17. Comparing equilibrium and kinetic protein unfolding using time-resolved electrospray-coupled ion mobility mass spectrometry.

    PubMed

    Liuni, Peter; Deng, Bin; Wilson, Derek J

    2015-10-21

    Protein unfolding intermediates are thought to play a critical role in conformational pathogenesis, acting as a 'gateway' to inactivation or pathogenic aggregation. Unfolding intermediates have long been studied either by populating partially-folded species at equilibrium using incresingly denaturing conditions, or by transiently populating 'kinetic' intermediates under fully denaturing conditions using a time-resolved approach (e.g. stopped-flow fluorescence). However, it is not clear that the folding intermediates populated under equilibrium conditions are comparable to intermediates transiently populated in kinetic experiments. In this work, we combine time-resolved electrospray (TRESI) with travelling wave Ion Mobility Spectrometry (IMS) for the first time to directly compare equilibrium and kinetic unfolding intermediates of cytochrome c. Our results show a high degree of correlation between all species populated under these substantially different regimes. PMID:26115375

  18. Accurate Estimation of Protein Folding and Unfolding Times: Beyond Markov State Models.

    PubMed

    Suárez, Ernesto; Adelman, Joshua L; Zuckerman, Daniel M

    2016-08-01

    Because standard molecular dynamics (MD) simulations are unable to access time scales of interest in complex biomolecular systems, it is common to "stitch together" information from multiple shorter trajectories using approximate Markov state model (MSM) analysis. However, MSMs may require significant tuning and can yield biased results. Here, by analyzing some of the longest protein MD data sets available (>100 μs per protein), we show that estimators constructed based on exact non-Markovian (NM) principles can yield significantly improved mean first-passage times (MFPTs) for protein folding and unfolding. In some cases, MSM bias of more than an order of magnitude can be corrected when identical trajectory data are reanalyzed by non-Markovian approaches. The NM analysis includes "history" information, higher order time correlations compared to MSMs, that is available in every MD trajectory. The NM strategy is insensitive to fine details of the states used and works well when a fine time-discretization (i.e., small "lag time") is used. PMID:27340835

  19. Thermal unfolding and aggregation of human complement protein C9: a differential scanning calorimetry study.

    PubMed

    Lohner, K; Esser, A F

    1991-07-01

    The thermotropic behavior of purified human complement protein C9 was investigated by high-sensitivity differential scanning calorimetry. When dissolved in physiological buffers (pH 7.2, 150 mM NaCl), C9 underwent three endothermic transitions with transition temperatures (Tm) centered at about 32, 48, and 53 degrees C, respectively, and one exothermic transition above 64 degrees C that correlated with protein aggregation. The associated calorimetric enthalpies of the three endothermic transitions were about 45, 60, and 161 kcal/mol with cooperative ratios (delta Hcal/delta HvH) close to unity. The total calorimetric enthalphy for the unfolding process was in the range of 260-280 kcal/mol under all conditions. The exothermic aggregation temperature was strongly pH dependent, changing from 60 degrees C at pH 6.6 to 81.4 degrees C at pH 8.0, whereas none of the three endothermic transitions was significantly affected by pH changes. They were, however, sensitive to addition of calcium ions; most affected was Tm1 which shifted from 32 to 35.8 degrees C in the presence of 3 mM calcium, i.e., the normal blood concentration. Kosmotropic ions stabilized the protein by shifting the endothermic transitions to slightly higher temperatures whereas inclusion of chaotropic ions (such as choline), removal of bound calcium by addition of EDTA, or proteolysis with thrombin lowered the transition temperatures. Previous studies had indicated the formation of at least three different forms of C9 during membrane insertion or during heat polymerization, and it is suggested that the three endothermic transitions reflect the formation of such C9 conformers. Choline, which is present at high concentrations on the surface of biological membranes, and calcium ions have the ability to shift the transition temperatures of the first two transitions to be either close to or below body temperature. Thus, it is very likely that C9 is present in vivo in a partially unfolded state when bound to a

  20. A simple two-state protein unfolds mechanically via multiple heterogeneous pathways at single-molecule resolution

    PubMed Central

    Schönfelder, Jörg; Perez-Jimenez, Raul; Muñoz, Victor

    2016-01-01

    A major drive in protein folding has been to develop experimental technologies to resolve the myriads of microscopic pathways and complex mechanisms that purportedly underlie simple two-state folding behaviour. This is key for cross-validating predictions from theory and modern computer simulations. Detecting such complexity experimentally has remained elusive even using methods with improved time, structural or single-molecule resolution. Here, we investigate the mechanical unfolding of cold shock protein B (Csp), a showcase two-state folder, using single-molecule force-spectroscopy. Under controlled-moderate pulling forces, the unfolding of Csp emerges as highly heterogeneous with trajectories ranging from single sweeps to different combinations of multiple long-lived mechanical intermediates that also vary in order of appearance. Steered molecular dynamics simulations closely reproduce the experimental observations, thus matching unfolding patterns with structural events. Our results provide a direct glimpse at the nanoscale complexity underlying two-state folding, and postulate these combined methods as unique tools for dissecting the mechanical unfolding mechanisms of such proteins. PMID:27248054

  1. Inactivation and unfolding of protein tyrosine phosphatase from Thermus thermophilus HB27 during urea and guanidine hydrochloride denaturation.

    PubMed

    Wang, Yejing; He, Huawei; Liu, Lina; Gao, Chunyan; Xu, Shui; Zhao, Ping; Xia, Qingyou

    2014-01-01

    The effects of urea and guanidine hydrochloride (GdnHCl) on the activity, conformation and unfolding process of protein tyrosine phosphatase (PTPase), a thermostable low molecular weight protein from Thermus thermophilus HB27, have been studied. Enzymatic activity assays showed both urea and GdnHCl resulted in the inactivation of PTPase in a concentration and time-dependent manner. Inactivation kinetics analysis suggested that the inactivation of PTPase induced by urea and GdnHCl were both monophasic and reversible processes, and the effects of urea and GdnHCl on PTPase were similar to that of mixed-type reversible inhibitors. Far-ultraviolet (UV) circular dichroism (CD), Tryptophan and 1-anilinonaphthalene -8-sulfonic acid (ANS) fluorescence spectral analyses indicated the existence of a partially active and an inactive molten globule-like intermediate during the unfolding processes induced by urea and GdnHCl, respectively. Based on the sequence alignment and the homolog Tt1001 protein structure, we discussed the possible conformational transitions of PTPase induced by urea and GdnHCl and compared the conformations of these unfolding intermediates with the transient states in bovine PTPase and its complex structures in detail. Our results may be able to provide some valuable clues to reveal the relationship between the structure and enzymatic activity, and the unfolding pathway and mechanism of PTPase. PMID:25255086

  2. A simple two-state protein unfolds mechanically via multiple heterogeneous pathways at single-molecule resolution

    NASA Astrophysics Data System (ADS)

    Schönfelder, Jörg; Perez-Jimenez, Raul; Muñoz, Victor

    2016-06-01

    A major drive in protein folding has been to develop experimental technologies to resolve the myriads of microscopic pathways and complex mechanisms that purportedly underlie simple two-state folding behaviour. This is key for cross-validating predictions from theory and modern computer simulations. Detecting such complexity experimentally has remained elusive even using methods with improved time, structural or single-molecule resolution. Here, we investigate the mechanical unfolding of cold shock protein B (Csp), a showcase two-state folder, using single-molecule force-spectroscopy. Under controlled-moderate pulling forces, the unfolding of Csp emerges as highly heterogeneous with trajectories ranging from single sweeps to different combinations of multiple long-lived mechanical intermediates that also vary in order of appearance. Steered molecular dynamics simulations closely reproduce the experimental observations, thus matching unfolding patterns with structural events. Our results provide a direct glimpse at the nanoscale complexity underlying two-state folding, and postulate these combined methods as unique tools for dissecting the mechanical unfolding mechanisms of such proteins.

  3. A simple two-state protein unfolds mechanically via multiple heterogeneous pathways at single-molecule resolution.

    PubMed

    Schönfelder, Jörg; Perez-Jimenez, Raul; Muñoz, Victor

    2016-01-01

    A major drive in protein folding has been to develop experimental technologies to resolve the myriads of microscopic pathways and complex mechanisms that purportedly underlie simple two-state folding behaviour. This is key for cross-validating predictions from theory and modern computer simulations. Detecting such complexity experimentally has remained elusive even u