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Sample records for unfolded protein response

  1. Proteostasis control by the unfolded protein response.

    PubMed

    Hetz, Claudio; Chevet, Eric; Oakes, Scott A

    2015-07-01

    Stress induced by accumulation of misfolded proteins in the endoplasmic reticulum is observed in many physiological and pathological conditions. To cope with endoplasmic reticulum stress, cells activate the unfolded protein response, a dynamic signalling network that orchestrates the recovery of homeostasis or triggers apoptosis, depending on the level of damage. Here we provide an overview of recent insights into the mechanisms that cells employ to maintain proteostasis and how the unfolded protein response determines cell fate under endoplasmic reticulum stress. PMID:26123108

  2. The unfolded protein response in multiple sclerosis

    PubMed Central

    Stone, Sarrabeth; Lin, Wensheng

    2015-01-01

    The unfolded protein response (UPR) occurs in response to endoplasmic reticulum (ER) stress caused by the accumulation of unfolded or misfolded proteins in the ER. The UPR is comprised of three signaling pathways that promote cytoprotective functions to correct ER stress; however, if ER stress cannot be resolved the UPR results in apoptosis of affected cells. The UPR is an important feature of various human diseases, including multiple sclerosis (MS). Recent studies have shown several components of the UPR are upregulated in the multiple cell types in MS lesions, including oligodendrocytes, T cells, microglia/macrophages, and astrocytes. Data from animal model studies, particularly studies of experimental autoimmune encephalomyelitis (EAE) and the cuprizone model, imply an important role of the UPR activation in oligodendrocytes in the development of MS. In this review we will cover current literature on the UPR and the evidence for its role in the development of MS. PMID:26283904

  3. Unfolded Protein Response Pathways in Neurodegenerative Diseases.

    PubMed

    Shah, Syed Zahid Ali; Zhao, Deming; Khan, Sher Hayat; Yang, Lifeng

    2015-12-01

    The aggregation of disease-specific misfolded proteins resulting in endoplasmic reticulum stress is associated with early pathological events in many neurodegenerative diseases, and apoptotic signaling is initiated when the stress goes beyond the maximum threshold level of endoplasmic reticulum stress sensors. All eukaryotic cells respond to the accumulation of unfolded proteins in the endoplasmic reticulum (ER) by signaling an adaptive pathway termed as unfolded protein response (UPR). Recently, the focus of research shifted from work on specific proteins as pathogenesis in these neurodegenerative diseases towards a more specific generic pathway known as UPR. ER is a major organelle for protein quality control, and cellular stress disrupts normal functioning of ER. The UPR acts as a protective mechanism during endoplasmic reticulum stress, but persistent long-term stress triggers UPR-mediated apoptotic pathways ultimately leading to cell death. Here in this review, we will briefly summarize the molecular events of endoplasmic reticulum stress-associated UPR signaling pathways and their potential therapeutic role in neurodegenerative diseases. PMID:26304853

  4. Druggable Sensors of the Unfolded Protein Response

    PubMed Central

    Maly, Dustin J.; Papa, Feroz R.

    2015-01-01

    The inability of cells to properly fold, modify, and assemble secretory and transmembrane proteins leads to accumulation of misfolded proteins in the endoplasmic reticulum (ER). Under these conditions of “ER stress,” cell survival depends on homeostatic benefits from an intracellular signaling pathway called the unfolded protein response (UPR). When activated, the UPR induces transcriptional and translational programs that restore ER homeostasis. However, under high-level/chronic ER stress, these adaptive changes ultimately become overshadowed by alternative “Terminal UPR” signals that actively commit cells to degeneration, culminating in programmed cell death. Chronic ER stress and maladaptive UPR signaling are implicated in the etiology and pathogenesis of myriad human diseases. Naturally, this has generated widespread interest in targeting key nodal components of the UPR as therapeutic strategies. Here we summarize the state of this field with emphasis placed on two of the master UPR regulators, PERK and IRE1?, which are both capable of being drugged with small molecules. PMID:25325700

  5. The impact of the unfolded protein response on human disease

    PubMed Central

    Wang, Shiyu

    2012-01-01

    A central function of the endoplasmic reticulum (ER) is to coordinate protein biosynthetic and secretory activities in the cell. Alterations in ER homeostasis cause accumulation of misfolded/unfolded proteins in the ER. To maintain ER homeostasis, eukaryotic cells have evolved the unfolded protein response (UPR), an essential adaptive intracellular signaling pathway that responds to metabolic, oxidative stress, and inflammatory response pathways. The UPR has been implicated in a variety of diseases including metabolic disease, neurodegenerative disease, inflammatory disease, and cancer. Signaling components of the UPR are emerging as potential targets for intervention and treatment of human disease. PMID:22733998

  6. Engineering of chaperone systems and of the unfolded protein response

    PubMed Central

    Khan, Saeed U.

    2008-01-01

    Production of recombinant proteins in mammalian cells is a successful technology that delivers protein pharmaceuticals for therapies and for diagnosis of human disorders. Cost effective production of protein biopharmaceuticals requires extensive optimization through cell and fermentation process engineering at the upstream and chemical engineering of purification processes at the downstream side of the production process. The majority of protein pharmaceuticals are secreted proteins. Accumulating evidence suggests that the folding and processing of these proteins in the endoplasmic reticulum (ER) is a general rate- and yield limiting step for their production. We will summarize our knowledge of protein folding in the ER and of signal transduction pathways activated by accumulation of unfolded proteins in the ER, collectively called the unfolded protein response (UPR). On the basis of this knowledge we will evaluate engineering approaches to increase cell specific productivities through engineering of the ER-resident protein folding machinery and of the UPR. PMID:19003179

  7. Delayed Ras/PKA signaling augments the unfolded protein response

    PubMed Central

    Pincus, David; Aranda-Díaz, Andrés; Walter, Peter; El-Samad, Hana

    2014-01-01

    During environmental, developmental, or genetic stress, the cell’s folding capacity can become overwhelmed, and misfolded proteins can accumulate in all cell compartments. Eukaryotes evolved the unfolded protein response (UPR) to counteract proteotoxic stress in the endoplasmic reticulum (ER). Although the UPR is vital to restoring homeostasis to protein folding in the ER, it has become evident that the response to ER stress is not limited to the UPR. Here, we used engineered orthogonal UPR induction, deep mRNA sequencing, and dynamic flow cytometry to dissect the cell’s response to ER stress comprehensively. We show that budding yeast augments the UPR with time-delayed Ras/PKA signaling. This second wave of transcriptional dynamics is independent of the UPR and is necessary for fitness in the presence of ER stress, partially due to a reduction in general protein synthesis. This Ras/PKA-mediated effect functionally mimics other mechanisms, such as translational control by PKR-like ER kinase (PERK) and regulated inositol-requiring enzyme 1 (IRE1)-dependent mRNA decay (RIDD), which reduce the load of proteins entering the ER in response to ER stress in metazoan cells. PMID:25275008

  8. 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 Sec61? (TbSec61?) failed to induce either response. Interestingly, cell growth halted within 16 h of silencing, but sufficient TbSec61? 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

  9. Unfolded Protein Response (UPR) is activated during normal lens development

    PubMed Central

    Firtina, Zeynep; Duncan, Melinda K.

    2010-01-01

    The lens of the eye is a transparent structure responsible for focusing light onto the retina. It is composed of two morphologically different cell types, epithelial cells found on the anterior surface and the fiber cells that are continuously formed by the differentiation of epithelial cells at the lens equator. The differentiation of an epithelial precursor cell into a fiber cell is associated with a dramatic increase in membrane protein synthesis. How the terminally differentiating fiber cells cope with the increased demand on the endoplasmic reticulum for this membrane protein synthesis is not known. In the present study, we have found evidence of Unfolded Protein Response (UPR) activation during normal lens development and differentiation in the mouse. The ER-resident chaperones, immunoglobulin heavy chain binding protein (BiP) and protein disulfide isomerase (PDI), were expressed at high levels in the newly forming fiber cells of embryonic lenses. These fiber cells also expressed the UPR-associated molecules; XBP1, ATF6, phospho-PERK and ATF4 during embryogenesis. Moreover, spliced XBP1, cleaved ATF6, and phospho-eIF2 were detected in embryonic mouse lenses suggesting that UPR pathways are active in this tissue. These results propose a role for UPR activation in lens fiber cell differentiation during embryogenesis. PMID:21044701

  10. 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

  11. 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

  12. 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

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

    PubMed Central

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

    2014-01-01

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

  14. 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

  15. Development of Antiproliferative Phenylmaleimides that Activate the Unfolded Protein Response

    PubMed Central

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

    2010-01-01

    The current paper presents the synthesis and evaluation of a series of maleimides that were designed to inhibit the Cdc25 phosphatase by alkylation of catalytically essential cysteine residues. Although in HepB3 cell culture assays the analogues did exhibit antiproliferative IC50 values ranging from sub-micromolar to greater than 100 µM, inhibition of Cdc25 through cysteine alkylation could not be demonstrated. It was also found that analysis using fluorescence activated cell sorting (FACS) following treatment with the most potent analogue (1t) did not provide data consistent with inhibition at one specific point in the cell cycle, as would be expected if Cdc25A were inhibited. Further studies with a subset of analogues resulted in a correlation of antiproliferative potencies with activation of the unfolded protein response (UPR). The UPR is a regulatory pathway that temporarily suspends protein production when misfolding of proteins occurs within the endoplastic reticulum (ER). In addition, ER chaperones that promote proper refolding become upregulated. If cellular damage cannot be resolved by these mechanisms, then the UPR can initiate apoptosis. The current study indicates that these maleimide analogues lead to UPR activation, which is predictive of the selective antiproliferative activity of the series. PMID:20472436

  16. 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

  17. Insulin demand regulates ? 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 ? cell number expands in response to an increase in insulin demand. Lineage tracing shows that new ? cells are generated from proliferation of mature, differentiated ? cells; however, the mechanism by which these mature cells sense systemic insulin demand and initiate a proliferative response remains unknown. Here, we identified the ? cell unfolded protein response (UPR), which senses insulin production, as a regulator of ? cell proliferation. Using genetic and physiologic models, we determined that among the population of ? cells, those with an active UPR are more likely to proliferate. Moreover, subthreshold endoplasmic reticulum stress (ER stress) drove insulin demand–induced ? cell proliferation, through activation of ATF6. We also confirmed that the UPR regulates proliferation of human ? cells, suggesting that therapeutic UPR modulation has potential to expand ? 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

  18. Insulin demand regulates ? 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 ? cell number expands in response to an increase in insulin demand. Lineage tracing shows that new ? cells are generated from proliferation of mature, differentiated ? cells; however, the mechanism by which these mature cells sense systemic insulin demand and initiate a proliferative response remains unknown. Here, we identified the ? cell unfolded protein response (UPR), which senses insulin production, as a regulator of ? cell proliferation. Using genetic and physiologic models, we determined that among the population of ? cells, those with an active UPR are more likely to proliferate. Moreover, subthreshold endoplasmic reticulum stress (ER stress) drove insulin demand-induced ? cell proliferation, through activation of ATF6. We also confirmed that the UPR regulates proliferation of human ? cells, suggesting that therapeutic UPR modulation has potential to expand ? 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

  19. 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 ?-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

  20. Review: Retinal degeneration: Focus on the unfolded protein response

    PubMed Central

    Gorbatyuk, Oleg

    2013-01-01

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

  1. 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

  2. Cyclosporin A induces the unfolded protein response in keratinocytes.

    PubMed

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

    2011-09-01

    Psoriasis vulgaris is a chronic inflammatory disorder of the skin, in which activation of keratinocytes and crosstalk between keratinocytes and T cells or dendritic cells are considered to be involved in the pathogenesis of psoriasis vulgaris. Cyclosporin (Cy) A, an immunomodulator, has been used for the treatment of psoriasis vulgaris, but the mechanism of its action on keratinocytes has not been well elucidated as its function on T cells is well known. Previous study indicated that the expression of the unfolded protein response (UPR) markers, GRP78/Bip and HRD1 were poorly expressed in psoriasis vulgaris. To investigate if the UPR in keratinocytes is involved in the pathogenesis of psoriasis vulgaris we assessed immunocytochemistry of normal human skin and psoriatic lesions, quantitative PCR of keratinocyte cell line (HaCaT) treated with TGF?. Moreover, to elucidate how CyA effects on the UPR in keratinocytes, we set out quantitative PCR and western blotting, HaCaT and squamous cell carcinoma cell lines (HSC-1) treated with CyA and CyA analog, cyclosporin D. Furthermore, the siRNA-mediated knockdown effect of cyclophilin (Cyp) A, Cyp B and Cyp C on HaCaT cells were also examined. As a result, the UPR was downregulated in keratinocytes from psoriatic lesions, characterized by immunocytochemical staining of GRP78/Bip, CHOP/GADD153, HRD1 and C/EBP?. TGF? induced UPR markers in HaCaT cells. CyA treatment and siRNA-mediated knockdown of Cyp B induced the UPR in HaCaT cells or HSC-1 cells. Altogether, we demonstrate that in psoriasis vulgaris CyA or reduction in Cyp B by RNA interference might induce the UPR in keratinocytes. PMID:21221615

  3. 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

  4. Review article The role of the unfolded protein response in the heart

    E-print Network

    Glembotski, Christopher

    , which requires correct folding. The bulk of cellular protein synthesis takes place on cytosolic free ri- bosomes. However, depending on the cell type, up to 35% of protein synthesis is in the rough endoplasmicReview article The role of the unfolded protein response in the heart Christopher C. Glembotski

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

    E-print Network

    Blackwell, Keith

    Integration of the Unfolded Protein and Oxidative Stress Responses through SKN-1/Nrf Kira M. Glover Protein Response (UPR) maintains homeostasis in the endoplasmic reticulum (ER) and defends against ER-established function in protection against oxidative and xenobiotic stress, we find that it also mobilizes

  6. NON-THEMATIC REVIEW The unfolded protein response during prostate

    E-print Network

    Walter, Peter

    (UPR). The UPR promotes cell survival by adjusting ER protein folding capacity but if homeostasis respond rapidly with adaptive programs to restore protein homeostasis, indicating a strong selection] re-establishes homeostasis by reducing protein translation thereby suppressing protein loading

  7. Interplay between unfolded protein response and autophagy promotes tumor drug resistance

    PubMed Central

    YAN, MING-MING; NI, JIANG-DONG; SONG, DEYE; DING, MULIANG; HUANG, JUN

    2015-01-01

    The endoplasmic reticulum (ER) is involved in the quality control of secreted protein via promoting the correct folding of nascent protein and mediating the degradation of unfolded or misfolded protein, namely ER-associated degradation. When the unfolded or misfolded proteins are abundant, the unfolded protein response (UPR) is elicited, an adaptive signaling cascade from the ER to the nucleus, which restores the homeostatic functions of the ER. Autophagy is a conserved catabolic process where cellular long-lived proteins and damaged organelles are engulfed and degraded for recycling to maintain homeostasis. The UPR and autophagy occur simultaneously and are involved in pathological processes, including tumorigenesis, chemoresistance of malignancies and neurodegeneration. Accumulative data has indicated that the UPR may induce autophagy and that autophagy is able to alleviate the UPR. However, the detailed mechanism of interplay between autophagy and UPR remains to be fully understood. The present review aimed to depict the core pathways of the two processes and to elucidate how autophagy and UPR are regulated. Moreover, the review also discusses the molecular mechanism of crosstalk between the UPR and autophagy and their roles in malignant survival and drug resistance.

  8. Plant ribosome-inactivating proteins type II induce the unfolded protein response in human cancer cells.

    PubMed

    Horrix, C; Raviv, Z; Flescher, E; Voss, C; Berger, M R

    2011-04-01

    Cytotoxic ribosome-inactivating proteins (RIPs) of type II such as ricin were investigated as anti-cancer agents, but also pose a threat as biological weapons. The molecular mechanism leading to their toxic effects is, however, not yet clear. The current paradigm, which states that the irreversible depurination of 28S rRNA results in a general translational arrest eventually leading to cell death, has been questioned. Using micro-array, qRT-PCR and Western blot, we identified the unfolded protein response (UPR), a cellular mechanism activated in response to endoplasmic reticulum stress, that is induced in HCT116 and MDA-MB-231 cells exposed to the plant type II RIPs ricin, riproximin and volkensin. Apoptosis was induced by concentrations at which translation of UPR-related genes still occurred, despite concomitant ribosomal depurination. We conclude that UPR induction represents a model that better describes the cellular effects of RIP exposure at concentrations at which selected proteins are translated despite ribosomal depurination. PMID:20844919

  9. 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

  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. 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

  12. The Unfolded Protein Response: A Pathway That Links Insulin Demand with ?-Cell Failure and Diabetes

    PubMed Central

    Scheuner, Donalyn; Kaufman, Randal J.

    2008-01-01

    The endoplasmic reticulum (ER) is the entry site into the secretory pathway for newly synthesized proteins destined for the cell surface or released into the extracellular milieu. The study of protein folding and trafficking within the ER is an extremely active area of research that has provided novel insights into many disease processes. Cells have evolved mechanisms to modulate the capacity and quality of the ER protein-folding machinery to prevent the accumulation of unfolded or misfolded proteins. These signaling pathways are collectively termed the unfolded protein response (UPR). The UPR sensors signal a transcriptional response to expand the ER folding capacity, increase degredation of malfolded proteins, and limit the rate of mRNA translation to reduce the client protein load. Recent genetic and biochemical evidence in both humans and mice supports a requirement for the UPR to preserve ER homeostasis and prevent the ?-cell failure that may be fundamental in the etiology of diabetes. Chronic or overwhelming ER stress stimuli associated with metabolic syndrome can disrupt protein folding in the ER, reduce insulin secretion, invoke oxidative stress, and activate cell death pathways. Therapeutic interventions to prevent polypeptide-misfolding, oxidative damage, and/or UPR-induced cell death have the potential to improve ?-cell function and/or survival in the treatment of diabetes. PMID:18436705

  13. The unfolded protein response is triggered following a single, unaccustomed resistance-exercise bout.

    PubMed

    Ogborn, Daniel I; McKay, Bryon R; Crane, Justin D; Parise, Gianni; Tarnopolsky, Mark A

    2014-09-15

    Endoplasmic reticulum (ER) stress results from an imbalance between the abundance of synthesized proteins and the folding capacity of the ER. In response, the unfolded protein response (UPR) attempts to restore ER function by attenuating protein synthesis and inducing chaperone expression. Resistance exercise (RE) stimulates protein synthesis; however, a postexercise accumulation of unfolded proteins may activate the UPR. Aging may impair protein folding, and the accumulation of oxidized and misfolded proteins may stimulate the UPR at rest in aged muscle. Eighteen younger (n = 9; 21 ± 3 yr) and older (n = 9; 70 ± 4 yr) untrained men completed a single, unilateral bout of RE using the knee extensors (four sets of 10 repetitions at 75% of one repetition maximum on the leg press and leg extension) to determine whether the UPR is increased in resting, aged muscle and whether RE stimulates the UPR. Muscle biopsies were taken from the nonexercised and exercised vastus lateralis at 3, 24, and 48 h postexercise. Age did not affect any of the proteins and transcripts related to the UPR. Glucose-regulated protein 78 (GRP78) and protein kinase R-like ER protein kinase (PERK) proteins were increased at 48 h postexercise, whereas inositol-requiring enzyme 1 alpha (IRE1?) was elevated at 24 h and 48 h. Despite elevated protein, GRP78 and PERK mRNA was unchanged; however, IRE1? mRNA was increased at 24 h postexercise. Activating transcription factor 6 (ATF6) mRNA increased at 24 h and 48 h, whereas ATF4, CCAAT/enhancer-binding protein homologous protein (CHOP), and growth arrest and DNA damage protein 34 mRNA were unchanged. These data suggest that RE activates specific pathways of the UPR (ATF6/IRE1?), whereas PERK/eukaryotic initiation factor 2 alpha/CHOP does not. In conclusion, acute RE results in UPR activation, irrespective of age. PMID:25009220

  14. Mitophagy and the mitochondrial unfolded protein response in neurodegeneration and bacterial infection.

    PubMed

    Pellegrino, Mark W; Haynes, Cole M

    2015-01-01

    Mitochondria are highly dynamic and structurally complex organelles that provide multiple essential metabolic functions. Mitochondrial dysfunction is associated with neurodegenerative conditions such as Parkinson's disease, as well as bacterial infection. Here, we explore the roles of mitochondrial autophagy (mitophagy) and the mitochondrial unfolded protein response (UPR(mt)) in the response to mitochondrial dysfunction, focusing in particular on recent evidence on the role of mitochondrial import efficiency in the regulation of these stress pathways and how they may interact to protect the mitochondrial pool while initiating an innate immune response to protect against bacterial pathogens. PMID:25857750

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

    PubMed Central

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

    2013-01-01

    BACKGROUND & AIMS Endoplasmic reticulum (ER) stress has been associated with development of inflammatory bowel disease. We examined the effects of ER stress–induced chaperone response and the orally active chemical chaperones tauroursodeoxycholate (TUDCA) and 4-phenylbutyrate (PBA), which facilitate protein folding and reduce ER stress, in mice with colitis. METHODS We used dextran sulfate sodium (DSS) to induce colitis in mice that do not express the transcription factor ATF6? or the protein chaperone P58IPK. We examined the effects of TUDCA and PBA in cultured intestinal epithelial cells (IECs); in wild-type, P58IPK?/?, and Atf6??/? mice with colitis; and in Il10?/? mice. RESULTS P58IPK?/? and Atf6??/? mice developed more severe colitis following administration of DSS than wild-type mice. IECs from P58IPK?/? mice had excessive ER stress, and apoptotic signaling was activated in IECs from Atf6??/? mice. Inflammatory stimuli induced ER stress signals in cultured IECs, which were reduced by incubation with TUDCA or PBA. Oral administration of either PBA or TUDCA reduced features of DSS-induced acute and chronic colitis in wild-type mice, the colitis that develops in Il10?/? mice, and DSS-induced colitis in P58IPK?/? and Atf6??/? mice. Reduced signs of colonic inflammation in these mice were associated with significantly decreased ER stress in colonic epithelial cells. CONCLUSIONS The unfolded protein response induces expression of genes that encode chaperones involved in ER protein folding; these factors prevent induction of colitis in mice. Chemical chaperones such as TUDCA and PBA alleviate different forms of colitis in mice and might be developed for treatment of inflammatory bowel diseases. PMID:23336977

  16. Estrogen receptor ? 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 ? (ER?)-positive breast and ovarian cancers are often therapy resistant. Using screening and functional validation, we identified BHPI, a potent noncompetitive small molecule ER? biomodulator that selectively blocks proliferation of drug-resistant ER?-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-ER?-regulated gene expression, BHPI is effective because it elicits sustained ER?-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-ER?: mild and transient UPR activation. In contrast, BHPI elicits massive and sustained UPR activation, converting the UPR from protective to toxic. In ER?(+) cancer cells, BHPI rapidly hyperactivates plasma membrane PLC?, 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 2? (eIF2?), 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

  17. 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.

  18. 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.

  19. Protein unfolding: Rigidity lost

    PubMed Central

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

    2002-01-01

    We relate the unfolding of a protein to its loss of structural stability or rigidity. Rigidity and flexibility are well defined concepts in mathematics and physics, with a body of theorems and algorithms that have been applied successfully to materials, allowing the constraints in a network to be related to its deformability. Here we simulate the weakening or dilution of the noncovalent bonds during protein unfolding, and identify the emergence of flexible regions as unfolding proceeds. The transition state is determined from the inflection point in the change in the number of independent bond-rotational degrees of freedom (floppy modes) of the protein as its mean atomic coordination decreases. The first derivative of the fraction of floppy modes as a function of mean coordination is similar to the fraction-folded curve for a protein as a function of denaturant concentration or temperature. The second derivative, a specific heat-like quantity, shows a peak around a mean coordination of ?r? = 2.41 for the 26 diverse proteins we have studied. As the protein denatures, it loses rigidity at the transition state, proceeds to a state where just the initial folding core remains stable, then becomes entirely denatured or flexible. This universal behavior for proteins of diverse architecture, including monomers and oligomers, is analogous to the rigid to floppy phase transition in network glasses. This approach provides a unifying view of the phase transitions of proteins and glasses, and identifies the mean coordination as the relevant structural variable, or reaction coordinate, along the unfolding pathway. PMID:11891336

  20. 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

  1. 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

  2. IRE1?/XBP1-mediated branch of the unfolded protein response regulates osteoclastogenesis

    PubMed Central

    Tohmonda, Takahide; Yoda, Masaki; Iwawaki, Takao; Matsumoto, Morio; Nakamura, Masaya; Mikoshiba, Katsuhiko; Toyama, Yoshiaki; Horiuchi, Keisuke

    2015-01-01

    The unfolded protein response (UPR) is a cellular adaptive mechanism that is activated in response to the accumulation of unfolded proteins in the endoplasmic reticulum. The inositol-requiring protein-1?/X-box–binding protein–mediated (IRE1?/XBP1-mediated) branch of the UPR is highly conserved and has also been shown to regulate various cell-fate decisions. Herein, we have demonstrated a crucial role for the IRE?/XBP1-mediated arm of the UPR in osteoclast differentiation. Using murine models, we found that the conditional abrogation of IRE1? in bone marrow cells increases bone mass as the result of defective osteoclastic bone resorption. In osteoclast precursors, IRE1? was transiently activated during osteoclastogenesis, and suppression of the IRE1?/XBP1 pathway in these cells substantially inhibited the formation of multinucleated osteoclasts in vitro. We determined that XBP1 directly binds the promoter and induces transcription of the gene encoding the master regulator of osteoclastogenesis nuclear factor of activated T cells cytoplasmic 1 (NFATc1). Moreover, activation of IRE1? was partially dependent on Ca2+ oscillation mediated by inositol 1,4,5-trisphosphate receptors 2 and 3 (ITPR2 and ITPR3) in the endoplasmic reticulum, as pharmacological inhibition or deletion of these receptors markedly decreased Xbp1 mRNA processing. The present study thus reveals an intracellular pathway that integrates the UPR and osteoclast differentiation through activation of the IRE1?/XBP1 pathway. PMID:26193638

  3. Effect of modulation of unfolded protein response pathway on dengue virus infection.

    PubMed

    Diwaker, Drishya; Mishra, Kamla Prasad; Ganju, Lilly

    2015-12-01

    The unfolded protein response (UPR) is a cascade of events that helps restoring cellular homeostasis under stressful conditions. It is activated when there is an imbalance in the protein load and protein folding capacity of the endoplasmic reticulum (ER) as a result of an increase in the naïve, unfolded, or misfolded protein content of the cell. Dengue virus (DENV) utilizes the host machinery to synthesize viral proteins and replicates in the cell. During DENV infection, up-regulation of viral proteins increases the protein pool of the cell, resulting in the induction of UPR pathway. In this study, we have tried to understand the consequence of UPR induction during DENV infection in human monocytic cells. To fulfill this objective, we have used VER-155008 (VER), a known inhibitor of the 78 kDa glucose-regulated protein (GRP78), which is the master regulator of the UPR pathway. After VER treatment, cells were infected with DENV, and the induction of the UPR elements and their downstream activation was studied by western blotting and RT-PCR analysis. Interestingly, inhibition of GRP78 via VER treatment led to the decreased expression of DENV envelope protein through the activation of the UPR elements, protein kinase-like ER resident kinase, activating transcription factor 6, and inositol-requiring enzyme 1 (IRE1), and then led to the activation of innate immune factors such as double-stranded RNA-activated protein kinase (PKR), interferon regulated factor 3 (IRF3), nuclear factor-?B (NF-?B) and interleukin 1? (IL-1?). This strategy may be used to decrease viral infection transiently. Thus UPR elements could be important therapeutic targets for decreasing DENV multiplication. PMID:26515795

  4. 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 IRE1? and PERK. While S-nitrosylation of IRE1? inhibited its ribonuclease activity, S-nitrosylation of PERK activated its kinase activity and downstream phosphorylation/inactivation or eIF2?. Site-directed mutagenesis of IRE1?(Cys931) prevented S-nitrosylation and inhibition of its ribonuclease activity, indicating that Cys931 is the predominant site of S-nitrosylation. Importantly, cells overexpressing mutant IRE1?(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

  5. Causal role of oxidative stress in unfolded protein response development in the hyperthyroid state.

    PubMed

    Videla, Luis A; Fernández, Virginia; Cornejo, Pamela; Vargas, Romina; Carrasco, Juan; Fernández, Javier; Varela, Nelson

    2015-12-01

    l-3,3',5-Triiodothyronine (T3)-induced liver oxidative stress underlies significant protein oxidation, which may trigger the unfolded protein response (UPR). Administration of daily doses of 0.1mg T3 for three consecutive days significantly increased the rectal temperature of rats and liver O2 consumption rate, with higher protein carbonyl and 8-isoprostane levels, glutathione depletion, and absence of morphological changes in liver parenchyma. Concomitantly, liver protein kinase RNA-like endoplasmic reticulum (ER) kinase and eukaryotic translation initiator factor 2? were phosphorylated in T3-treated rats compared to controls, with increased protein levels of binding immunoglobulin protein and activating transcription factor 4. In addition, higher mRNA levels of C/EBP homologous protein, growth arrest and DNA damage 34, protein disulfide isomerase, and ER oxidoreductin 1? were observed, changes that were suppressed by N-acetylcysteine (0.5g/kg) given before each dose of T3. In conclusion, T3-induced liver oxidative stress involving higher protein oxidation status has a causal role in UPR development, a response that is aimed to alleviate ER stress and promote cell survival. PMID:26434419

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

    PubMed

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

    2014-07-01

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

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

    PubMed

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

    2013-10-01

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

  8. 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

  9. 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

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

    PubMed Central

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

    2013-01-01

    The endoplasmic reticulum (ER) is a dynamic intracellular organelle with multiple functions essential for cellular homeostasis, development, and stress responsiveness. In response to cellular stress, a well-established signaling cascade, the unfolded protein response (UPR), is activated. This intricate mechanism is an important means of reestablishing cellular homeostasis and alleviating the inciting stress. Now, emerging evidence has demonstrated that the UPR influences cellular metabolism through diverse mechanisms, including calcium and lipid transfer, raising the prospect of involvement of these processes in the pathogenesis of disease, including neurodegeneration, cancer, diabetes mellitus and cardiovascular disease. Here, we review the distinct functions of the ER and UPR from a metabolic point of view, highlighting their association with prevalent pathologies. PMID:23317820

  11. Surveillance-activated defenses block the ROS-induced mitochondrial unfolded protein response.

    PubMed

    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 (UPR(mt)) to a nuclear chaperone promoter. We used the ROS generating complex I inhibitor paraquat to induce the UPR(mt), 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 UPR(mt). Activation of the UPR(mt), 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 UPR(mt), suggesting that surveillance-activated defenses specifically inhibit the UPR(mt) 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

  12. 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

  13. Stressed-out B cells? Plasma-cell differentiation and the unfolded protein response.

    PubMed

    Gass, Jennifer N; Gunn, Kathryn E; Sriburi, Rungtawan; Brewer, Joseph W

    2004-01-01

    Plasma cells operate as factories where large quantities of Ig heavy and light chains are made and assembled into functional antibodies. The finished products are shipped out with impressive efficiency. A major component of the machinery necessary for high-rate antibody secretion is an elaborate network of endoplasmic reticulum (ER), the site of antibody biosynthesis. Recent discoveries have provided insights into how this expansive secretory machinery is built, equipped and maintained. The unfolded protein response (UPR) pathway, a stress-induced signaling cascade emanating from the ER, regulates the expression and activity of X-box binding protein 1, a transcription factor required for plasma-cell development. The UPR pathway therefore senses conditions in the ER--the very compartment where antibodies are formed--and directs events required for humoral immunity. PMID:14698280

  14. Role of Oxidative Stress in Modulating Unfolded Protein Response Activity in Chronic Myeloid Leukemia Cell Line

    PubMed Central

    Bazi, Ali; Keramati, Mohammad Reza; Gholamin, Mehran

    2016-01-01

    Background: Recently, it has been revealed that tyrosine kinase inhibitors (TKIs) act through inducing both oxidative and endoplasmic reticulum (ER) stress in chronic myeloid leukemia cells. However, ER stress signaling triggers both apoptotic and survival processes within cells. Nevertheless, mechanisms by which TKIs avoid the pro-survival effects are not clear. The aim of this study was to evaluate the potential role of oxidative stress in activity of unfolded protein response (UPR) survival pathway within K562 cell line. Methods: The expression of UPR survival target genes, Xbp1, and Grp94 (glucose requiring protein 94) was studied in single and combined exposure to oxidative and ER stress in K562 cell line by quantitative and qualitative PCR. Results: The expression of UPR-related survival gene Grp94 was hampered by exposing to oxidative stress in cell induced with ER stress. Conclusion: Interaction of oxidative and ER stress may role as a mediator influencing UPR signaling activity. PMID:26432458

  15. Gene expression profiling in tibial muscular dystrophy reveals unfolded protein response and altered autophagy.

    PubMed

    Screen, Mark; Raheem, Olayinka; Holmlund-Hampf, Jeanette; Jonson, Per Harald; Huovinen, Sanna; Hackman, Peter; Udd, Bjarne

    2014-01-01

    Tibial muscular dystrophy (TMD) is a late onset, autosomal dominant distal myopathy that results from mutations in the two last domains of titin. The cascade of molecular events leading from the causative Titin mutations to the preterm death of muscle cells in TMD is largely unknown. In this study we examined the mRNA and protein changes associated with the myopathology of TMD. To identify these components we performed gene expression profiling using muscle biopsies from TMD patients and healthy controls. The profiling results were confirmed through quantitative real-time PCR and protein level analysis. One of the pathways identified was activation of endoplasmic reticulum (ER) stress response. ER stress activates the unfolded protein response (UPR) pathway. UPR activation was supported by elevation of the marker genes HSPA5, ERN1 and the UPR specific XBP1 splice form. However, UPR activation appears to be insufficient to correct the protein abnormalities causing its activation because degenerative TMD muscle fibres show an increase in ubiquitinated protein inclusions. Abnormalities of VCP-associated degradation pathways are also suggested by the presence of proteolytic VCP fragments in western blotting, and VCP's accumulation within rimmed vacuoles in TMD muscle fibres together with p62 and LC3B positive autophagosomes. Thus, pathways controlling turnover and degradation, including autophagy, are distorted and lead to degeneration and loss of muscle fibres. PMID:24618559

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

    PubMed Central

    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

  17. 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-eIF2?, ATF4 and CHOP. The expression of these proteins is altered both at transcriptional and translational levels. Pretreatment with chemical chaperon, 4-phenylbutyric acid (PBA) attenuated the ATO-induced activation in UPR signaling and afforded protection against ATO-induced disruption of macrophage functions. This treatment also reduced ATO-mediated reactive oxygen species (ROS) generation. Interestingly, treatment with antioxidant N-acetylcysteine (NAC) prior to ATO exposure, not only reduced ROS production and UPR signaling but also improved macrophage functions. These data demonstrate that UPR signaling and ROS generation are interdependent and are involved in the arsenic-induced pathobiology of macrophage. These data also provide a novel strategy to block the ATO-dependent impairment in innate immune responses. - 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.

  18. A Dynamic Unfolded Protein Response Contributes to the Control of Cortical Neurogenesis.

    PubMed

    Laguesse, Sophie; Creppe, Catherine; Nedialkova, Danny D; Prévot, Pierre-Paul; Borgs, Laurence; Huysseune, Sandra; Franco, Bénédicte; Duysens, Guérin; Krusy, Nathalie; Lee, Gabsang; Thelen, Nicolas; Thiry, Marc; Close, Pierre; Chariot, Alain; Malgrange, Brigitte; Leidel, Sebastian A; Godin, Juliette D; Nguyen, Laurent

    2015-12-01

    The cerebral cortex contains layers of neurons sequentially generated by distinct lineage-related progenitors. At the onset of corticogenesis, the first-born progenitors are apical progenitors (APs), whose asymmetric division gives birth directly to neurons. Later, they switch to indirect neurogenesis by generating intermediate progenitors (IPs), which give rise to projection neurons of all cortical layers. While a direct lineage relationship between APs and IPs has been established, the molecular mechanism that controls their transition remains elusive. Here we show that interfering with codon translation speed triggers ER stress and the unfolded protein response (UPR), further impairing the generation of IPs and leading to microcephaly. Moreover, we demonstrate that a progressive downregulation of UPR in cortical progenitors acts as a physiological signal to amplify IPs and promotes indirect neurogenesis. Thus, our findings reveal a contribution of UPR to cell fate acquisition during mammalian brain development. PMID:26651292

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

    PubMed

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

    2012-10-26

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

  20. 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

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

    PubMed

    Hempstead, Andrew D; Isberg, Ralph R

    2015-12-01

    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

  2. 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

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

    PubMed Central

    Lenna, Stefania; Trojanowska, Maria

    2013-01-01

    Purpose of review To review the present knowledge of the role of endoplasmic reticulum (ER) stress and unfolded protein response (UPR) in the pathogenesis of fibrotic diseases. Recent findings ER stress and UPR occur in a number of diseases associated with organ fibrosis; however, the contribution of these pathways to the fibrotic process has not been systematically investigated. Current studies suggest that prolonged ER stress may lead to fibrosis through activation of CCAAT/enhancer-binding homologous protein-mediated apoptosis, followed by an inflammatory response and release of profibrotic cytokines. A direct profibrotic role of UPR mediators in activation of TGF-? signaling has been shown in lung fibroblasts. In addition, activation of ER stress and UPR pathways in immune cells contributes to increased production of proinflammatory cytokines. Summary Although limited in scope, current studies strongly suggest that ER stress and UPR may play an important role during development of fibrosis. Further studies are warranted to gain additional insights into the relationship between these processes. PMID:22918530

  4. 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 link the UPR to chemoresistance possibly via enhanced metabolism. Given the role of the UPR in the balance between cell survival and apoptosis, targeting the UPR and/or controlling metabolic activity may prove beneficial for malignant glioma therapeutics. PMID:24039668

  5. 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

  6. 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

  7. 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, pIRE1? 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

  8. 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

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

    PubMed

    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, pIRE1? 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

  10. 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 LC3?, 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 (LC3? 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 (LC3? 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 LC3? in HBVNeg and HBVPos revealed that majority of HBV-infected hepatocytes display strong positive staining for LC3?. 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 apoptosis, autophagy, and UPR. CONCLUSION: The results of this study indicate that HCV and HBV infection activates apoptosis, autophagy and UPR, but slightly differently by each virus. Further studies are warranted to elucidate the interconnections between these pathways in relation to pathology of HCV and HBV in the liver tissue. PMID:26715805

  11. Targeting HSP90 and monoclonal protein trafficking modulates the unfolded protein response, chaperone regulation and apoptosis in myeloma cells

    PubMed Central

    Born, E J; Hartman, S V; Holstein, S A

    2013-01-01

    Multiple myeloma is characterized by the production of substantial quantities of monoclonal protein. We have previously demonstrated that select inhibitors of the isoprenoid biosynthetic pathway (IBP) induce apoptosis of myeloma cells via inhibition of Rab geranylgeranylation, leading to disruption of monoclonal protein trafficking and induction of the unfolded protein response (UPR) pathway. Heat-shock protein 90 (HSP90) inhibitors disrupt protein folding and are currently under clinical investigation in myeloma. The effects of combining IBP and HSP90 inhibitors on cell death, monoclonal protein trafficking, the UPR and chaperone regulation were investigated in monoclonal protein-producing cells. An enhanced induction of cell death was observed following treatment with IBP and HSP90 inhibitors, which occurred through both ER stress and non-ER stress pathways. The HSP90 inhibitor 17-AAG abrogated the effects of the IBP inhibitors on intracellular monoclonal protein levels and localization as well as induction of the UPR in myeloma cells. Disparate effects on chaperone expression were observed in myeloma vs amyloid light chain cells. Here we demonstrate that the novel strategy of targeting MP trafficking in concert with HSP90 enhances myeloma cell death via a complex modulation of ER stress, UPR, and cell death pathways. PMID:24317089

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

    PubMed Central

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

    2014-01-01

    Abstract Aims: Age-related macular degeneration (AMD), a major cause of legal blindness in the elderly, is associated with genetic and environmental risk factors, such as cigarette smoking. Recent evidence shows that cigarette smoke (CS) that contains high levels of potent oxidants preferably targets retinal pigment epithelium (RPE) leading to oxidative damage and apoptosis; however, the mechanisms are poorly understood. The present study aimed to investigate the role of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in CS-related RPE apoptosis. Results: ER stress and proapoptotic gene C/EBP homologous protein (CHOP) were induced in the RPE/choroid complex from mice exposed to CS for 2 weeks and in human RPE cells treated with hydroquinone, a potent oxidant found at high concentrations in CS. Suppressing ER stress or inhibiting CHOP activation by pharmacological chaperones or genetic approaches attenuated hydroquinone-induced RPE cell apoptosis. In contrast to enhanced CHOP activation, protein level of active X-box binding protein 1 (XBP1), a major regulator of the adaptive UPR, was reduced in hydroquinone-treated cells. Conditional knockout of XBP1 gene in the RPE resulted in caspase-12 activation, increased CHOP expression, and decreased antiapoptotic gene Bcl-2. Furthermore, XBP1-deficient RPE cells are more sensitive to oxidative damage induced by hydroquinone or NaIO3, a CS-unrelated chemical oxidant. Conversely, overexpressing XBP1 protected RPE cells and attenuated oxidative stress-induced RPE apoptosis. Innovation and Conclusion: These findings provide strong evidence suggesting an important role of ER stress and the UPR in CS-related oxidative injury of RPE cells. Thus, the modulation of the UPR signaling may provide a promising target for the treatment of AMD. Antioxid. Redox Signal. 20, 2091–2106. PMID:24053669

  13. 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

  14. 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.

  15. Integration of Hippo signalling and the unfolded protein response to restrain liver overgrowth and tumorigenesis.

    PubMed

    Wu, Hongtan; Wei, Luyao; Fan, Fuqin; Ji, Suyuan; Zhang, Shihao; Geng, Jing; Hong, Lixin; Fan, Xin; Chen, Qinghua; Tian, Jing; Jiang, Mingting; Sun, Xiufeng; Jin, Changnan; Yin, Zhen-Yu; Liu, Qingxu; Zhang, Jinjia; Qin, Funiu; Lin, Kwang-Huei; Yu, Jau-Song; Deng, Xianming; Wang, Hong-Rui; Zhao, Bin; Johnson, Randy L; Chen, Lanfen; Zhou, Dawang

    2015-01-01

    The role of the unfolded protein response (UPR) in tissue homeostasis remains largely unknown. Here we find that loss of Mst1/2, the mammalian Hippo orthologues, or their regulator WW45, leads to a remarkably enlarged endoplasmic reticulum (ER) size-associated UPR. Intriguingly, attenuation of the UPR by tauroursodeoxycholic acid (TUDCA) diminishes Mst1/2 mutant-driven liver overgrowth and tumorigenesis by promoting nuclear exit and degradation of Hippo downstream effector Yap. Yap is required for UPR activity and ER expansion to alleviate ER stress. During the adaptive stage of the UPR, PERK kinase-eIF2? axis activates Yap, while prolonged ER stress-induced Hippo signalling triggers assembly of the GADD34/PP1 complex in a negative feedback loop to inhibit Yap and promote apoptosis. Significantly, the deregulation of UPR signals associated with Yap activation is found in a substantial fraction of human hepatocellular carcinoma (HCC). Thus, we conclude Yap integrates Hippo and UPR signalling to control liver size and tumorigenesis. PMID:25695629

  16. Maintenance of Pdx1 mRNA Translation in Islet ?-Cells During the Unfolded Protein Response

    PubMed Central

    Templin, Andrew T.; Maier, Bernhard; Tersey, Sarah A.; Hatanaka, Masayuki

    2014-01-01

    In type 1 diabetes, proinflammatory cytokines secreted by infiltrating immune cells activate the unfolded protein response (UPR) in islet ?-cells, which leads to attenuation of global mRNA translation. Under such conditions, privileged mRNAs required for adaptation to the prevailing stress are maintained in an actively translated state. Pdx1 is a ?-cell transcription factor that is required for the adaptive UPR, but it is not known how translation of its mRNA is maintained under these conditions. To study translation, we established conditions in vitro with MIN6 cells and mouse islets and a mixture of proinflammatory cytokines (IL-1?, TNF-?, and IFN-?) that mimicked the UPR conditions seen in type 1 diabetes. Cell extracts were then subjected to polyribosome profiling to monitor changes to mRNA occupancy by ribosomes. Similar to other privileged mRNAs (Atf4 and Chop), Pdx1 mRNA remained partitioned in actively translating polyribosomes under the UPR, whereas the mRNA encoding a proinsulin-processing enzyme (Cpe) and others partitioned into inactively translating monoribosomes. Bicistronic luciferase reporter analyses revealed that the distal portion of the 5?-untranslated region of mouse Pdx1 (between bp ?105 to ?280) contained elements that promoted translation under both normal and UPR conditions, and this region exhibited conserved sequences and secondary structure similar to those of other known internal ribosome entry sites. Our findings suggest that Pdx1 protein levels are maintained in the setting of the UPR, in part, through elements in the 5?-untranslated region that confer privileged mRNA translation in a 5?-7-methylguanylate cap–independent manner. PMID:25251389

  17. 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 1? (IRE1?) 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 IRE1?-TRAF2-ASK1 complex and FADD. In vivo overexpression of a FADD dominant negative (FADD-DN) mutant lacking the death effector domain disrupted diabetes-induced IRE1? 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 IRE1? 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 IRE1? 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

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

    PubMed

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

    2014-07-01

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

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

    PubMed

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

    2014-05-27

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

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

    PubMed Central

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

    2014-01-01

    The unfolded protein response (UPR) pathway, a stress-induced signaling cascade emanating from the endoplasmic reticulum (ER), regulates the expression and activity of molecules including BiP (HSPA5), IRE1 (ERN1), Blimp-1 (PRDM1), and X-box binding protein 1 (XBP1). These molecules are required for terminal differentiation of B cells into plasma cells and expressed at high levels in plasma cell-derived multiple myeloma. Although these molecules have no known role at early stages of B-cell development, here we show that their expression transiently peaks at the pre–B-cell receptor checkpoint. Inducible, Cre-mediated deletion of Hspa5, Prdm1, and Xbp1 consistently induces cellular stress and cell death in normal pre-B cells and in pre–B-cell acute lymphoblastic leukemia (ALL) driven by BCR-ABL1- and NRASG12D oncogenes. Mechanistically, expression and activity of the UPR downstream effector XBP1 is regulated positively by STAT5 and negatively by the B-cell–specific transcriptional repressors BACH2 and BCL6. In two clinical trials for children and adults with ALL, high XBP1 mRNA levels at the time of diagnosis predicted poor outcome. A small molecule inhibitor of ERN1-mediated XBP1 activation induced selective cell death of patient-derived pre-B ALL cells in vitro and significantly prolonged survival of transplant recipient mice in vivo. Collectively, these studies reveal that pre-B ALL cells are uniquely vulnerable to ER stress and identify the UPR pathway and its downstream effector XBP1 as novel therapeutic targets to overcome drug resistance in pre-B ALL. PMID:24821775

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

    PubMed

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

    2014-02-01

    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

  2. The flavonoid tangeretin activates the unfolded protein response and synergizes with imatinib in the erythroleukemia cell line K562.

    PubMed

    Lust, Sofie; Vanhoecke, Barbara; Van Gele, Mireille; Philippé, Jan; Bracke, Marc; Offner, Fritz

    2010-06-01

    We explored the mechanism of cell death of the polymethoxyflavone tangeretin (TAN) in K562 breakpoint cluster region-abelson murine leukemia (Bcr-Abl+) cells. Flow cytometric analysis showed that TAN arrested the cells in the G(2)/M phase and stimulated an accumulation of the cells in the sub-G(0) phase. TAN-induced cell death was evidenced by poly(ADP)-ribose polymerase cleavage, DNA laddering fragmentation, activation of the caspase cascade and downregulation of the antiapoptotic proteins Mcl-1 and Bcl-x(L). Pretreatment with the pancaspase inhibitor Z-VAD-FMK_blocked caspase activation and cell cycle arrest but did not inhibit apoptosis which suggest that other cell killing mechanisms like endoplasmic reticulum (ER)-associated cell death pathways could be involved. We demonstrated that TAN-induced apoptosis was preceded by a rapid activation of the proapoptotic arm of the unfolded protein response, namely PKR-like ER kinase. This was accompanied by enhanced levels of glucose-regulated protein of 78 kDa and of spliced X-box binding protein 1. Furthermore, TAN sensitized K562 cells to the cell killing effects of imatinib via an apoptotic mechanism. In conclusion, our results suggest that TAN is able to induce apoptosis in Bcr-Abl+ cells via cell cycle arrest and the induction of the unfolded protein response, and has synergistic cytotoxicity with imatinib. PMID:20077418

  3. Multiple ways to die: delineation of the unfolded protein response and apoptosis induced by Surfactant Protein C BRICHOS mutants.

    PubMed

    Maguire, Jean Ann; Mulugeta, Surafel; Beers, Michael F

    2012-01-01

    Epithelial cell dysfunction is now recognized as an important mechanism in the pathogenesis of interstitial lung diseases. Surfactant Protein C (SP-C), an alveolar type II cell specific protein, has contributed to this concept with the observation that heterozygous expression of SFTPC gene mutations are associated with chronic interstitial lung disease. We have shown that transient expression of aggregation prone mutant SP-C isoforms (SP-C BRICHOS) destabilize ER quality control mechanisms resulting in the intracellular accumulation of aggregating propeptide, inhibition of the ubiquitin/proteasome system, and activation of apoptosis. The goal of the present study was to define signaling pathways linking the unfolded protein response (UPR) and subsequent ER stress with intrinsic apoptosis events observed following mutant SP-C expression. In vitro expression of the SP-C BRICHOS mutant, SP-C(?exon4), was used as a model system. Here we show stimulation of a broad ER stress response in both transfected A549 and HEK293 cells with activation of all 3 canonical sensing pathways, IRE1/XBP-1, ATF6, and PERK/eIF2?. SP-C(?exon4) expression also resulted in activation of caspase 3, but failed to stimulate expression of the apoptosis mediating transcription factors ATF4/CHOP. However, inhibition of either caspase 4 or c-jun kinase (JNK) each blocked caspase 3 mediated cell death. Taken together, these results suggest that expression of SP-C BRICHOS mutants induce apoptosis through multiple UPR signaling pathways, and provide new therapeutic targets for the amelioration of ER stress induced cytotoxicity observed in fibrotic lung remodeling. PMID:22016030

  4. The Unfolded Protein Response Is Necessary but Not Sufficient to Compensate for Defects in Disulfide Isomerization*S?

    PubMed Central

    Kim, Jai-Hyun; Zhao, Yinsuo; Pan, Xuewen; He, Xiangwei; Gilbert, Hiram F.

    2009-01-01

    Pdi1p (protein-disulfide isomerase) is a folding assistant of the endoplasmic reticulum (ER) that catalyzes disulfide formation and the isomerization of incorrect disulfides. Its disulfide forming activity is its essential function in Saccharomyces cerevisiae. A truncation mutant (Pdi1a?) that is competent in disulfide formation but deficient in catalyzing isomerization has only a small effect on growth, although the maturation of isomerase-requiring substrates (carboxypeptidase Y) is impaired (Xiao, R., Wilkinson, B., Solovyov, A., Winther, J. R., Holmgren, A., Lundstrom-Ljung, J., and Gilbert, H. F. (2004) J. Biol. Chem. 279, 49780–49786). We show here that there are multiple ways to compensate for defects in disulfide formation and isomerization in the ER. Genes of the unfolded protein response are induced, and deletions of the nonessential IRE1 or HAC1 genes are synthetically lethal. Diploid synthetic lethality analysis by microarray (dSLAM) using PDIa? and a temperature-sensitive mutant of PDIa? as query mutations reveals a group of 130 synthetically lethal genes. Only 10 of these correspond to genes clearly associated with the unfolded protein response. More than half are involved in vesicle traffic, not only out of and into the ER but anterograde and retrograde traffic from most cellular compartments. This suggests that defects in protein maturation in one intracellular compartment may be compensated for by adjusting vesicular traffic patterns throughout the cell. PMID:19233841

  5. 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 plant immunity. PMID:22359644

  6. Activation of unfolded protein response pathway during infectious salmon anemia virus (ISAV) infection in vitro an in vivo.

    PubMed

    Kavaliauskis, Arturas; Arnemo, Marianne; Rishovd, Anne-Lise; Gjøen, Tor

    2016-01-01

    Infectious salmon anemia virus (ISAV) is a salmon pathogen causing serious outbreaks in fish farms world-wide. There is currently no effective commercially available vaccine and there is a need for better understanding of host pathogen interactions with this virus. Various strains can cause both acute and persistent infections and therefore establish a balance with the host immune responses. We have studied host responses to this infection by analyzing the main branches of the unfolded protein response (UPR) in salmon cells in vitro and in tissues from infected fish to gain a better understanding of virus-host interactions. ISAV induce the main symptoms and signaling pathways of UPR (ATF6, PERK and IRE1) without inducing translational attenuation. This may be due to concomitant induction of an important negative feedback loop via the phosphatase regulator GADD34. The host cells can therefore respond with translation of cytokine and antiviral proteins to curb or control infection. PMID:26303456

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

    PubMed Central

    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.

    2015-01-01

    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 eIF2? 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

  8. Induction of the unfolded protein response and cell death pathway in Alzheimer's disease, but not in aged Tg2576 mice

    PubMed Central

    Lee, Jin Hwan; Won, Sun Mi; Suh, Jaehong; Son, Sun Joo; Moon, Gyeong Joon; Park, Ui-Jin

    2010-01-01

    The endoplasmic reticulum (ER) stress results from disrupted protein folding triggered by protein mutation or oxidation, reduced proteasome activity, and altered Ca2+ homeostasis. ER stress is accompanied by activation of the unfolded protein response (UPR) and cell death pathway. We examined if the UPR and cell death pathway would be activated in Alzheimer's disease (AD). RT-PCR experiments revealed increased splicing of X-box binding protein-1 (XBP-1), an UPR transcription factor, in AD compared with age-matched control. Among target genes of XBP-1, expression of protein disulfide isomerase (PDI), but not glucose-regulated protein 78 (GRP78), was increased in AD, suggesting disturbed activation of the UPR in AD. C/EBP homologous protein (CHOP), caspase-3, caspase-4, and caspase-12, downstream mediators of cell death pathway, were activated in AD. Neither the UPR nor cell death pathway was induced in aged Tg2576 mice, a transgenic mouse model of Alzheimer's disease that reveals both plaque pathology and some cognitive deficits. The present study suggests that disturbed induction of the UPR and activation of the pro-apoptotic proteins contribute to neuropathological process in AD irrespective of amyloid ? and senile plaque. PMID:20368688

  9. Neurodegeneration and Unfolded-Protein Response in Mice Expressing a Membrane-Tethered Flexible Tail of PrP

    PubMed Central

    Dametto, Paolo; Lakkaraju, Asvin K. K.; Bridel, Claire; Villiger, Lukas; O’Connor, Tracy; Herrmann, Uli S.; Pelczar, Pawel; Rülicke, Thomas; McHugh, Donal; Adili, Arlind; Aguzzi, Adriano

    2015-01-01

    The cellular prion protein (PrPC) consists of a flexible N-terminal tail (FT, aa 23–128) hinged to a membrane-anchored globular domain (GD, aa 129–231). Ligation of the GD with antibodies induces rapid neurodegeneration, which is prevented by deletion or functional inactivation of the FT. Therefore, the FT is an allosteric effector of neurotoxicity. To explore its mechanism of action, we generated transgenic mice expressing the FT fused to a GPI anchor, but lacking the GD (PrP?141–225, or “FTgpi”). Here we report that FTgpi mice develop a progressive, inexorably lethal neurodegeneration morphologically and biochemically similar to that triggered by anti-GD antibodies. FTgpi was mostly retained in the endoplasmic reticulum, where it triggered a conspicuous unfolded protein response specifically activating the PERK pathway leading to phosphorylation of eIF2? and upregulation of CHOP ultimately leading to neurodegeration similar to what was observed in prion infection. PMID:25658480

  10. Crosstalk between the Unfolded Protein Response and NF-?B-Mediated Inflammation in the Progression of Chronic Kidney Disease

    PubMed Central

    Cruz, Gaile L.; Dickhout, Jeffrey G.

    2015-01-01

    The chronic inflammatory response is emerging as an important therapeutic target in progressive chronic kidney disease. A key transcription factor in the induction of chronic inflammation is NF-?B. Recent studies have demonstrated that sustained activation of the unfolded protein response (UPR) can initiate this NF-?B signaling phenomenon and thereby induce chronic kidney disease progression. A key factor influencing chronic kidney disease progression is proteinuria and this condition has now been demonstrated to induce sustained UPR activation. This review details the crosstalk between the UPR and NF-?B pathways as pertinent to chronic kidney disease. We present potential tools to study this phenomenon as well as potential therapeutics that are emerging to regulate the UPR. These therapeutics may prevent inflammation specifically induced in the kidney due to proteinuria-induced sustained UPR activation. PMID:25977931

  11. 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

  12. Limitation of individual folding resources in the ER leads to outcomes distinct from the unfolded protein response

    PubMed Central

    Eletto, Davide; Maganty, Avinash; Eletto, Daniela; Dersh, Devin; Makarewich, Catherine; Biswas, Chhanda; Paton, James C.; Paton, Adrienne W.; Doroudgar, Shirin; Glembotski, Christopher C.; Argon, Yair

    2012-01-01

    Summary ER stress leads to upregulation of multiple folding and quality control components, known as the unfolded protein response (UPR). Glucose Regulated Protein 78 (GRP78) (also known as binding immunoglobulin protein, BiP, and HSPA5) and GRP94 are often upregulated coordinately as part of this homeostatic response. Given that endoplasmic reticulum (ER) chaperones have distinct sets of clients, we asked how cells respond to ablation of individual chaperones. The cellular responses to silencing BiP, GRP94, HSP47, PDIA6 and OS-9, were distinct. When BiP was silenced, a widespread UPR was observed, but when GRP94 was either inhibited or depleted by RNA interference (RNAi), the expression of only some genes was induced, notably those encoding BiP and protein disulfide isomerase A6 (PDIA6). Silencing of HSP47 or OS-9 did not lead to any compensatory induction of other genes. The selective response to GRP94 depletion was distinct from a typical ER stress response, both because other UPR target genes were not affected and because the canonical UPR signaling branches were not activated. The response to silencing of GRP94 did not preclude further UPR induction when chemical stress was imposed. Importantly, re-expression of wild-type GRP94 in the silenced cells prevented the upregulation of BiP and PDIA6, whereas re-expression of an ATPase-deficient GRP94 mutant did not, indicating that cells monitor the activity state of GRP94. These findings suggest that cells are able to distinguish among folding resources and generate distinct responses. PMID:22854046

  13. MicroRNA-30c-2* limits expression of proadaptive factor XBP1 in the unfolded protein response

    PubMed Central

    Byrd, Andrew E.; Aragon, Ileana V.

    2012-01-01

    Stress in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), a multifaceted signaling system coordinating translational control and gene transcription to promote cellular adaptation and survival. Microribonucleic acids (RNAs; miRNAs), single-stranded RNAs that typically function as posttranscriptional modulators of gene activity, have been shown to inhibit translation of certain secretory pathway proteins during the UPR. However, it remains unclear whether miRNAs regulate UPR signaling effectors directly. In this paper, we report that a star strand miRNA, miR-30c-2* (recently designated miR-30c-2-3p), is induced by the protein kinase RNA activated–like ER kinase (PERK) pathway of the UPR and governs expression of XBP1 (X-box binding protein 1), a key transcription factor that augments secretory capacity and promotes cell survival in the adaptive UPR. These data provide the first link between an miRNA and direct regulation of the ER stress response and reveal a novel molecular mechanism by which the PERK pathway, via miR-30c-2*, influences the scale of XBP1-mediated gene expression and cell fate in the UPR. PMID:22431749

  14. 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 3?, 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

  15. Vitiligo-inducing phenols activate the unfolded protein response in melanocytes resulting in upregulation of IL6 and IL8.

    PubMed

    Toosi, Siavash; Orlow, Seth J; Manga, Prashiela

    2012-11-01

    Vitiligo is characterized by depigmented skin patches caused by loss of epidermal melanocytes. Oxidative stress may have a role in vitiligo onset, while autoimmunity contributes to disease progression. In this study, we sought to identify mechanisms that link disease triggers and spreading of lesions. A hallmark of melanocytes at the periphery of vitiligo lesions is dilation of the endoplasmic reticulum (ER). We hypothesized that oxidative stress results in redox disruptions that extend to the ER, causing accumulation of misfolded peptides, which activates the unfolded protein response (UPR). We used 4-tertiary butyl phenol and monobenzyl ether of hydroquinone, known triggers of vitiligo. We show that expression of key UPR components, including the transcription factor X-box-binding protein 1 (XBP1), is increased following exposure of melanocytes to phenols. XBP1 activation increases production of immune mediators IL6 and IL8. Co-treatment with XBP1 inhibitors reduced IL6 and IL8 production induced by phenols, while overexpression of XBP1 alone increased their expression. Thus, melanocytes themselves produce cytokines associated with activation of an immune response following exposure to chemical triggers of vitiligo. These results expand our understanding of the mechanisms underlying melanocyte loss in vitiligo and pathways linking environmental stressors and autoimmunity. PMID:22696056

  16. 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-1? (IRE-1?) and the PKR-like ER Kinase (PERK)-dependent arms of the Unfolded Protein Response (UPR). The inhibition of IRE-1? 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

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

    PubMed

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

    2012-01-01

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

  18. Gamma-Tocotrienol induced Apoptosis is Associated with Unfolded Protein Response in Human Breast Cancer Cells

    PubMed Central

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

    2011-01-01

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

  19. The unfolded protein response controls induction and activation of ADAM17/TACE by severe hypoxia and ER stress.

    PubMed

    Rzymski, T; Petry, A; Kra?un, D; Rieß, F; Pike, L; Harris, A L; Görlach, A

    2012-08-01

    The family of ADAM (a disintegrin and metalloproteinase) proteins has been implicated in tumor initiation and progression. ADAM17/tumor necrosis factor-? (TNF?)-converting enzyme (TACE) has been initially recognized to release TNF? as well as its receptors (TNFRs) from the membrane. ADAM17, TNF? and TNFR have been found upregulated in cancer patients, although the underlying mechanisms remain largely unknown. As hypoxia is a hallmark of cancer that can lead to severe stress conditions accumulating in endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), we investigated the role of these stress conditions in the regulation of ADAM17 and release of TNFR1.We found that severe hypoxia induced ADAM17 expression and activity. Although hypoxia-inducible factor 1? (HIF1?) was important to maintain basal ADAM17 mRNA levels during moderate hypoxia, it was not sufficient to induce ADAM17 levels under severe hypoxia. Instead, we found that ADAM17 induction by severe hypoxia can be mimicked by ER stressors such as Thapsigargin and occurs as a consequence of the activation of the PERK/eIF2?/ATF4 and activating transcription factor 6 (ATF6) arms of UPR in several tumor cell lines. ADAM17 expression was also increased in xenografts displaying ER stress because of treatment with the vascular endothelial growth factor (VEGF) inhibitory antibody Bevacizumab. Additionally, severe hypoxia and ER stress activated ADAM17 and ectodomain shedding of TNFR1 involving mitogen-activated protein (MAP) kinases and reactive oxygen species (ROS). Collectively, these results show that ADAM17 is a novel UPR-regulated gene in response to severe hypoxia and ER stress, which is actively involved in the release of TNFR1 under these conditions. These data provide a novel link between severe hypoxic stress conditions and inflammation in the tumor environment. PMID:22105359

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

    PubMed

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

    2013-09-01

    Secretory pathway dysfunction and lipid accumulation (steatosis) are the two most common responses of hepatocytes to ethanol exposure and are major factors in the pathophysiology of alcoholic liver disease (ALD). However, the mechanisms by which ethanol elicits these cellular responses are not fully understood. Recent data indicates that activation of the unfolded protein response (UPR) in response to secretory pathway dysfunction can cause steatosis. Here, we examined the relationship between alcohol metabolism, oxidative stress, secretory pathway stress and steatosis using zebrafish larvae. We found that ethanol was immediately internalized and metabolized by larvae, such that the internal ethanol concentration in 4-day-old larvae equilibrated to 160 mM after 1 hour of exposure to 350 mM ethanol, with an average ethanol metabolism rate of 56 ?mol/larva/hour over 32 hours. Blocking alcohol dehydrogenase 1 (Adh1) and cytochrome P450 2E1 (Cyp2e1), the major enzymes that metabolize ethanol, prevented alcohol-induced steatosis and reduced induction of the UPR in the liver. Thus, we conclude that ethanol metabolism causes ALD in zebrafish. Oxidative stress generated by Cyp2e1-mediated ethanol metabolism is proposed to be a major culprit in ALD pathology. We found that production of reactive oxygen species (ROS) increased in larvae exposed to ethanol, whereas inhibition of the zebrafish CYP2E1 homolog or administration of antioxidants reduced ROS levels. Importantly, these treatments also blocked ethanol-induced steatosis and reduced UPR activation, whereas hydrogen peroxide (H2O2) acted as a pro-oxidant that synergized with low doses of ethanol to induce the UPR. Collectively, these data demonstrate that ethanol metabolism and oxidative stress are conserved mechanisms required for the development of steatosis and hepatic dysfunction in ALD, and that these processes contribute to ethanol-induced UPR activation and secretory pathway stress in hepatocytes. PMID:23798569

  1. 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. Cancer Res; 75(24); 5367-77. ©2015 AACR. PMID:26573792

  2. Carbon ions induce autophagy effectively through stimulating the unfolded protein response and subsequent inhibiting Akt phosphorylation in tumor cells.

    PubMed

    Jin, Xiaodong; Li, Feifei; Zheng, Xiaogang; Liu, Yan; Hirayama, Ryoichi; Liu, Xiongxiong; Li, Ping; Zhao, Ting; Dai, Zhongying; Li, Qiang

    2015-01-01

    Heavy ion beams have advantages over conventional radiation in radiotherapy due to their superb biological effectiveness and dose conformity. However, little information is currently available concerning the cellular and molecular basis for heavy ion radiation-induced autophagy. In this study, human glioblastoma SHG44 and cervical cancer HeLa cells were irradiated with carbon ions of different linear energy transfers (LETs) and X-rays. Our results revealed increased LC3-II and decreased p62 levels in SHG44 and HeLa cells post-irradiation, indicating marked induction of autophagy. The autophagic level of tumor cells after irradiation increased in a LET-dependent manner and was inversely correlated with the sensitivity to radiations of various qualities. Furthermore, we demonstrated that high-LET carbon ions stimulated the unfolded protein response (UPR) and mediated autophagy via the UPR-eIF2?-CHOP-Akt signaling axis. High-LET carbon ions more severely inhibited Akt-mTOR through UPR to effectively induce autophagy. Thus, the present data could serve as an important radiobiological basis to further understand the molecular mechanisms by which high-LET radiation induces cell death. PMID:26338671

  3. Carbon ions induce autophagy effectively through stimulating the unfolded protein response and subsequent inhibiting Akt phosphorylation in tumor cells

    PubMed Central

    Jin, Xiaodong; Li, Feifei; Zheng, Xiaogang; Liu, Yan; Hirayama, Ryoichi; Liu, Xiongxiong; Li, Ping; Zhao, Ting; Dai, Zhongying; Li, Qiang

    2015-01-01

    Heavy ion beams have advantages over conventional radiation in radiotherapy due to their superb biological effectiveness and dose conformity. However, little information is currently available concerning the cellular and molecular basis for heavy ion radiation-induced autophagy. In this study, human glioblastoma SHG44 and cervical cancer HeLa cells were irradiated with carbon ions of different linear energy transfers (LETs) and X-rays. Our results revealed increased LC3-II and decreased p62 levels in SHG44 and HeLa cells post-irradiation, indicating marked induction of autophagy. The autophagic level of tumor cells after irradiation increased in a LET-dependent manner and was inversely correlated with the sensitivity to radiations of various qualities. Furthermore, we demonstrated that high-LET carbon ions stimulated the unfolded protein response (UPR) and mediated autophagy via the UPR-eIF2?-CHOP-Akt signaling axis. High-LET carbon ions more severely inhibited Akt-mTOR through UPR to effectively induce autophagy. Thus, the present data could serve as an important radiobiological basis to further understand the molecular mechanisms by which high-LET radiation induces cell death. PMID:26338671

  4. Investigating the role of the Caenorhabditis elegans unfolded protein response in immunity and development

    E-print Network

    Richardson, Claire E

    2012-01-01

    Proteins destined for the secretory pathway are folded, posttranslationally modified, and assembled into complexes in the endoplasmic reticulum (ER). To maintain ER proteostasis, the rate of nascent peptide influx into the ...

  5. 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.

  6. Cell Host & Microbe The Unfolded Protein Response Element IRE1a

    E-print Network

    Higgins, Darren

    -acid inducible gene 1 (RIG-I)-like receptor fam- ilies of proteins that survey various subcellular compartments to survey the extracellular environment, the cytosol, various endosomes, mitochondria, and peroxisomes traffic through these organ- elles en route to the cytosol. Cholera toxin (CT) is the major virulence

  7. 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.

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

    PubMed

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

    2013-09-01

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

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

    PubMed

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

    2014-05-01

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

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

    PubMed

    Yu, Chao; Achazi, Katharina; Niedrig, Matthias

    2013-12-26

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

  11. Differential unfolded protein response during Chikungunya and Sindbis virus infection: CHIKV nsP4 suppresses eIF2? 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 (eIF2?), which in turn regulates the PERK pathway. This study provides insight into a mechanism by which CHIKV replication responds to overcome the host UPR machinery. PMID:23356742

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

    PubMed Central

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

    2013-01-01

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

  13. 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.

  14. Hypoxic preconditioning and cell death from oxygen/glucose deprivation co-opt a subset of the unfolded protein response in hippocampal neurons.

    PubMed

    Bickler, P E; Clark, J P; Gabatto, P; Brosnan, H

    2015-12-01

    The state of protein folding in the endoplasmic reticulum (ER), via the unfolded protein response (UPR), regulates a pro- or anti-apoptotic cell fate. Hypoxic preconditioning (HPC) is a potent anti-apoptotic stimulus, wherein ischemic neural injury is averted by a non-damaging exposure to hypoxia. We tested if UPR modulation contributes to the pro-survival/anti-apoptotic phenotype in neurons preconditioned with hypoxia, using organotypic cultures of rat hippocampus as a model system. Pharmacologic induction of the UPR with tunicamycin increased mRNA of 79 of 84 UPR genes and replicated the pro-survival phenotype of HPC, whereas only small numbers of the same mRNAs were upregulated at 0, 6 and 24h after HPC. During the first 24h after HPC, protein signals in all 3 UPR pathways increased at various times: increased ATF4, phosphorylation of eif2? and IRE1, cleavage of xbb1 mRNA and cleavage of ATF6. Pharmacologic inhibition of ATF6 and IRE1 blocked HPC. Ischemia-like conditions (oxygen/glucose deprivation, OGD) caused extensive neuron cell damage and involved some of the same UPR protein signals as HPC. In distinction to HPC and tunicamycin, OGD caused widespread suppression of UPR genes: 55 of 84 UPR gene mRNAs were numerically downregulated. We conclude that although HPC and ischemic cell death in hippocampal neurons involve protein-based signaling in all 3 UPR pathways, these processes co-opt only a subset of the genomic response elicited by agents known to cause protein misfolding, possibly because of persistent transcription/translation arrest induced by hypoxia and especially OGD. PMID:26404874

  15. 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

    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; Courtoy, P J

    2015-06-01

    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 endolysosomal trafficking and iodo-Tg processing. The Ctns(-/-) thyroid is useful to study disease progression and evaluate novel therapies. PMID:25811319

  16. CREB is activated by ER stress and modulates the unfolded protein response by regulating the expression of IRE1? and PERK.

    PubMed

    Kikuchi, Daisuke; Tanimoto, Kousuke; Nakayama, Koh

    2016-01-01

    Living cells are frequently exposed to various stresses. Hypoxic conditions induce endoplasmic reticulum (ER) stress, and activate the unfolded protein response (UPR) to maintain homeostasis. We previously reported that CREB has an important role in the proper response to prolonged hypoxia. To further understand the role of CREB in the hypoxic response, CREB stable knock-down (CREB-KD) cells were established from breast cancer MDA-MB231 cells and analyzed. CREB was activated by ER stress, and activation of CREB and the UPR pathway occurred in a coordinated manner in response to different stimuli, including ER stress-inducing chemicals, prolonged hypoxia, and oxygen-glucose deprivation (OGD). Depletion of CREB decreased the expression of IRE1? and PERK, two critical UPR signaling molecules. Promoter analysis and a chromatin immunoprecipitation assay indicated that CREB binds to the promoter region of these genes and regulates their expression. ER stress induced by hypoxia was reduced in CREB-KD cells, leading to reduced tumor metastasis to the lung. Finally, OGD strongly activated the UPR and induced cell death in control cells, whereas the UPR was moderately activated in CREB-KD cells, which were more resistant to cell death. This study demonstrates a new role for CREB as a regulator of ER stress, which is required to properly respond to stressful conditions, such as hypoxia. PMID:26642955

  17. IRE1 inhibition perturbs the unfolded protein response in a pancreatic ?-cell line expressing mutant proinsulin, but does not sensitize the cells to apoptosis

    PubMed Central

    2014-01-01

    Background The Akita mutation (C96Y) in the insulin gene results in early onset diabetes in both humans and mice. Expression of mutant proinsulin (C96Y) causes endoplasmic reticulum (ER) stress in pancreatic ?-cells and consequently the cell activates the unfolded protein response (UPR). Since the proinsulin is terminally misfolded ER stress is irremediable and chronic activation of the UPR eventually activates apoptosis in some cells. Here we analyzed the IRE1-dependent activation of genes in response to misfolded proinsulin production in an inducible mutant proinsulin (C96Y) insulinoma cell line. Results The IRE1 endoribonuclease inhibitors 4?8c and MKC-3946 prevented the splicing of the XBP1 mRNA in response to ER stress caused by mutant proinsulin production. Microarray expression analysis and qPCR validation of select genes revealed that maximal upregulation of many UPR genes in response to mutant proinsulin production required IRE1, although most were still increased above control. Interestingly, neither degradation of misfolded proinsulin via ER-associated degradation (ERAD), nor apoptosis induced by prolonged misfolded proinsulin expression were affected by inhibiting IRE1. Conclusions Although maximal induction of most UPR genes requires IRE1, inhibition of IRE1 does not affect ERAD of misfolded proinsulin or predispose pancreatic ?-cells expressing misfolded proinsulin to chronic ER stress-induced apoptosis. PMID:25011481

  18. Anticipatory estrogen activation of the unfolded protein response is linked to cell proliferation and poor survival in estrogen receptor ?-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 ? (ER?), 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-ER?-induced cell proliferation. In ER?-containing cancer cells, the estrogen, 17?-estradiol (E2) activates the UPR through a phospholipase C ? (PLC?)-mediated opening of EnR IP3R calcium channels, enabling passage of calcium from the lumen of the EnR into the cytosol. siRNA knockdown of ER? blocked the estrogen-mediated increase in cytosol calcium and UPR activation. Knockdown or inhibition of PLC?, or of IP3R, strongly inhibited the estrogen-mediated increases in cytosol calcium, UPR activation and cell proliferation. E2-ER? activates all three arms of the UPR in breast and ovarian cancer cells in culture and in a mouse xenograft. Knockdown of ATF6?, which regulates UPR chaperones, blocked estrogen induction of BiP and strongly inhibited E2-ER?-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 ER?(+) 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-ER? proliferation program, the mitogen estrogen, drives rapid anticipatory activation of the UPR. Anticipatory activation of the UPR is a new role for estrogens in cancer cell proliferation and resistance to therapy. PMID:25263449

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

    E-print Network

    Jwa, Miri

    Poly(ADP-ribose) polymerases (PARPs; also known as ADP-ribosyl transferase D proteins) modify acceptor proteins with ADP-ribose modifications of varying length (reviewed in refs 1, 2, 3). PARPs regulate key stress response ...

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

    PubMed

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

    2013-11-01

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

  1. Small-Molecule RA-9 Inhibits Proteasome-Associated DUBs and Ovarian Cancer in Vitro and in Vivo Via Exacerbating Unfolded Protein Responses

    PubMed Central

    Coughlin, Kathleen; Anchoori, Ravi; Iizuka, Yoshie; Meints, Joyce; MacNeill, Lauren; Vogel, Rachel Isaksson; Orlowski, Robert Z.; Lee, Michael K.; Roden, Richard BS; Bazzaro, Martina

    2014-01-01

    Purpose Ovarian cancer is the deadliest of the gynecological malignancies. Carcinogenic progression is accompanied by up-regulation of ubiquitin-dependent protein degradation machinery as a mechanism to compensate with elevated endogenous proteotoxic stress. Recent studies support the notion that deubiquitinating enzymes (DUBs) are essential factors in proteolytic degradation and that their aberrant activity is linked to cancer progression and chemoresistance. Thus, DUBs are an attractive therapeutic target for ovarian cancer. Experimental Design The potency and selectivity of RA-9 inhibitor for proteasome-associated DUBs was determined in ovarian cancer cell lines and primary cells. The anticancer activity of RA-9 and its mechanism of action was evaluated in multiple cancer cell lines in vitro and in vivo in immunodeficient mice bearing an intra-peritoneal ES-2 xenograft model of human ovarian cancer. Results Here we report the characterization of RA-9 as a small-molecule inhibitor of proteasome-associated DUBs. Treatment with RA-9 selectively induces onset of apoptosis, in ovarian cancer cell lines and primary cultures derived from donors. Loss of cell viability following RA-9 exposure is associated with an Unfolded Protein Response (UPR) as mechanism to compensate for unsustainable levels of proteotoxic stress. In vivo treatment with RA-9 retards tumor growth, increases overall survival and was well tolerated by the host. Conclusions Our preclinical studies support further evaluation of RA-9 as an ovarian cancer therapeutic. PMID:24727327

  2. Thermal unfolding dynamics of proteins probed by nonlinear infrared spectroscopy

    E-print Network

    Chung, Hoi Sung

    2007-01-01

    This thesis presents spectroscopic approaches to study the thermal unfolding dynamics of proteins. The spectroscopic tool is nonlinear infrared (IR) spectroscopy of the protein amide I band. Among various nonlinear IR ...

  3. 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

  4. 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.

  5. 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

  6. 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…

  7. The Malat1 long non-coding RNA is upregulated by signalling through the PERK axis of unfolded protein response during flavivirus infection

    PubMed Central

    Bhattacharyya, Sankar; Vrati, Sudhanshu

    2015-01-01

    Flavivirus infection causes host cell death by initiation of an unfolded protein response (UPR). UPR is initiated following activation of three ER-membrane resident sensors, PERK, IRE1? and ATF6, which are otherwise kept inactive through association with the ER-chaperone GRP78. Activation precedes cellular and molecular changes that act to restore homeostasis but might eventually initiate apoptosis. These changes involve influencing function of multiple genes by either transcriptional or post-transcriptional or post-translational mechanisms. Transcriptional control includes expression of transcription factor cascades, which influence cognate gene expression. Malat1 is a long non-coding RNA which is over-expressed in many human oncogenic tissues and regulates cell cycle and survival. In this report, for the first time we show activation of Malat1 following infection by two flaviviruses, both of which activate the UPR in host cells. The temporal kinetics of expression was restricted to later time points. Further, Malat1 was also activated by pharmacological inducer of UPR, to a similar degree. Using drugs that specifically inhibit or activate the PERK or IRE1? sensors, we demonstrate that signalling through the PERK axis activates this expression, through a transcriptional mechanism. To our knowledge, this is the first report of an UPR pathway regulating the expression of an lncRNA. PMID:26634309

  8. Effect of dexamethasone on unfolded protein response genes (MTJ1, Grp78, Grp94, CHOP, HMOX-1) in HEp2 cell line.

    PubMed

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

    2013-12-01

    The endoplasmic reticulum (ER) is related to the various signal routes that are activated in unfolded protein response (UPR). The Grp78, Grp94, CHOP, MTJ1 and HMOX1 genes expressions demonstrate UPR activity. In this study, we investigated the UPR gene expressions in larynx epidermoid carcinoma (HEp2) to which dexamethasone (dex) was applied. HEp2 cells were administered for 48 h with different combinations using 0.1 microM and 1 microM dex, 1 mM phenyl butyric acid (PBA) and 100 ng/ml lipopolysaccharide (LPS). The Grp78, Grp94, CHOP, MTJ1 and HMOX1 genes expression was determined using quantitative RT-PCR. The Grp78, MTJ1 and HMOX1 gene expression increased with the administration of 1 microM dex. CHOP expression, on the other hand, decreased with 0.1 microM dex. When dex was combined with LPS, nearly all gene expressions decreased. The increase in Grp78, Grp94, HMOX1 and MTJ1 gene expression was greater in groups in which dex was administered in combination with PBA than in groups in which dex was administered alone. Dex in low dose (0.1 microM) caused a decrease in CHOP expression in HEp2 cells and an increase in Grp78 expression, in particular. The changes in UPR genes expressions may lead to the extended survival of the cells. PMID:24772975

  9. The Malat1 long non-coding RNA is upregulated by signalling through the PERK axis of unfolded protein response during flavivirus infection.

    PubMed

    Bhattacharyya, Sankar; Vrati, Sudhanshu

    2015-01-01

    Flavivirus infection causes host cell death by initiation of an unfolded protein response (UPR). UPR is initiated following activation of three ER-membrane resident sensors, PERK, IRE1? and ATF6, which are otherwise kept inactive through association with the ER-chaperone GRP78. Activation precedes cellular and molecular changes that act to restore homeostasis but might eventually initiate apoptosis. These changes involve influencing function of multiple genes by either transcriptional or post-transcriptional or post-translational mechanisms. Transcriptional control includes expression of transcription factor cascades, which influence cognate gene expression. Malat1 is a long non-coding RNA which is over-expressed in many human oncogenic tissues and regulates cell cycle and survival. In this report, for the first time we show activation of Malat1 following infection by two flaviviruses, both of which activate the UPR in host cells. The temporal kinetics of expression was restricted to later time points. Further, Malat1 was also activated by pharmacological inducer of UPR, to a similar degree. Using drugs that specifically inhibit or activate the PERK or IRE1? sensors, we demonstrate that signalling through the PERK axis activates this expression, through a transcriptional mechanism. To our knowledge, this is the first report of an UPR pathway regulating the expression of an lncRNA. PMID:26634309

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

    PubMed Central

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

    2012-01-01

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

  11. Unfolded protein response is required for the definitive endodermal specification of mouse embryonic stem cells via Smad2 and ?-catenin signaling.

    PubMed

    Xu, Huiming; Tsang, Kam Sze; Wang, Yonghui; Chan, Juliana C N; Xu, Gang; Gao, Wei-Qiang

    2014-09-19

    Tremendous efforts have been made to elucidate the molecular mechanisms that control the specification of definitive endoderm cell fate in gene knockout mouse models and ES cell (ESC) differentiation models. However, the impact of the unfolded protein response (UPR), because of the stress of the endoplasmic reticulum on endodermal specification, is not well addressed. We employed UPR-inducing agents, thapsigargin and tunicamycin, in vitro to induce endodermal differentiation of mouse ESCs. Apart from the endodermal specification of ESCs, Western blotting demonstrated the enhanced phosphorylation of Smad2 and nuclear translocation of ?-catenin in ESC-derived cells. The inclusion of the endoplasmic reticulum stress inhibitor tauroursodeoxycholic acid to the induction cultures prevented the differentiation of ESCs into definitive endodermal cells even when Activin A was supplemented. Also, the addition of the TGF-? inhibitor SB431542 and the Wnt/?-catenin antagonist IWP-2 negated the endodermal differentiation of ESCs mediated by thapsigargin and tunicamycin. These data suggest that the activation of the UPR appears to orchestrate the induction of the definitive endodermal cell fate of ESCs via both the Smad2 and ?-catenin signaling pathways. The prospective regulatory machinery may be helpful for directing ESCs to differentiate into definitive endodermal cells for cellular therapy in the future. PMID:25092289

  12. Unfolded Protein Response Is Required for the Definitive Endodermal Specification of Mouse Embryonic Stem Cells via Smad2 and ?-Catenin Signaling*

    PubMed Central

    Xu, Huiming; Tsang, Kam Sze; Wang, Yonghui; Chan, Juliana CN; Xu, Gang; Gao, Wei-Qiang

    2014-01-01

    Tremendous efforts have been made to elucidate the molecular mechanisms that control the specification of definitive endoderm cell fate in gene knockout mouse models and ES cell (ESC) differentiation models. However, the impact of the unfolded protein response (UPR), because of the stress of the endoplasmic reticulum on endodermal specification, is not well addressed. We employed UPR-inducing agents, thapsigargin and tunicamycin, in vitro to induce endodermal differentiation of mouse ESCs. Apart from the endodermal specification of ESCs, Western blotting demonstrated the enhanced phosphorylation of Smad2 and nuclear translocation of ?-catenin in ESC-derived cells. The inclusion of the endoplasmic reticulum stress inhibitor tauroursodeoxycholic acid to the induction cultures prevented the differentiation of ESCs into definitive endodermal cells even when Activin A was supplemented. Also, the addition of the TGF-? inhibitor SB431542 and the Wnt/?-catenin antagonist IWP-2 negated the endodermal differentiation of ESCs mediated by thapsigargin and tunicamycin. These data suggest that the activation of the UPR appears to orchestrate the induction of the definitive endodermal cell fate of ESCs via both the Smad2 and ?-catenin signaling pathways. The prospective regulatory machinery may be helpful for directing ESCs to differentiate into definitive endodermal cells for cellular therapy in the future. PMID:25092289

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

    PubMed

    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

    Given the safety concerns expressed over negative cardiovascular outcomes resulting from the clinical use of rosiglitazone, and the view that rosiglitazone exerts PPAR?-independent effects alongside its insulin-sensitising PPAR?-dependent effects, we hypothesised that rosiglitazone may trigger Unfolded Protein Responses (UPRs) due to disruptions in [Ca(2+)](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 (30min) brought about concentration-dependent PPAR?-independent inhibition of Ca(2+)ATPase activity (IC(50) ?2?M). Fluo-3 fluorimetric data demonstrated in intact cells that 1h treatment with 1 or 10?M rosiglitazone caused Ca(2+) ions to leak into the cytoplasm. Gene expression analysis showed that within 4h of rosiglitazone exposure, the UPR transcription factor XBP-1 was activated (likely due to corresponding ER Ca(2+) depletion), and the UPR target genes BiP and SERCA2b were subsequently upregulated within 24-72h. After 72h 1 or 10?M rosiglitazone treatment, microsomal Ca(2+)ATPase activity increased to >2-fold of that seen in control microsomes, while [Ca(2+)](i) returned to basal, indicating that UPR-triggered SERCA2b upregulation was responsible for enhanced enzymatic Ca(2+) sequestration within the ER. This appeared to be sufficient to replenish ER Ca(2+) stores and restore normal cell physiology, as cell viability levels were not decreased due to rosiglitazone treatment throughout a 2-week study. Thus, incubation with 1-10?M rosiglitazone triggers the UPR, but does not prove cytotoxic, in cells of the cardiovascular system. This observation provides an important contribution to the current debate over the use of rosiglitazone in the clinical treatment of Type-2 Diabetes. PMID:20816668

  14. Activation of the Unfolded Protein Response Is Required for Defenses against Bacterial Pore-Forming Toxin In

    E-print Network

    Aroian, Raffi V.

    -Forming Toxin In Vivo Larry J. Bischof1¤ , Cheng-Yuan Kao1 , Ferdinand C. O. Los1 , Manuel R. Gonzalez2-forming toxins (PFTs) constitute the single largest class of proteinaceous bacterial virulence factors and are made by many of the most important bacterial pathogens. Host responses to these toxins are complex

  15. 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

  16. 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??8C) 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

  17. High Glucose-Repressed CITED2 Expression Through miR-200b Triggers the Unfolded Protein Response and Endoplasmic Reticulum Stress.

    PubMed

    Gu, Hui; Yu, Jingwen; Dong, Daoyin; Zhou, Qun; Wang, Jian-Ying; Fang, Shengyun; Yang, Peixin

    2016-01-01

    High glucose in vivo and in vitro induces neural tube defects (NTDs). CITED2 (CBP/p300-interacting transactivator with ED-rich tail 2) is essential for neural tube closure. We explored the regulatory mechanism underlying CITED2 expression and its relationship with miRNA and endoplasmic reticulum (ER) stress. miR-200b levels were increased by maternal diabetes or high glucose in vitro, and this increase was abrogated by transgenic overexpression of superoxide dismutase 1 (SOD1) or an SOD1 mimetic. CITED2 was the target of miR-200b and was downregulated by high glucose. Two miR-200b binding sites in the 3'-untranslated region of the CITED2 mRNA were required for inhibiting CITED2 expression. The miR-200b mimic and a CITED2 knockdown mimicked the stimulative effect of high glucose on unfolded protein response (UPR) and ER stress, whereas the miR-200b inhibitor and CITED2 overexpression abolished high glucose-induced UPR signaling, ER stress, and apoptosis. The ER stress inhibitor, 4-phenylbutyrate, blocked CITED2 knockdown-induced apoptosis. Furthermore, the miR-200b inhibitor reversed high glucose-induced CITED2 downregulation, ER stress, and NTDs in cultured embryos. Thus, we showed a novel function of miR-200b and CITED2 in high glucose-induced UPR and ER stress, suggesting that miR-200b and CITED2 are critical for ER homeostasis and NTD formation in the developing embryo. PMID:26450995

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

    PubMed

    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??8C) 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. 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

  20. Ultrafast hydration dynamics in protein unfolding: Human serum albumin

    PubMed Central

    Kamal, J. K. Amisha; Zhao, Liang; Zewail, Ahmed H.

    2004-01-01

    We report studies of unfolding and ultrafast hydration dynamics of the protein human serum albumin. Unique in this study is our ability to examine different domains of the same protein and the intermediate on the way to the unfolded state. With femtosecond resolution and site-selective labeling, we isolate the dynamics of domains I and II of the native protein, domain I of the intermediate at 2 M guanidine hydrochloride, and the unfolded state at 6 M of the denaturant. For studies of unfolding, we used the fluorophores, acrylodan (covalently bound to Cys-34 in domain I) and the intrinsic tryptophan (domain II), whereas for hydration dynamics, we probed acrylodan and prodan; the latter is bound to domain II. From the time-dependent spectra and the correlation functions, we obtained the time scale of dynamically ordered water: 57 ps for the more stable domain I and 32 ps for the less stable domain II, in contrast to ?0.8 ps for labile, bulk-type water. This trend suggests an increased hydrophilic residues–water interaction of domain I, contrary to some packing models. In the intermediate state, which is characterized by essentially intact domain I and unfolded domain II, the dynamics of ordered water around domain I is nearly the same (61 ps) as that of native state (57 ps), whereas that in the unfolded protein is much shorter (13 ps). We discuss the role of this fluidity in the correlation between stability and function of the protein. PMID:15353599

  1. Statistical mechanics of warm and cold unfolding in proteins

    E-print Network

    Alex Hansen; Mogens H. Jensen; Kim Sneppen; Giovanni Zocchi

    1997-11-27

    We present a statistical mechanics treatment of the stability of globular proteins which takes explicitly into account the coupling between the protein and water degrees of freedom. This allows us to describe both the cold and the warm unfolding, thus qualitatively reproducing the known thermodynamics of proteins.

  2. First Passage Times, Lifetimes, and Relaxation Times of Unfolded Proteins.

    PubMed

    Dai, Wei; Sengupta, Anirvan M; Levy, Ronald M

    2015-07-24

    The dynamics of proteins in the unfolded state can be quantified in computer simulations by calculating a spectrum of relaxation times which describes the time scales over which the population fluctuations decay to equilibrium. If the unfolded state space is discretized, we can evaluate the relaxation time of each state. We derive a simple relation that shows the mean first passage time to any state is equal to the relaxation time of that state divided by the equilibrium population. This explains why mean first passage times from state to state within the unfolded ensemble can be very long but the energy landscape can still be smooth (minimally frustrated). In fact, when the folding kinetics is two-state, all of the unfolded state relaxation times within the unfolded free energy basin are faster than the folding time. This result supports the well-established funnel energy landscape picture and resolves an apparent contradiction between this model and the recently proposed kinetic hub model of protein folding. We validate these concepts by analyzing a Markov state model of the kinetics in the unfolded state and folding of the miniprotein NTL9 (where NTL9 is the N-terminal domain of the ribosomal protein L9), constructed from a 2.9 ms simulation provided by D. E. Shaw Research. PMID:26252709

  3. 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

  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 appeared to be sufficient to replenish ER Ca{sup 2+} stores and restore normal cell physiology, as cell viability levels were not decreased due to rosiglitazone treatment throughout a 2-week study. Thus, incubation with 1-10 {mu}M rosiglitazone triggers the UPR, but does not prove cytotoxic, in cells of the cardiovascular system. This observation provides an important contribution to the current debate over the use of rosiglitazone in the clinical treatment of Type-2 Diabetes.

  5. 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

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

    PubMed Central

    Jamin, Marc; Jacob-Dubuisson, Françoise

    2013-01-01

    The parallel ?helix is a common fold among extracellular proteins, however its mechanical properties remain unexplored. In Gram-negative bacteria, extracellular proteins of diverse functions of the large ‘TpsA’ family all fold into long ?helices. Here, single-molecule atomic force microscopy and steered molecular dynamics simulations were combined to investigate the mechanical properties of a prototypic TpsA protein, FHA, the major adhesin of Bordetella pertussis. Strong extension forces were required to fully unfold this highly repetitive protein, and unfolding occurred along a stepwise, hierarchical process. Our analyses showed that the extremities of the ?helix unfold early, while central regions of the helix are more resistant to mechanical unfolding. In particular, a mechanically resistant subdomain conserved among TpsA proteins and critical for secretion was identified. This nucleus harbors structural elements packed against the ?helix that might contribute to stabilizing the N-terminal region of FHA. Hierarchical unfolding of the ?helix in response to a mechanical stress may maintain ?-helical portions that can serve as templates for regaining the native structure after stress. The mechanical properties uncovered here might apply to many proteins with ?-helical or related folds, both in prokaryotes and in eukaryotes, and play key roles in their structural integrity and functions. PMID:24009757

  7. 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

  8. Power-law rheology and mechano-sensing in a cytoskeleton model with forced protein unfolding

    E-print Network

    Brenton D. Hoffman; Gladys Massiera; John C. Crocker

    2006-03-18

    We describe a model of cytoskeletal mechanics based on the force-induced conformational change of protein cross-links in a stressed polymer network. Slow deformation of simulated networks containing cross-links that undergo repeated, serial domain unfolding leads to an unusual state--with many cross-links accumulating near the critical force for further unfolding. Thermal activation of these links gives rise to power-law rheology resembling the previously unexplained mechanical response of living cells. Moreover, we hypothesize that such protein cross-links function as biochemical mechano-sensors of cytoskeletal deformation.

  9. Protein unfolding accounts for the unusual mechanical behavior of fibrin networks

    E-print Network

    Purohit, Prashant

    Protein unfolding accounts for the unusual mechanical behavior of fibrin networks Prashant K and thrombosis. Abstract 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

  10. 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 C? 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

  11. 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

  12. Quality control for unfolded proteins at the plasma membrane

    PubMed Central

    Apaja, Pirjo M.; Xu, Haijin

    2010-01-01

    Cellular protein homeostasis profoundly depends on the disposal of terminally damaged polypeptides. To demonstrate the operation and elucidate the molecular basis of quality control of conformationally impaired plasma membrane (PM) proteins, we constructed CD4 chimeras containing the wild type or a temperature-sensitive bacteriophage ? domain in their cytoplasmic region. Using proteomic, biochemical, and genetic approaches, we showed that thermal unfolding of the ? domain at the PM provoked the recruitment of Hsp40/Hsc70/Hsp90 chaperones and the E2–E3 complex. Mixed-chain polyubiquitination, monitored by bioluminescence resonance energy transfer and immunoblotting, is responsible for the nonnative chimera–accelerated internalization, impaired recycling, and endosomal sorting complex required for transport–dependent lysosomal degradation. A similar paradigm prevails for mutant dopamine D4.4 and vasopressin V2 receptor removal from the PM. These results outline a peripheral proteostatic mechanism in higher eukaryotes and its potential contribution to the pathogenesis of a subset of conformational diseases. PMID:20974815

  13. [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

  14. Direction dependent mechanical unfolding and Green Fluorescent Protein as a force sensor

    E-print Network

    M. Caraglio; A. Imparato; A. Pelizzola

    2011-07-21

    An Ising--like model of proteins is used to investigate the mechanical unfolding of the Green Fluorescent Protein along different directions. When the protein is pulled from its ends, we recover the major and minor unfolding pathways observed in experiments. Upon varying the pulling direction, we find the correct order of magnitude and ranking of the unfolding forces. Exploiting the direction dependence of the unfolding force at equilibrium, we propose a force sensor whose luminescence depends on the applied force.

  15. Small-angle neutron scattering study of protein unfolding and refolding.

    PubMed

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

    2009-07-01

    Small-angle neutron scattering has been used to study protein unfolding and refolding in protein bovine serum albumin (BSA) due to perturbation in its native structure as induced by three different protein denaturating agents: urea, surfactant, and pressure. The BSA protein unfolds for urea concentrations greater than 4 M and is observed to be independent of the protein concentration. The addition of surfactant unfolds the protein by the formation of micellelike aggregates of surfactants along the unfolded polypeptide chains of the protein and depends on the ratio of surfactant to protein concentration. We make use of the dilution method to show the refolding of unfolded proteins in the presence of urea and surfactant. BSA does not show any protein unfolding up to the pressure of 450 MPa. The presence of urea and surfactant (for concentrations prior to inducing their own unfolding) has been used to examine pressure-induced unfolding of the protein at lower pressures. The protein unfolds at 200 MPa pressure in the presence of urea; however, no unfolding is observed with surfactant. The protein unfolding is shown to be reversible in all the above denaturating methods. PMID:19658746

  16. Energetically significant networks of coupled interactions within an unfolded protein

    PubMed Central

    Cho, Jae-Hyun; Meng, Wenli; Sato, Satoshi; Kim, Eun Young; Schindelin, Hermann; Raleigh, Daniel P.

    2014-01-01

    Unfolded and partially unfolded proteins participate in a wide range of biological processes from pathological aggregation to the regulation of normal cellular activity. Unfolded states can be populated under strongly denaturing conditions, but the ensemble which is relevant for folding, stability, and aggregation is that populated under physiological conditions. Characterization of nonnative states is critical for the understanding of these processes, yet comparatively little is known about their energetics and their structural propensities under native conditions. The standard view is that energetically significant coupled interactions involving multiple residues are generally not present in the denatured state ensemble (DSE) or in intrinsically disordered proteins. Using the N-terminal domain of the ribosomal protein L9, a small ?-? protein, as an experimental model system, we demonstrate that networks of energetically significant, coupled interactions can form in the DSE of globular proteins, and can involve residues that are distant in sequence and spatially well separated in the native structure. X-ray crystallography, NMR, dynamics studies, native state pKa measurements, and thermodynamic analysis of more than 25 mutants demonstrate that residues are energetically coupled in the DSE. Altering these interactions by mutation affects the stability of the domain. Mutations that alter the energetics of the DSE can impact the analysis of cooperativity and folding, and may play a role in determining the propensity to aggregate. PMID:25099351

  17. Energetically significant networks of coupled interactions within an unfolded protein.

    PubMed

    Cho, Jae-Hyun; Meng, Wenli; Sato, Satoshi; Kim, Eun Young; Schindelin, Hermann; Raleigh, Daniel P

    2014-08-19

    Unfolded and partially unfolded proteins participate in a wide range of biological processes from pathological aggregation to the regulation of normal cellular activity. Unfolded states can be populated under strongly denaturing conditions, but the ensemble which is relevant for folding, stability, and aggregation is that populated under physiological conditions. Characterization of nonnative states is critical for the understanding of these processes, yet comparatively little is known about their energetics and their structural propensities under native conditions. The standard view is that energetically significant coupled interactions involving multiple residues are generally not present in the denatured state ensemble (DSE) or in intrinsically disordered proteins. Using the N-terminal domain of the ribosomal protein L9, a small ?-? protein, as an experimental model system, we demonstrate that networks of energetically significant, coupled interactions can form in the DSE of globular proteins, and can involve residues that are distant in sequence and spatially well separated in the native structure. X-ray crystallography, NMR, dynamics studies, native state pKa measurements, and thermodynamic analysis of more than 25 mutants demonstrate that residues are energetically coupled in the DSE. Altering these interactions by mutation affects the stability of the domain. Mutations that alter the energetics of the DSE can impact the analysis of cooperativity and folding, and may play a role in determining the propensity to aggregate. PMID:25099351

  18. 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.

  19. Do Protein Molecules Unfold in a Simple Shear Flow?

    PubMed Central

    Jaspe, Juan; Hagen, Stephen J.

    2006-01-01

    Protein molecules typically unfold (denature) when subjected to extremes of heat, cold, pH, solvent composition, or mechanical stress. One might expect that shearing forces induced by a nonuniform fluid flow would also destabilize proteins, as when a protein solution flows rapidly through a narrow channel. However, although the protein literature contains many references to shear denaturation, we find little quantitative evidence for the phenomenon. We have investigated whether a high shear can destabilize a small globular protein to any measurable extent. We study a protein (horse cytochrome c, 104 amino acids) whose fluorescence increases sharply upon unfolding. By forcing the sample through a silica capillary (inner diameter 150–180 ?m) at speeds approaching 10 m/s, we subject the protein to shear rates dvz/dr as large as ?2 × 105 s?1 while illuminating it with an ultraviolet laser. We can readily detect fluorescence changes of <1%, corresponding to shifts of protein. A simple model suggests that extraordinary shear rates, ?107 s?1, would be required to denature typical small, globular proteins in water. PMID:16891374

  20. Unfoldings

    E-print Network

    Johnson, Caryn L. (Caryn Lindani)

    1992-01-01

    Huizinga's analysis of play, described in his text Homo Ludens, is compared to the creative process in art-making and to the creative response of the viewer. The play process is examined through questionnaire responses and ...

  1. 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

  2. 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.

  3. 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 ?-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.

  4. 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

  5. Natively unfolded proteins: A point where biology waits for physics

    PubMed Central

    Uversky, Vladimir N.

    2002-01-01

    The experimental material accumulated in the literature on the conformational behavior of intrinsically unstructured (natively unfolded) proteins was analyzed. Results of this analysis showed that these proteins do not possess uniform structural properties, as expected for members of a single thermodynamic entity. Rather, these proteins may be divided into two structurally different groups: intrinsic coils, and premolten globules. Proteins from the first group have hydrodynamic dimensions typical of random coils in poor solvent and do not possess any (or almost any) ordered secondary structure. Proteins from the second group are essentially more compact, exhibiting some amount of residual secondary structure, although they are still less dense than native or molten globule proteins. An important feature of the intrinsically unstructured proteins is that they undergo disorder–order transition during or prior to their biological function. In this respect, the Protein Quartet model, with function arising from four specific conformations (ordered forms, molten globules, premolten globules, and random coils) and transitions between any two of the states, is discussed. PMID:11910019

  6. Unfolding the Role of Stress Response Signaling in Endocrine Resistant Breast Cancers

    PubMed Central

    Clarke, Robert; Cook, Katherine L.

    2015-01-01

    The unfolded protein response (UPR) is an ancient stress response that enables a cell to manage the energetic stress that accompanies protein folding. There has been a significant recent increase in our understanding of the UPR, how it integrates physiological processes within cells, and how this integration can affect cancer cells and cell fate decisions. Recent publications have highlighted the role of UPR signaling components on mediating various cell survival pathways, cellular metabolism and bioenergenics, and autophagy. We address the role of UPR on mediating endocrine therapy resistance and estrogen receptor-positive breast cancer cell survival. PMID:26157705

  7. 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.

  8. 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

  9. Single-Molecule Spectroscopy of Cold Denaturation and the Temperature-Induced Collapse of Unfolded Proteins

    E-print Network

    Schuler, Ben

    Single-Molecule Spectroscopy of Cold Denaturation and the Temperature-Induced Collapse of Unfolded that cold- denatured proteins are more expanded than heat- denatured proteins. To clarify the connection between these observations, we investigated the unfolded state of yeast frataxin, whose cold denaturation

  10. 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…

  11. Single-Molecule Protein Unfolding and Translocation by an ATP-Fueled Proteolytic Machine

    E-print Network

    Aubin-Tam, Marie-Eve

    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 ...

  12. Optimizing the calculation of energy landscape parameters from single-molecule protein unfolding experiments

    NASA Astrophysics Data System (ADS)

    Tych, Katarzyna M.; Hughes, Megan L.; Bourke, James; Taniguchi, Yukinori; Kawakami, Masaru; Brockwell, David J.; Dougan, Lorna

    2015-01-01

    Single-molecule force spectroscopy using an atomic force microscope (AFM) can be used to measure the average unfolding force of proteins in a constant velocity experiment. In combination with Monte Carlo simulations and through the application of the Zhurkov-Bell model, information about the parameters describing the underlying unfolding energy landscape of the protein can be obtained. Using this approach, we have completed protein unfolding experiments on the polyprotein (I27 ) 5 over a range of pulling velocities. In agreement with previous work, we find that the observed number of protein unfolding events observed in each approach-retract cycle varies between one and five, due to the nature of the interactions between the polyprotein, the AFM tip, and the substrate, and there is an unequal unfolding probability distribution. We have developed a Monte Carlo simulation that incorporates the impact of this unequal unfolding probability distribution on the median unfolding force and the calculation of the protein unfolding energy landscape parameters. These results show that while there is a significant, unequal unfolding probability distribution, the unfolding energy landscape parameters obtained from use of the Zhurkov-Bell model are not greatly affected. This result is important because it demonstrates that the minimum acceptance criteria typically used in force extension experiments are justified and do not skew the calculation of the unfolding energy landscape parameters. We further validate this approach by determining the error in the energy landscape parameters for two extreme cases, and we provide suggestions for methods that can be employed to increase the level of accuracy in single-molecule experiments using polyproteins.

  13. Assaying the kinetics of protein denaturation catalyzed by AAA+ unfolding machines and proteases

    E-print Network

    Baytshtok, Vladimir

    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 ...

  14. Rate of Intrachain Contact Formation in an Unfolded Protein: Temperature and Denaturant Effects

    E-print Network

    Hagen, Stephen J.

    Rate of Intrachain Contact Formation in an Unfolded Protein: Temperature and Denaturant Effects Box 118440 Gainesville, FL 32611, USA We have measured the effect of temperature and denaturant concen and unfolding conditions, we ®nd a signi®cant effect of denaturant on the chain dynamics. The diffusion

  15. Truncated HSPB1 causes axonal neuropathy and impairs tolerance to unfolded protein stress

    PubMed Central

    Ylikallio, Emil; Konovalova, Svetlana; Dhungana, Yogesh; Hilander, Taru; Junna, Nella; Partanen, Juhani V.; Toppila, Jussi P.; Auranen, Mari; Tyynismaa, Henna

    2015-01-01

    Background HSPB1 belongs to the family of small heat shock proteins (sHSP) that have importance in protection against unfolded protein stress, in cancer cells for escaping drug toxicity stress and in neurons for suppression of protein aggregates. sHSPs have a conserved ?-crystalline domain (ACD), flanked by variable N- and C-termini, whose functions are not fully understood. Dominant missense variants in HSPB1, locating mostly to the ACD, have been linked to inherited neuropathy. Methods Patients underwent detailed clinical and neurophysiologic characterization. Disease causing variants were identified by exome or gene panel sequencing. Primary patient fibroblasts were used to investigate the effects of the dominant defective HSPB1 proteins. Results Frameshift variant predicting ablation of the entire C-terminus p.(Met169Cfs2*) of HSPB1 and a missense variant p.(Arg127Leu) were identified in patients with dominantly inherited motor-predominant axonal Charcot–Marie–Tooth neuropathy. We show that the truncated protein is stable and binds wild type HSPB1. Both mutations impaired the heat stress tolerance of the fibroblasts. This effect was particularly pronounced for the cells with the truncating variant, independent of heat-induced nuclear translocation and induction of global transcriptional heat response. Furthermore, the truncated HSPB1 increased cellular sensitivity to protein misfolding. Conclusion Our results suggest that truncation of the non-conserved C-terminus impairs the function of HSPB1 in cellular stress response. General significance sHSPs have important roles in prevention of protein aggregates that induce toxicity. We showed that C-terminal part of HSPB1 is critical for tolerance of unfolded protein stress, and when lacking causes axonal neuropathy in patients. PMID:26675522

  16. 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

  17. 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

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

    E-print Network

    Bura, Efstathia

    , and mapping the biomolecular free energy landscape. The inference of the unknown probability distribution of green fluo- rescent protein GFP revealed the bifurcation in the GFP unfolding pathways.27 A similar

  19. Wild type, mutant protein unfolding and phase transition detected by single-nanopore recording.

    PubMed

    Merstorf, Céline; Cressiot, Benjamin; Pastoriza-Gallego, Manuela; Oukhaled, Abdelghani; Betton, Jean-Michel; Auvray, Loïc; Pelta, Juan

    2012-04-20

    Understanding protein folding remains a challenge. A difficulty is to investigate experimentally all the conformations in the energy landscape. Only single molecule methods, fluorescence and force spectroscopy, allow observing individual molecules along their folding pathway. Here we observe that single-nanopore recording can be used as a new single molecule method to explore the unfolding transition and to examine the conformational space of native or variant proteins. We show that we can distinguish unfolded states from partially folded ones with the aerolysin pore. The unfolding transition curves of the destabilized variant are shifted toward the lower values of the denaturant agent compared to the wild type protein. The dynamics of the partially unfolded wild type protein follows a first-order transition. The denaturation curve obtained with the aerolysin pore is similar to that obtained with the ?-hemolysin pore. The nanopore geometry or net charge does not influence the folding transition but changes the dynamics. PMID:22260417

  20. Assaying the kinetics of protein denaturation catalyzed by AAA+ unfolding machines and proteases

    PubMed Central

    Baytshtok, Vladimir; Baker, Tania A.; Sauer, Robert T.

    2015-01-01

    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

  1. pE-DB: a database of structural ensembles of intrinsically disordered and of unfolded proteins

    E-print Network

    Sussman, Joel L.

    pE-DB: a database of structural ensembles of intrinsically disordered and of unfolded proteins, 3 CEA, CNRS, UJF-Grenoble 1, Protein Dynamics and Flexibility, Institut de Biologie Structurale Jean ensembles of intrinsically disordered proteins (IDPs) and of denatured proteins based on nuclear magnetic

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

    PubMed

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

    2013-02-01

    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

  3. Computational approaches to study protein unfolding: hen egg white lysozyme as a case study.

    PubMed

    Hünenberger, P H; Mark, A E; van Gunsteren, W F

    1995-03-01

    Four methods are compared to drive the unfolding of a protein: (1) high temperature (T-run), (2) high pressure (P-run), (3) by imposing a gradual increase in the mean radius of the protein using a penalty function added to the physical interaction function (F-run, radial force driven unfolding), and (4) by weak coupling of the difference between the temperature of the radially outward moving atoms and the radially inward moving atoms to an external temperature bath (K-run, kinetic energy driven unfolding). The characteristic features of the four unfolding pathways are analyzed in order to detect distortions due to the size or the type of the applied perturbation, as well as the features that are common to all of them. Hen egg white lysozyme is used as a test system. The simulations are analyzed and compared to experimental data like 1H-NMR amide proton exchange-folding competition, heat capacity, and compressibility measurements. PMID:7784424

  4. 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.

  5. A 22 bp cis-acting element is necessary and sufficient for the induction of the yeast KAR2 (BiP) gene by unfolded proteins.

    PubMed Central

    Mori, K; Sant, A; Kohno, K; Normington, K; Gething, M J; Sambrook, J F

    1992-01-01

    The KAR2 gene of Saccharomyces cerevisiae codes for an essential chaperone protein (BiP) that is localized in the lumen of the endoplasmic reticulum (ER). The high basal rate of transcription of KAR2 is increased transiently by heat shock: prolonged induction occurs when unfolded proteins accumulate in the ER. Three cis-acting elements in the KAR2 promoter control expression of KAR2: (i) a GC-rich region that contributes to the high level of constitutive expression, (ii) a functional heat shock element (HSE) and (iii) an element (UPR) that is involved in the induction of BiP mRNA by unfolded proteins. By analyzing internal deletion mutants of the KAR2 promoter, we demonstrate here that these three elements regulate transcription of KAR2 independently. Furthermore, the 22 bp UPR element causes a heterologous (CYC1) promoter to respond to the presence of unfolded proteins in the ER. Extracts of both stressed and unstressed yeast cells contain proteins that bind specifically to synthetic HSE and UPR elements and retard their migration through gels. Binding proteins specific for the UPR element can be fractionated by ammonium sulfate precipitation. Two of the proteins UPRF-1 and UPRF-2 (which is apparently a proteolytic degradation product of UPRF-1) bind inefficiently to mutant versions of the UPR that are unable to confer responsiveness to unfolded proteins to the (CYC1) promoter. UPRF-1 therefore displays the properties expected of a transcription factor that is involved in the sustained response of the KAR2 promoter to unfolded proteins in the ER. These experiments show that yeast cells can activate a transcription factor that stimulates expression of a nuclear gene in response to the accumulation of unfolded proteins in another cellular compartment. Images PMID:1628622

  6. Solvation in protein (un)folding of melittin tetramer–monomer transition

    PubMed Central

    Othon, Christina M.; Kwon, Oh-Hoon; Lin, Milo M.; Zewail, Ahmed H.

    2009-01-01

    Protein structural integrity and flexibility are intimately tied to solvation. Here, we examine the effect that changes in bulk and local solvent properties have on protein structure and stability. We observe the change in solvation of an unfolding of the protein model, melittin, in the presence of a denaturant, trifluoroethanol. The peptide system displays a well defined transition in that the tetramer unfolds without disrupting the secondary or tertiary structure. In the absence of local structural perturbation, we are able to reveal exclusively the role of solvation dynamics in protein structure stabilization and the (un)folding pathway. A sudden retardation in solvent dynamics, which is coupled to the change in protein structure, is observed at a critical trifluoroethanol concentration. The large amplitude conformational changes are regulated by the local solvent hydrophobicity and bulk solvent viscosity. PMID:19622745

  7. 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…

  8. Direct prediction of NMR residual dipolar couplings from the primary sequence of unfolded proteins.

    PubMed

    Huang, Jie-rong; Ozenne, Valéry; Jensen, Malene Ringkjøbing; Blackledge, Martin

    2013-01-01

    Conformational analysis: an approach to the prediction of RDCs from disordered protein chains, integrating the effect of nearest neighbors and the alignment characteristics of the statistical coil, is reported. NMR residual dipolar couplings (RDC) are sensitive probes of conformational sampling in unfolded proteins. PMID:23192984

  9. Single-Molecule Protein Unfolding and Translocation by an ATP-Fueled

    E-print Network

    Lang, Matthew

    Single-Molecule Protein Unfolding and Translocation by an ATP-Fueled Proteolytic Machine Marie ATP-powered proteases for protein- quality control and regulation. In the ClpXP protease, Clp with ClpXP. INTRODUCTION In all organisms, AAA+ family molecular machines harness the energy of ATP binding

  10. A Water-Explicit Lattice Model of Heat-, Cold-, and Pressure-Induced Protein Unfolding

    E-print Network

    A Water-Explicit Lattice Model of Heat-, Cold-, and Pressure-Induced Protein Unfolding Bryan A captures many aspects of water ther- modynamics, including the existence of density anomalies, and we exposure to water (6). Second, natural water-soluble proteins are unable to fold into their native states

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

    SciTech Connect

    Patananan, Alexander; Goheen, Steven C.

    2008-12-01

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

  12. 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, ?-glucosidase, and ovalbumin were investigated by adsorbing the globular proteins to non-porous cationic polymer beads. The protein fractions were adsorbed at different residence times (0, 9, 10, 20, and 30 min) at near-physiological conditions using a gradient elution system similar to that in high-performance liquid chromatography. The elution profi les and retention times were obtained by ultraviolet/visible spectrophotometry. A decrease in recovery was observed with time for almost all proteins and was attributed to irreversible protein unfolding on the non-porous surfaces. These data, and those of previous studies, fi t a positively increasing linear trend between percent unfolding after a fi xed (9 min) residence time (71.8%, 31.1%, and 32.1% of myoglobin, ?-glucosidase, and ovalbumin, respectively) and molecular weight. Of all the proteins examined so far, only myoglobin deviated from this trend with higher than predicted unfolding rates. Myoglobin also exhibited an increase in retention time over a wide temperature range (0°C and 55°C, 4.39 min and 5.74 min, respectively) whereas ovalbumin and ?-glucosidase did not. Further studies using a larger set of proteins are required to better understand the physiological and physiochemical implications of protein unfolding kinetics. This study confi rms that surface-mediated unfolding can be described by experimental techniques, thereby allowing for the better elucidation of the relationships between the structure and function of soluble proteins as well as other macromolecules.

  13. A hypothesis to reconcile the physical and chemical unfolding of proteins.

    PubMed

    de Oliveira, Guilherme A P; Silva, Jerson L

    2015-05-26

    High pressure (HP) or urea is commonly used to disturb folding species. Pressure favors the reversible unfolding of proteins by causing changes in the volumetric properties of the protein-solvent system. However, no mechanistic model has fully elucidated the effects of urea on structure unfolding, even though protein-urea interactions are considered to be crucial. Here, we provide NMR spectroscopy and 3D reconstructions from X-ray scattering to develop the "push-and-pull" hypothesis, which helps to explain the initial mechanism of chemical unfolding in light of the physical events triggered by HP. In studying MpNep2 from Moniliophthora perniciosa, we tracked two cooperative units using HP-NMR as MpNep2 moved uphill in the energy landscape; this process contrasts with the overall structural unfolding that occurs upon reaching a threshold concentration of urea. At subdenaturing concentrations of urea, we were able to trap a state in which urea is preferentially bound to the protein (as determined by NMR intensities and chemical shifts); this state is still folded and not additionally exposed to solvent [fluorescence and small-angle X-ray scattering (SAXS)]. This state has a higher susceptibility to pressure denaturation (lower p1/2 and larger ?Vu); thus, urea and HP share concomitant effects of urea binding and pulling and water-inducing pushing, respectively. These observations explain the differences between the molecular mechanisms that control the physical and chemical unfolding of proteins, thus opening up new possibilities for the study of protein folding and providing an interpretation of the nature of cooperativity in the folding and unfolding processes. PMID:25964355

  14. A hypothesis to reconcile the physical and chemical unfolding of proteins

    PubMed Central

    de Oliveira, Guilherme A. P.; Silva, Jerson L.

    2015-01-01

    High pressure (HP) or urea is commonly used to disturb folding species. Pressure favors the reversible unfolding of proteins by causing changes in the volumetric properties of the protein–solvent system. However, no mechanistic model has fully elucidated the effects of urea on structure unfolding, even though protein–urea interactions are considered to be crucial. Here, we provide NMR spectroscopy and 3D reconstructions from X-ray scattering to develop the “push-and-pull” hypothesis, which helps to explain the initial mechanism of chemical unfolding in light of the physical events triggered by HP. In studying MpNep2 from Moniliophthora perniciosa, we tracked two cooperative units using HP-NMR as MpNep2 moved uphill in the energy landscape; this process contrasts with the overall structural unfolding that occurs upon reaching a threshold concentration of urea. At subdenaturing concentrations of urea, we were able to trap a state in which urea is preferentially bound to the protein (as determined by NMR intensities and chemical shifts); this state is still folded and not additionally exposed to solvent [fluorescence and small-angle X-ray scattering (SAXS)]. This state has a higher susceptibility to pressure denaturation (lower p1/2 and larger ?Vu); thus, urea and HP share concomitant effects of urea binding and pulling and water-inducing pushing, respectively. These observations explain the differences between the molecular mechanisms that control the physical and chemical unfolding of proteins, thus opening up new possibilities for the study of protein folding and providing an interpretation of the nature of cooperativity in the folding and unfolding processes. PMID:25964355

  15. Forced protein unfolding leads to highly elastic and tough protein hydrogels

    NASA Astrophysics Data System (ADS)

    Fang, Jie; Mehlich, Alexander; Koga, Nobuyasu; Huang, Jiqing; Koga, Rie; Gao, Xiaoye; Hu, Chunguang; Jin, Chi; Rief, Matthias; Kast, Juergen; Baker, David; Li, Hongbin

    2013-12-01

    Protein-based hydrogels usually do not exhibit high stretchability or toughness, significantly limiting the scope of their potential biomedical applications. Here we report the engineering of a chemically cross-linked, highly elastic and tough protein hydrogel using a mechanically extremely labile, de novo-designed protein that assumes the classical ferredoxin-like fold structure. Due to the low mechanical stability of the ferredoxin-like fold structure, swelling of hydrogels causes a significant fraction of the folded domains to unfold. Subsequent collapse and aggregation of unfolded ferredoxin-like domains leads to intertwining of physically and chemically cross-linked networks, entailing hydrogels with unusual physical and mechanical properties: a negative swelling ratio, high stretchability and toughness. These hydrogels can withstand an average strain of 450% before breaking and show massive energy dissipation. Upon relaxation, refolding of the ferredoxin-like domains enables the hydrogel to recover its massive hysteresis. This novel biomaterial may expand the scope of hydrogel applications in tissue engineering.

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

    PubMed

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

    2012-03-01

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

  17. 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 (?/?)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

  18. Human defensins facilitate local unfolding of thermodynamically unstable regions of bacterial protein toxins.

    PubMed

    Kudryashova, Elena; Quintyn, Royston; Seveau, Stephanie; Lu, Wuyuan; Wysocki, Vicki H; Kudryashov, Dmitri S

    2014-11-20

    Defensins are short cationic, amphiphilic, cysteine-rich peptides that constitute the front-line immune defense against various pathogens. In addition to exerting direct antibacterial activities, defensins inactivate several classes of unrelated bacterial exotoxins. To date, no coherent mechanism has been proposed to explain defensins' enigmatic efficiency toward various toxins. In this study, we showed that binding of neutrophil ?-defensin HNP1 to affected bacterial toxins caused their local unfolding, potentiated their thermal melting and precipitation, exposed new regions for proteolysis, and increased susceptibility to collisional quenchers without causing similar effects on tested mammalian structural and enzymatic proteins. Enteric ?-defensin HD5 and ?-defensin hBD2 shared similar toxin-unfolding effects with HNP1, albeit to different degrees. We propose that protein susceptibility to inactivation by defensins is contingent to their thermolability and conformational plasticity and that defensin-induced unfolding is a key element in the general mechanism of toxin inactivation by human defensins. PMID:25517613

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

    SciTech Connect

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

    2014-05-28

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

  20. Diffuse transition state structure for the unfolding of a leucine-rich repeat protein

    PubMed Central

    Kelly, Sadie E.; Meisl, Georg; Rowling, Pamela J. E.; McLaughlin, Stephen H.; Knowles, Tuomas; Itzhaki, Laura S.

    2015-01-01

    Tandem-repeat proteins, such as leucine-rich repeats, comprise arrays of small structural motifs that pack in a linear fashion to produce elongated architectures. They lack contacts between residues that are distant in primary sequence, a feature that distinguishes them from the complex topologies of globular proteins. Here we have investigated the unfolding pathway of the leucine-rich repeat domain of the mRNA export protein TAP (TAPLRR) using ?-value analysis. Whereas most of the tandem-repeat proteins studied to date have been found to unfold via a polarised mechanism in which only a small, localised number of repeats are structured in the transition state, the unfolding mechanism of TAPLRR is more diffuse in nature. In the transition state for unfolding of TAPLRR, three of the four LRRs are highly structured and non-native interactions are formed within the N-terminal ?-helical cap and the first LRR. Thus, the ?-helical cap plays an important role in which non-native interactions are required to provide a scaffold for the LRRs to pack against in the folding reaction. PMID:24535093

  1. Protein Unfolding, and the "Tuning In" of Reversible Intermediate States, in Protic Ionic Liquid Media

    E-print Network

    Angell, C. Austen

    Protein Unfolding, and the "Tuning In" of Reversible Intermediate States, in Protic Ionic Liquid February 2008 Protic ionic liquids (PILs) are currently being shown to be as interesting and valuable mole fraction of a protic ionic liquid (PIL).7 Thus, where previous studies have been obliged to report

  2. John von Neumann Institute for Computing Protein Aggregation and Unfolding Studied

    E-print Network

    Irbäck, Anders

    John von Neumann Institute for Computing Protein Aggregation and Unfolding Studied Using an All H.E. Hansmann (Editors) John von Neumann Institute for Computing, J¨ulich, NIC Series, Vol. 34, ISBN-10: 3-9810843-0-6, ISBN-13: 978-3-9810843-0-6, pp. 15-20 , 2006. c 2006 by John von Neumann Institute

  3. UV Resonance Raman Determination of Protein Acid Denaturation: Selective Unfolding of Helical Segments of Horse Myoglobin

    E-print Network

    Asher, Sanford A.

    UV Resonance Raman Determination of Protein Acid Denaturation: Selective Unfolding of Helical, 1997 ABSTRACT: We have used UV resonance Raman spectroscopy to study the acid denaturation of horse the Tyr residues do not melt. The dramatic heme absorption acid denaturation changes indicate major

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

    PubMed

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

    2009-11-24

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

  5. 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

  6. 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:22004751

  7. 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

  8. 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 (PrP(Sc)) converted from a normal host cellular prion protein (PrP(C)). Experimental studies suggest that PrP(C) is enriched with ?-helical structure, whereas PrP(Sc) contains a high proportion of ?-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 PrP(C) 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

  9. 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 ?s, 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

  10. Protein unfolding an important process in vivo? Andreas Matouschek

    E-print Network

    Matouschek, Andreas

    is an important step in several cellular processes, most interestingly protein degradation by ATP translocation across some membranes; protein degradation by ATP-dependent proteases; and the pas- sive, chloroplasts, peroxisomes and the endoplasmic reticulum (ER) occurs through membrane channels, which are lined

  11. Thermodynamic analysis of an antagonistic folding-unfolding equilibrium between two protein domains.

    PubMed

    Cutler, Thomas A; Loh, Stewart N

    2007-08-10

    A simple model is formulated for analyzing the coupled folding-unfolding equilibrium present in a unique class of molecular switch proteins. We previously fused two single-domain proteins, barnase and ubiquitin, such that the free energy stored in the folded structure of one subunit is used to drive unfolding of the other. Here, we present a thermodynamic test of that mechanism. The antagonistic interaction is represented by a coupling free energy term DeltaGX. DeltaGX is the penalty imposed on folding of one domain by the native structure of the other. If DeltaGX=0, then neither domain senses the other and they fold and unfold independently. If DeltaGX>0, then destabilizing one domain will stabilize the other, and vice versa. In the limit where DeltaGX is greater than the intrinsic stability of either protein, then only one domain can be folded at any given time. We estimate DeltaGX by measuring stability parameters for a series of mutants that destabilize either the barnase or ubiquitin domains. Fitting the data to the model leads to a DeltaGX value of approximately 4 kcal mol(-1). DeltaGX is proposed to depend on both the length of the linker peptides used to join the two proteins, and on the inherent structural plasticity of each domain. We predict that shortening the linkers from their current lengths of two and three amino acid residues will increase structural and thermodynamic coupling. PMID:17572441

  12. ?-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 ?-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 ?-helical structure to the ?-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.

  13. 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 ?-sheet in PrP^Sc, and Prion protein's conformational conversion may involve an unfolding of H1 and refolding into ?-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 ?-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.

  14. Genetic regulation of spy gene expression in Escherichia coli in the presence of protein unfolding agent ethanol.

    PubMed

    Srivastava, Santosh Kumar; Lambadi, Paramesh Ramulu; Ghosh, Tamoghna; Pathania, Ranjana; Navani, Naveen Kumar

    2014-09-10

    In a living cell, folding of proteins is assisted by molecular chaperones and other folding helpers. In Escherichia coli (E. coli), recently an ATP independent chaperon 'Spy' was discovered which is highly up-regulated in the presence of protein unfolding agents like ethanol, butanol and tannic acid. Two response regulators; BaeR and CpxR have been recognized as transcriptional regulators of spy gene. However, the mechanism of genetic regulation of spy under protein denaturants like ethanol has not been studied in detail so far. Based on a combination of genetic, molecular biology and biochemical experimental data, we propose that BaeR protein is the primary regulator of spy gene in response to ethanol stress in E. coli. In addition, we expanded the experimental spectrum and validated that regulation of spy gene in the presence of zinc and copper metal stress is primarily via BaeR and CpxR regulators respectively. We also performed in-silico analysis to identify the homologs of Spy protein and their cognate regulatory elements in bacterial species belonging to enterobacteriaceae family. Based on the unique ATP-independent chaperone nature and genetic regulation of spy we also propose its importance in biosensor development and facilitated production of properly folded recombinant proteins. PMID:24999585

  15. 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

  16. 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 ?-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

  17. Mechanism of Protein Denaturation: Partial Unfolding of the P22 Coat Protein I-Domain by Urea Binding.

    PubMed

    Newcomer, Rebecca L; Fraser, LaTasha C R; Teschke, Carolyn M; Alexandrescu, Andrei T

    2015-12-15

    The I-domain is an insertion domain of the bacteriophage P22 coat protein that drives rapid folding and accounts for over half of the stability of the full-length protein. We sought to determine the role of hydrogen bonds (H-bonds) in the unfolding of the I-domain by examining (3)JNC' couplings transmitted through H-bonds, the temperature and urea-concentration dependence of (1)HN and (15)N chemical shifts, and native-state hydrogen exchange at urea concentrations where the domain is predominantly folded. The native-state hydrogen-exchange data suggest that the six-stranded ?-barrel core of the I-domain is more stable against unfolding than a smaller subdomain comprised of a short ?-helix and three-stranded ?-sheet. H-bonds, separately determined from solvent protection and (3)JNC' H-bond couplings, are identified with an accuracy of 90% by (1)HN temperature coefficients. The accuracy is improved to 95% when (15)N temperature coefficients are also included. In contrast, the urea dependence of (1)HN and (15)N chemical shifts is unrelated to H-bonding. The protein segments with the largest chemical-shift changes in the presence of urea show curved or sigmoidal titration curves suggestive of direct urea binding. Nuclear Overhauser effects to urea for these segments are also consistent with specific urea-binding sites in the I-domain. Taken together, the results support a mechanism of urea unfolding in which denaturant binds to distinct sites in the I-domain. Disordered segments bind urea more readily than regions in stable secondary structure. The locations of the putative urea-binding sites correlate with the lower stability of the structure against solvent exchange, suggesting that partial unfolding of the structure is related to urea accessibility. PMID:26682823

  18. 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

  19. 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

  20. Recognition and Binding of Human Telomeric G-Quadruplex DNA by Unfolding Protein 1

    PubMed Central

    2015-01-01

    The specific recognition by proteins of G-quadruplex structures provides evidence of a functional role for in vivo G-quadruplex structures. As previously reported, the ribonucleoprotein, hnRNP Al, and it is proteolytic derivative, unwinding protein 1 (UP1), bind to and destabilize G-quadruplex structures formed by the human telomeric repeat d(TTAGGG)n. UP1 has been proposed to be involved in the recruitment of telomerase to telomeres for chain extension. In this study, a detailed thermodynamic characterization of the binding of UP1 to a human telomeric repeat sequence, the d[AGGG(TTAGGG)3] G-quadruplex, is presented and reveals key insights into the UP1-induced unfolding of the G-quadruplex structure. The UP1–G-quadruplex interactions are shown to be enthalpically driven, exhibiting large negative enthalpy changes for the formation of both the Na+ and K+ G-quadruplex–UP1 complexes (?H values of ?43 and ?19 kcal/mol, respectively). These data reveal three distinct enthalpic contributions from the interactions of UP1 with the Na+ form of G-quadruplex DNA. The initial interaction is characterized by a binding affinity of 8.5 × 108 M–1 (strand), 200 times stronger than the binding of UP1 to a single-stranded DNA with a comparable but non-quadruplex-forming sequence [4.1 × 106 M–1 (strand)]. Circular dichroism spectroscopy reveals the Na+ form of the G-quadruplex to be completely unfolded by UP1 at a binding ratio of 2:1 (UP1:G-quadruplex DNA). The data presented here demonstrate that the favorable energetics of the initial binding event are closely coupled with and drive the unfolding of the G-quadruplex structure. PMID:24831962

  1. Long-Lived States to Monitor Protein Unfolding by Proton NMR

    PubMed Central

    Bornet, Aurélien; Ahuja, Puneet; Sarkar, Riddhiman; Fernandes, Laetitia; Hadji, Sonia; Lee, Shirley Y.; Haririnia, Aydin; Fushman, David; Bodenhausen, Geoffrey; Vasos, Paul R.

    2012-01-01

    The relaxation of long-lived states (LLS) corresponds to the slow return to statistical thermal equilibrium between symmetric and antisymmetric proton spin states. This process is remarkably sensitive to the presence of external spins and can be used to obtain information about partial unfolding of proteins. We detected the appearance of a destabilized conformer of ubiquitin when urea is added to the protein in its native state. This conformer shows increased mobility in the C-terminus, which significantly extends the lifetimes of proton LLS magnetisation in Ser-65. These changes could not be detected by conventional measurements of T1 and T2 relaxation times of protons, and would hardly be sensed by carbon-13 or nitrogen-15 relaxation measurements. Conformers with similar dynamic and structural features, as revealed by LLS relaxation times, could be observed, in the absence of urea, in two ubiquitin mutants, L67S and L69S. PMID:21882334

  2. Single-molecule chemo-mechanical unfolding reveals multiple transition state barriers in a small single-domain protein

    NASA Astrophysics Data System (ADS)

    Guinn, Emily J.; Jagannathan, Bharat; Marqusee, Susan

    2015-04-01

    A fundamental question in protein folding is whether proteins fold through one or multiple trajectories. While most experiments indicate a single pathway, simulations suggest proteins can fold through many parallel pathways. Here, we use a combination of chemical denaturant, mechanical force and site-directed mutations to demonstrate the presence of multiple unfolding pathways in a simple, two-state folding protein. We show that these multiple pathways have structurally different transition states, and that seemingly small changes in protein sequence and environment can strongly modulate the flux between the pathways. These results suggest that in vivo, the crowded cellular environment could strongly influence the mechanisms of protein folding and unfolding. Our study resolves the apparent dichotomy between experimental and theoretical studies, and highlights the advantage of using a multipronged approach to reveal the complexities of a protein's free-energy landscape.

  3. Ice-induced partial unfolding and aggregation of an integral membrane protein.

    PubMed

    Garber Cohen, Iona P; Castello, Pablo R; González Flecha, F Luis

    2010-11-01

    Although the deleterious effects of ice on water-soluble proteins are well established, little is known about the freeze stability of membrane proteins. Here we explore this issue through a combined kinetic and spectroscopic approach using micellar-purified plasma membrane calcium pump as a model. The ATPase activity of this protein significantly diminished after freezing using a slow-cooling procedure, with the decrease in the activity being an exponential function of the storage time at 253K, with t(½)=3.9±0.6h. On the contrary, no significant changes on enzyme activity were detected when a fast cooling procedure was performed. Regardless of the cooling rate, successive freeze-thaw cycles produced an exponential decrease in the Ca(2+)-ATPase activity, with the number of cycles at which the activity was reduced to half being 9.2±0.3 (fast cooling) and 3.7±0.2 (slow cooling). PAGE analysis showed that neither degradation nor formation of SDS-stable aggregates of the protein takes place during protein inactivation. Instead, the inactivation process was found to be associated with the irreversible partial unfolding of the polypeptide chain, as assessed by Trp fluorescence, far UV circular dichroism, and 1-anilino-naphtalene-8-sulfonate binding. This inactive protein undergoes, in a later stage, a further irreversible transformation leading to large aggregates. PMID:20691147

  4. 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 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. Electronic supplementary information (ESI) available: The schematic diagram of environment change, the comparison of F-E curves obtained at various stretching velocities, the details of the QM-WLC model and the process of generation of fitting curves, the comparison of F-E curves of polylysine obtained in PBS and I278 obtained in 6 M GdnHCl, the normalized F-E curves of polylysine obtained in a PBS solution, MD simulation movies, the hydrophobicity change of PAN/20S and HsIU. See DOI: 10.1039/c4nr07140a

  5. How does reorganization energy change upon protein unfolding? Monitoring the structural perturbations in the heme cavity of cytochrome c.

    PubMed

    Shafiey, Hassan; Ghourchian, Hedayatollah; Mogharrab, Navid

    2008-05-01

    In several classes of proteins the redox center provides an additional intrinsic biophysical probe that could be used to study the protein structure and function. In present report reorganization energy (lambda, as a parameter describing electron transfer properties) was used to study the protein structural changes around the heme prosthetic group in cytochrome c (cyt c). We attempted to monitor the value of this parameter upon the unfolding process of cyt c by urea, during which it was increased sigmoidally from about 0.52 to 0.82 eV for native and unfold protein, respectively. Results indicate that by structural changes in the heme site, lambda provides a complementary tool for following the unfolding process. Assuming a reversible two-state model for cyt c unfolding, Delta G(H2O), Cm and m values were determined to be 8.32+/-0.7 kcal mol(-1), 1.53+/-0.19 kcalmol(-1)M(-1) and 5.03 M, respectively. PMID:18325656

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

    E-print Network

    Tian, Weidong

    in protein science. Various physical and chemical perturbations such as temperature, pH, or chemical of advantages compared with other physical or chemical perturbations. It provides valuable information studied to date. The free energy (DGu) and volume change (DVu) for the unfolding are 5.9 kcal/mol and ÿ160

  7. The C-terminal domain of the HIV-1 Vif protein is natively unfolded in its unbound state

    E-print Network

    Lebendiker, Mario

    and indicate that it may gain structure upon binding its natural ligands. Keywords: biophysics/HIV-1/nativelyThe C-terminal domain of the HIV-1 Vif protein is natively unfolded in its unbound state Tali H of Chemistry and 2 The Wolfson Centre for Applied Structural Biology, Hebrew University of Jerusalem, Safra

  8. On the Mechanism of Protein Unfolding by Pressure A Molecular Dynamics Simulation Study

    NASA Astrophysics Data System (ADS)

    Grigera, J. Raul; McCarthy, Andres; Ferrara, Carlos

    2007-03-01

    Proteins are denaturized at high pressure and the mechanism of such a denaturation is still on debate. We have studied lyzozyme and apomyoglobin, under pressure up to 0.3GPa using molecular dynamics simulation. Lysozyme shows more stability, although it cannot retain the native structure. On the other hand apomyoglobin shows a continuing unfolding process during the 180 ns simulation time. The analysis of the hydrophilic and hydrophobic proteins Solvent Accessed Surface clearly shows the increment of the hydrophobic exposed area in the formation of crevices and in the appearing of hydrophobic `spikes' around the overall surface. The observation of the final state, within the simulation time, shows a clear effect on the conformational state of the proteins. Comparing the behavior of the proteins with de aggregation state of simple non-polar solutes at different pressures we have been able to conclude that the driving force of the denaturation is the change in the hydrophobic contribution to the native folding due to the changes of water structure under pressure, which have been shown both experimental and by computer simulation.

  9. 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

  10. The thermal unfolding of the ribosome-inactivating protein saporin-S6 characterized by infrared spectroscopy.

    PubMed

    Sánchez, Marina; Scirè, Andrea; Tanfani, Fabio; Ausili, Alessio

    2015-10-01

    Saporin-S6 is a plant toxin belonging to the type 1 ribosome-inactivating protein (RIP) family. Since it was extracted and isolated from Saponaria officinalis for the first time almost thirty years ago, the protein has been widely studied mainly for its potential applications in anti-tumour and anti-viral infection therapy. Like other RIPs, saporin-S6 is particularly effective in the form of immunotoxin conjugated with monoclonal antibodies and its chemico-physical characteristics made the protein a perfect candidate for the synthesis, development and use of saporin-S6-based chimeric toxins. The high stability of the protein against different denaturing agents has been broadly demonstrated, however, its complete thermal unfolding characterization has not already been performed. In this work we analyse in detail structure, thermostability and unfolding features by means of infrared spectroscopy coupled with two-dimensional correlation spectroscopy. Our data showed that saporin-S6 in solution at neutral pH exhibits a secondary structure analogue to that of the crystal and confirmed its good stability at moderately high temperatures, with a temperature of melting of 58°C. Our results also demonstrated that the thermal unfolding process is non-cooperative and occurs in two steps, and revealed the sequence of the events that take place during the denaturation, showing a higher stability of the N-terminal domain of the protein. PMID:26096917

  11. Lipid-dependent regulation of the unfolded protein response

    E-print Network

    Volmer, Romain; Ron, David

    2014-12-25

    , Bellenger J, Tessier C, Narce M, Rialland M: Inhibition of stearoyl-CoA desaturase 1 expression induces CHOP- dependent cell death in human cancer cells. PLoS ONE 2010, 5:e14363. 12. Sorli SC, Colie S, Albinet V, Dubrac A, Touriol C, Guilbaud N, Bedia C... , Fabrias G, Casas J, Segui B et al.: The nonlysosomal beta-glucosidase GBA2 promotes endoplasmic reticulum stress and impairs tumorigenicity of human melanoma cells. FASEB J 2013, 27:489-498. 13. Spassieva SD, Mullen TD, Townsend DM, Obeid LM: Disruption...

  12. Energy functions for protein design: adjustment with protein-protein complex affinities, models for the unfolded state, and negative design of solubility and specificity.

    PubMed

    Pokala, Navin; Handel, Tracy M

    2005-03-18

    The development of the EGAD program and energy function for protein design is described. In contrast to most protein design methods, which require several empirical parameters or heuristics such as patterning of residues or rotamers, EGAD has a minimalist philosophy; it uses very few empirical factors to account for inaccuracies resulting from the use of fixed backbones and discrete rotamers in protein design calculations, and describes the unfolded state, aggregates, and alternative conformers explicitly with physical models instead of fitted parameters. This approach unveils important issues in protein design that are often camouflaged by heuristic-emphasizing methods. Inter-atom energies are modeled with the OPLS-AA all-atom forcefield, electrostatics with the generalized Born continuum model, and the hydrophobic effect with a solvent-accessible surface area-dependent term. Experimental characterization of proteins designed with an unmodified version of the energy function revealed problems with under-packing, stability, aggregation, and structural specificity. Under-packing was addressed by modifying the van der Waals function. By optimizing only three parameters, the effects of >400 mutations on protein-protein complex formation were predicted to within 1.0 kcal mol(-1). As an independent test, this modified energy function was used to predict the stabilities of >1500 mutants to within 1.0 kcal mol(-1); this required a physical model of the unfolded state that includes more interactions than traditional tripeptide-based models. Solubility and structural specificity were addressed with simple physical approximations of aggregation and conformational equilibria. The complete energy function can design protein sequences that have high levels of identity with their natural counterparts, and have predicted structural properties more consistent with soluble and uniquely folded proteins than the initial designs. PMID:15733929

  13. Protein unfolding from free-energy calculations: Integration of the Gaussian network model with bond binding energies

    NASA Astrophysics Data System (ADS)

    Srivastava, Amit; Granek, Rony

    2015-02-01

    Motivated by single molecule experiments, we study thermal unfolding pathways of four proteins, chymotrypsin inhibitor, barnase, ubiquitin, and adenylate kinase, using bond network models that combine bond energies and elasticity. The protein elasticity is described by the Gaussian network model (GNM), to which we add prescribed bond binding energies that are assigned to all (nonbackbone) connecting bonds in the GNM of native state and assumed identical for simplicity. Using exact calculation of the Helmholtz free energy for this model, we consider bond rupture single events. The bond designated for rupture is chosen by minimizing the free-energy difference for the process, over all (nonbackbone) bonds in the network. Plotting the free-energy profile along this pathway at different temperatures, we observe a few major partial unfolding, metastable or stable, states, that are separated by free-energy barriers and change role as the temperature is raised. In particular, for adenylate kinase we find three major partial unfolding states, which is consistent with single molecule FRET experiments [Pirchi et al., Nat. Commun. 2, 493 (2011), 10.1038/ncomms1504] for which hidden Markov analysis reveals between three and five such states. Such states can play a major role in enzymatic activity.

  14. An alternative explanation for the collapse of unfolded proteins in an aqueous mixture of urea and guanidinium chloride

    NASA Astrophysics Data System (ADS)

    Graziano, Giuseppe

    2014-09-01

    Molecular dynamics simulations have shown that a totally unfolded protein in aqueous 8 M urea undergoes a collapse transition on replacing urea molecules by guanidinium chloride, GdmCl, assuming a compact conformation in 4 M urea + 4 M GdmCl [J. Am. Chem. Soc. 134 (2012) 18266]. This is unexpected because GdmCl is a denaturant stronger than urea. It is shown that such collapse can originate from an increase in the magnitude of the solvent-excluded volume effect due the high density of urea + GdmCl mixtures, coupled to their low water number density that pushes denaturant molecules toward the protein surface.

  15. Peptides as Model Systems for the Unfolded State of Proteins Explored By Vibrational Spectroscopy

    NASA Astrophysics Data System (ADS)

    Schweitzer-Stenner, Reinhard; Measey, Thomas; Hagarman, Andrew

    2008-11-01

    Unfolded proteins are generally thought to be structurally random with a minimum of non-local interactions. This concept implies that with the exception of glycine and proline the conformational propensities of amino acid residues in polypeptides should be comparable in that they all sample the statistically allowed region of the Ramachandran plot. However, over the last ten years experimental and computational evidence has emerged for the notion that the conformational space of residues might be more restricted than predicted by random or statistical coil models. We have developed several algorithms which can be used to simulate the amide I band profile of the IR, isotropic Raman, anisotropic Raman and Vibrational Circular Dichroism (VCD) spectra of polypeptides based on assumed ensembles of side chain conformations. The simulations are generally restricted by 3Jc?HNH coupling constants obtained from NMR spectroscopy. A comparison with experimental results reveals that e.g. alanine has a clear preference for the so called polyproline II (PPII) conformation in short peptides. The situation becomes more complex if longer polyalanines are doped with negatively charged residues. For the so-called XAO-peptide (X2A7O2, X: diaminobutyric acid, O;ornithine) we found a more compact structure owing to multiple turn conformations sampled by the X2A7 interfaces. For Salmon Calcitonin, a 32-residue hormone, we identified a mixture of PPII, ?-strand and helical conformations. Currently, we are in the process of investigating short GxG (x; different natural amino acid residues) peptides in terms of conformational distributions obtained from coil libraries. This will enable us obtain the conformational preferences of amino acid residues in the absence of nearest neighbor interactions.

  16. Structural Characteristic of the Initial Unfolded State on Refolding Determines Catalytic Efficiency of the Folded Protein in Presence of Osmolytes

    PubMed Central

    Warepam, Marina; Sharma, Gurumayum Suraj; Dar, Tanveer Ali; Khan, Md. Khurshid Alam; Singh, Laishram Rajendrakumar

    2014-01-01

    Osmolytes are low molecular weight organic molecules accumulated by organisms to assist proper protein folding, and to provide protection to the structural integrity of proteins under denaturing stress conditions. It is known that osmolyte-induced protein folding is brought by unfavorable interaction of osmolytes with the denatured/unfolded states. The interaction of osmolyte with the native state does not significantly contribute to the osmolyte-induced protein folding. We have therefore investigated if different denatured states of a protein (generated by different denaturing agents) interact differently with the osmolytes to induce protein folding. We observed that osmolyte-assisted refolding of protein obtained from heat-induced denatured state produces native molecules with higher enzyme activity than those initiated from GdmCl- or urea-induced denatured state indicating that the structural property of the initial denatured state during refolding by osmolytes determines the catalytic efficiency of the folded protein molecule. These conclusions have been reached from the systematic measurements of enzymatic kinetic parameters (Km and kcat), thermodynamic stability (Tm and ?Hm) and secondary and tertiary structures of the folded native proteins obtained from refolding of various denatured states (due to heat-, urea- and GdmCl-induced denaturation) of RNase-A in the presence of various osmolytes. PMID:25313668

  17. Mistargeted mitochondrial proteins activate a proteostatic response in the cytosol.

    PubMed

    Wrobel, Lidia; Topf, Ulrike; Bragoszewski, Piotr; Wiese, Sebastian; Sztolsztener, Malgorzata E; Oeljeklaus, Silke; Varabyova, Aksana; Lirski, Maciej; Chroscicki, Piotr; Mroczek, Seweryn; Januszewicz, Elzbieta; Dziembowski, Andrzej; Koblowska, Marta; Warscheid, Bettina; Chacinska, Agnieszka

    2015-08-27

    Most of the mitochondrial proteome originates from nuclear genes and is transported into the mitochondria after synthesis in the cytosol. Complex machineries which maintain the specificity of protein import and sorting include the TIM23 translocase responsible for the transfer of precursor proteins into the matrix, and the mitochondrial intermembrane space import and assembly (MIA) machinery required for the biogenesis of intermembrane space proteins. Dysfunction of mitochondrial protein sorting pathways results in diminishing specific substrate proteins, followed by systemic pathology of the organelle and organismal death. The cellular responses caused by accumulation of mitochondrial precursor proteins in the cytosol are mainly unknown. Here we present a comprehensive picture of the changes in the cellular transcriptome and proteome in response to a mitochondrial import defect and precursor over-accumulation stress. Pathways were identified that protect the cell against mitochondrial biogenesis defects by inhibiting protein synthesis and by activation of the proteasome, a major machine for cellular protein clearance. Proteasomal activity is modulated in proportion to the quantity of mislocalized mitochondrial precursor proteins in the cytosol. We propose that this type of unfolded protein response activated by mistargeting of proteins (UPRam) is beneficial for the cells. UPRam provides a means for buffering the consequences of physiological slowdown in mitochondrial protein import and for counteracting pathologies that are caused or contributed by mitochondrial dysfunction. PMID:26245374

  18. Insight into the Unfolding Properties of Chd64, a Small, Single Domain Protein with a Globular Core and Disordered Tails.

    PubMed

    Tarczewska, Aneta; Koz?owska, Ma?gorzata; Dobryszycki, Piotr; Kaus-Drobek, Magdalena; Dadlez, Micha?; O?yhar, Andrzej

    2015-01-01

    Two major lipophilic hormones, 20-hydroxyecdysone (20E) and juvenile hormone (JH), govern insect development and growth. While the mode of action of 20E is well understood, some understanding of JH-dependent signalling has been attained only in the past few years, and the crosstalk of the two hormonal pathways remains unknown. Two proteins, the calponin-like Chd64 and immunophilin FKBP39 proteins, have recently been found to play pivotal roles in the formation of dynamic, multiprotein complex that cross-links these two signalling pathways. However, the molecular mechanism of the interaction remains unexplored. The aim of this work was to determine structural elements of Chd64 to provide an understanding of molecular basis of multiple interactions. We analysed Chd64 in two unrelated insect species, Drosophila melanogaster (DmChd64) and Tribolium castaneum (TcChd64). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS), we showed that both Chd64 proteins have disordered tails that outflank the globular core. The folds of the globular cores of both Chd64 resemble the calponin homology (CH) domain previously resolved by crystallography. Monitoring the unfolding of DmChd64 and TcChd64 by far-ultraviolet (UV) circular dichroism (CD) spectroscopy, fluorescence spectroscopy and size-exclusion chromatography (SEC) revealed a highly complex process. Chd64 unfolds and forms of a molten globule (MG)-like intermediate state. Furthermore, our data indicate that in some conditions, Chd64 may exists in discrete structural forms, indicating that the protein is pliable and capable of easily acquiring different conformations. The plasticity of Chd64 and the existence of terminal intrinsically disordered regions (IDRs) may be crucial for multiple interactions with many partners. PMID:26325194

  19. Insight into the Unfolding Properties of Chd64, a Small, Single Domain Protein with a Globular Core and Disordered Tails

    PubMed Central

    Dobryszycki, Piotr; Kaus-Drobek, Magdalena; Dadlez, Micha?; O?yhar, Andrzej

    2015-01-01

    Two major lipophilic hormones, 20-hydroxyecdysone (20E) and juvenile hormone (JH), govern insect development and growth. While the mode of action of 20E is well understood, some understanding of JH-dependent signalling has been attained only in the past few years, and the crosstalk of the two hormonal pathways remains unknown. Two proteins, the calponin-like Chd64 and immunophilin FKBP39 proteins, have recently been found to play pivotal roles in the formation of dynamic, multiprotein complex that cross-links these two signalling pathways. However, the molecular mechanism of the interaction remains unexplored. The aim of this work was to determine structural elements of Chd64 to provide an understanding of molecular basis of multiple interactions. We analysed Chd64 in two unrelated insect species, Drosophila melanogaster (DmChd64) and Tribolium castaneum (TcChd64). Using hydrogen-deuterium exchange mass spectrometry (HDX-MS), we showed that both Chd64 proteins have disordered tails that outflank the globular core. The folds of the globular cores of both Chd64 resemble the calponin homology (CH) domain previously resolved by crystallography. Monitoring the unfolding of DmChd64 and TcChd64 by far-ultraviolet (UV) circular dichroism (CD) spectroscopy, fluorescence spectroscopy and size-exclusion chromatography (SEC) revealed a highly complex process. Chd64 unfolds and forms of a molten globule (MG)—like intermediate state. Furthermore, our data indicate that in some conditions, Chd64 may exists in discrete structural forms, indicating that the protein is pliable and capable of easily acquiring different conformations. The plasticity of Chd64 and the existence of terminal intrinsically disordered regions (IDRs) may be crucial for multiple interactions with many partners. PMID:26325194

  20. Unfolding stabilities of two structurally similar proteins as probed by temperature-induced and force-induced molecular dynamics simulations.

    PubMed

    Gorai, Biswajit; Prabhavadhni, Arasu; Sivaraman, Thirunavukkarasu

    2015-09-01

    Unfolding stabilities of two homologous proteins, cardiotoxin III and short-neurotoxin (SNTX) belonging to three-finger toxin (TFT) superfamily, have been probed by means of molecular dynamics (MD) simulations. Combined analysis of data obtained from steered MD and all-atom MD simulations at various temperatures in near physiological conditions on the proteins suggested that overall structural stabilities of the two proteins were different from each other and the MD results are consistent with experimental data of the proteins reported in the literature. Rationalization for the differential structural stabilities of the structurally similar proteins has been chiefly attributed to the differences in the structural contacts between C- and N-termini regions in their three-dimensional structures, and the findings endorse the 'CN network' hypothesis proposed to qualitatively analyse the thermodynamic stabilities of proteins belonging to TFT superfamily of snake venoms. Moreover, the 'CN network' hypothesis has been revisited and the present study suggested that 'CN network' should be accounted in terms of 'structural contacts' and 'structural strengths' in order to precisely describe order of structural stabilities of TFTs. PMID:25387083

  1. "Invisible" conformers of an antifungal disulfide protein revealed by constrained cold and heat unfolding, CEST-NMR experiments, and molecular dynamics calculations.

    PubMed

    Fizil, Ádám; Gáspári, Zoltán; Barna, Terézia; Marx, Florentine; Batta, Gyula

    2015-03-23

    Transition between conformational states in proteins is being recognized as a possible key factor of function. In support of this, hidden dynamic NMR structures were detected in several cases up to populations of a few percent. Here, we show by two- and three-state analysis of thermal unfolding, that the population of hidden states may weight 20-40?% at 298?K in a disulfide-rich protein. In addition, sensitive (15) N-CEST NMR experiments identified a low populated (0.15?%) state that was in slow exchange with the folded PAF protein. Remarkably, other techniques failed to identify the rest of the NMR "dark matter". Comparison of the temperature dependence of chemical shifts from experiments and molecular dynamics calculations suggests that hidden conformers of PAF differ in the loop and terminal regions and are most similar in the evolutionary conserved core. Our observations point to the existence of a complex conformational landscape with multiple conformational states in dynamic equilibrium, with diverse exchange rates presumably responsible for the completely hidden nature of a considerable fraction. PMID:25676351

  2. Urea denaturation by stronger dispersion interactions with proteins than water implies a 2-stage unfolding

    E-print Network

    Thirumalai, Devarajan

    Urea denaturation by stronger dispersion interactions with proteins than water implies a 2-stage of denaturation of proteins by urea is explored by using all-atom microseconds molecular dynamics simulations mechanism'' whereby urea has a stronger dispersion interaction with protein than water. denaturing mechanism

  3. Refinement of non-calorimetric determination of the change in heat capacity, ?Cp, of protein unfolding and validation across a wide temperature range

    PubMed Central

    Singh, Deepika Talla; Stites, Wesley E.

    2008-01-01

    The change in heat capacity, ?Cp, upon protein unfolding has been usually determined by calorimetry. A non-calorimetric method which employs the Gibbs-Helmholtz relationship to determine ?Cp has seen some use. Generally, the free energy change upon unfolding of the protein is determined at a variety of temperatures and the temperature at which ?G is zero, Tm, and change in enthalpy at Tm are determined by thermal denaturation and ?Cp is then calculated using the Gibbs-Helmholtz equation. We show here that an abbreviated method with stability determinations at just two temperatures gives values of ?Cp consistent with values from free energy change upon unfolding determination at a much wider range of temperatures. Further, even the free energy change upon unfolding from a single solvent denaturation at the proper temperature, when coupled with the melting temperature, Tm, and the van’t Hoff enthalpy, ?HvH, from a thermal denaturation, gives a reasonable estimate of ?Cp, albeit with greater uncertainty than solvent denaturations at two temperatures. We also find that non-linear regression of the Gibbs-Helmholtz equation as a function of stability and temperature while simultaneously fitting ?Cp, Tm, and ?HvH gives values for the last two parameters that are in excellent agreement with experimental values. PMID:18384147

  4. Halophilic Protein Adaptation Results from Synergistic Residue-Ion Interactions in the Folded and Unfolded States.

    PubMed

    Ortega, Gabriel; Diercks, Tammo; Millet, Oscar

    2015-12-17

    Halophilic organisms thrive in environments with extreme salt concentrations and have adapted by allowing molar quantities of cosolutes, mainly KCl, to accumulate in their cytoplasm. To cope with this high intracellular salinity, halophilic organisms modified the chemical composition of their proteins to enrich their surface with acidic and short polar side chains, while lysines and bulky hydrophobic residues got depleted. We have emulated the evolutionary process of haloadaptation with natural and designed halophilic polypeptides and applied novel nuclear magnetic resonance (NMR) methodology to study the different mechanisms contributing to protein haloadaptation at a per residue level. Our analysis of an extensive set of NMR observables, determined over several proteins, allowed us to disentangle the synergistic contributions of protein haloadaptation: cation exclusion and electrostatic repulsion between negatively charged residues destabilize the denatured state ensemble while cumulative weak cation-protein interactions stabilize the folded conformations. PMID:26628359

  5. G protein-coupled receptors show unusual patterns of intrinsic unfolding.

    PubMed

    Jaakola, Veli-Pekka; Prilusky, Jaime; Sussman, Joel L; Goldman, Adrian

    2005-02-01

    Intrinsically unstructured proteins (IUPs) or IUP-like regions often play key roles in controlling processes ranging from transcription to the cell cycle. In silico such proteins can be identified by their sequence properties; they have low hydrophobicity and high net charge. In this study, we applied the FoldIndex (http://bioportal.weizmann.ac.il/fldbin/findex) program to analyze human G protein-coupled receptors and compared them with membrane proteins of known structure and with IUPs. We show that human G protein-coupled receptor (GPCR) extramembranous domains include long (>50 residues) disordered segments, unlike membrane proteins of known structure. The predicted disorder occurred primarily in the N-terminal, C-terminal and third intracellular domain regions: 55, 69 and 56% of the human GPCRs were disordered in these regions, respectively. This increased flexibility may therefore be critical for GPCR function. Surprisingly, however, the kinds of residues used in GPCR unstructured regions were different than in hitherto-identified IUPs. The GPCR third intracellular loop domains contain very high percentages of Arg, Lys and His residues, especially Arg, but the percentage of Glu, Asp and Pro is no higher than in folded proteins. We propose that this has structural and functional consequences. PMID:15790574

  6. A review of methods available to estimate solvent-accessible surface areas of soluble proteins in the folded and unfolded states.

    PubMed

    Ali, Syed Ausaf; Hassan, Md Imtaiyaz; Islam, Asimul; Ahmad, Faizan

    2014-01-01

    Solvent accessible surface area (SASA) of proteins has always been considered as a decisive factor in protein folding and stability studies. It is defined as the surface characterized around a protein by a hypothetical centre of a solvent sphere with the van der Waals contact surface of the molecule. Based on SASA values, amino acid residues of a protein can be classified as buried or exposed. There are various types of SASAs starting from relative solvent accessibility to absolute surface areas. Direct estimation of accurate SASAs of folded proteins experimentally at the atomic level is not possible. However, the SASA of a native protein can be estimated computationally from the atomic coordinates. Similarly, various simulation methods are available to compute the SASA of a protein in its unfolded state. In efforts to estimate the changes in SASA related to the protein folding, a number of the unfolded state models have been proposed. In this review, we have summarized different algorithms and computational tools for SASA estimations. Furthermore, online resources for SASA calculations and representations have also been discussed in detail. This review will be useful for protein chemists and biologists for the accurate measurements of SASA and its subsequent applications for the calculation of various biophysical and thermodynamic properties of proteins. PMID:24678666

  7. Cooperative Unfolding of Residual Structure in Heat Denatured Proteins by Urea and Guanidinium Chloride

    PubMed Central

    Singh, Ritu; Hassan, Md. Imtaiyaz; Islam, Asimul; Ahmad, Faizan

    2015-01-01

    The denatured states of proteins have always attracted our attention due to the fact that the denatured state is the only experimentally achievable state of a protein, which can be taken as initial reference state for considering the in vitro folding and defining the native protein stability. It is known that heat and guanidinium chloride (GdmCl) give structurally different states of RNase-A, lysozyme, ?-chymotrypsinogen A and ?-lactalbumin. On the contrary, differential scanning calorimetric (DSC) and isothermal titration calorimetric measurements, reported in the literature, led to the conclusion that heat denatured and GdmCl denatured states are thermodynamically and structurally identical. In order to resolve this controversy, we have measured changes in the far-UV CD (circular dichroism) of these heat-denatured proteins on the addition of different concentrations of GdmCl. The observed sigmoidal curve of each protein was analyzed for Gibbs free energy change in the absence of the denaturant (?G0X?D) associated with the process heat denatured (X) state ? GdmCl denatured (D) state. To confirm that this thermodynamic property represents the property of the protein alone and is not a manifestation of salvation effect, we measured urea-induced denaturation curves of these heat denatured proteins under the same experimental condition in which GdmCl-induced denaturation was carried out. In this paper we report that (a) heat denatured proteins contain secondary structure, and GdmCl (or urea) induces a cooperative transition between X and D states, (b) for each protein at a given pH and temperature, thermodynamic cycle connects quantities, ?G0N?X (native (N) state ? X state), ?G0X?D and ?G0N?D (N state ? D state), and (c) there is not a good enthalpy difference between X and D states, which is the reason for the absence of endothermic peak in DSC scan for the transition, X state ? D state. PMID:26046628

  8. Single-Molecule Protein Unfolding in Solid State Nanopores David S. Talaga*,

    E-print Network

    Li, Jiali

    with that predicted by a simple sum of the partial volumes of the amino acids in the polypeptide segment inside is a common event in trafficking of proteins between eukaryotic organelles. Emerging technolo- gies have, the signal depends on the amino acid sequence through the volume and charge of the polypeptide segment

  9. ClpXP, an ATP-powered unfolding and protein-degradation machine

    E-print Network

    Sauer, Robert T.

    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 ...

  10. pE-DB: a database of structural ensembles of intrinsically disordered and of unfolded proteins.

    PubMed

    Varadi, Mihaly; Kosol, Simone; Lebrun, Pierre; Valentini, Erica; Blackledge, Martin; Dunker, A Keith; Felli, Isabella C; Forman-Kay, Julie D; Kriwacki, Richard W; Pierattelli, Roberta; Sussman, Joel; Svergun, Dmitri I; Uversky, Vladimir N; Vendruscolo, Michele; Wishart, David; Wright, Peter E; Tompa, Peter

    2014-01-01

    The goal of pE-DB (http://pedb.vib.be) is to serve as an openly accessible database for the deposition of structural ensembles of intrinsically disordered proteins (IDPs) and of denatured proteins based on nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and other data measured in solution. Owing to the inherent flexibility of IDPs, solution techniques are particularly appropriate for characterizing their biophysical properties, and structural ensembles in agreement with these data provide a convenient tool for describing the underlying conformational sampling. Database entries consist of (i) primary experimental data with descriptions of the acquisition methods and algorithms used for the ensemble calculations, and (ii) the structural ensembles consistent with these data, provided as a set of models in a Protein Data Bank format. PE-DB is open for submissions from the community, and is intended as a forum for disseminating the structural ensembles and the methodologies used to generate them. While the need to represent the IDP structures is clear, methods for determining and evaluating the structural ensembles are still evolving. The availability of the pE-DB database is expected to promote the development of new modeling methods and leads to a better understanding of how function arises from disordered states. PMID:24174539

  11. pE-DB: a database of structural ensembles of intrinsically disordered and of unfolded proteins

    PubMed Central

    Varadi, Mihaly; Kosol, Simone; Lebrun, Pierre; Valentini, Erica; Blackledge, Martin; Dunker, A. Keith; Felli, Isabella C.; Forman-Kay, Julie D.; Kriwacki, Richard W.; Pierattelli, Roberta; Sussman, Joel; Svergun, Dmitri I.; Uversky, Vladimir N.; Vendruscolo, Michele; Wishart, David; Wright, Peter E.; Tompa, Peter

    2014-01-01

    The goal of pE-DB (http://pedb.vib.be) is to serve as an openly accessible database for the deposition of structural ensembles of intrinsically disordered proteins (IDPs) and of denatured proteins based on nuclear magnetic resonance spectroscopy, small-angle X-ray scattering and other data measured in solution. Owing to the inherent flexibility of IDPs, solution techniques are particularly appropriate for characterizing their biophysical properties, and structural ensembles in agreement with these data provide a convenient tool for describing the underlying conformational sampling. Database entries consist of (i) primary experimental data with descriptions of the acquisition methods and algorithms used for the ensemble calculations, and (ii) the structural ensembles consistent with these data, provided as a set of models in a Protein Data Bank format. PE-DB is open for submissions from the community, and is intended as a forum for disseminating the structural ensembles and the methodologies used to generate them. While the need to represent the IDP structures is clear, methods for determining and evaluating the structural ensembles are still evolving. The availability of the pE-DB database is expected to promote the development of new modeling methods and leads to a better understanding of how function arises from disordered states. PMID:24174539

  12. Trapping, unfolding, identifying, and binding single proteins using the double-nanohole optical trap

    NASA Astrophysics Data System (ADS)

    Wheaton, Skyler; Kotnala, Abhay; Al Balushi, Ahmed; Gefald, Ryan M.; Zehtabi-Oskuie, Ana; Rajashekara, Yashaswini; Gordon, Reuven

    2014-05-01

    In this paper we describe the double nanohole laser tweezer system used to trap single nanoparticles. We cover the basic theory behind the DNH and what makes it more powerful than traditional laser tweezers commonly used for larger particles. We outline the basic setup used to reliably trap several different types of particles ranging in size from 1 nm to 40 nm. Data from several experiments is shown which displays exactly how a particle is confirmed to be trapped. We will discuss the use of autocorrelation as well as other information that can be extracted from the optical transmission in our setup and how it has been applied to the identification of protein small molecule interactions and protein binding. Other uses of the data collected from our setup will be discussed including the observation of protein folding. Finally we discuss the current developments of the process and its possible uses as a drug discovery tool, a new type of single particle nanopipette and new bio-sensors.

  13. Verification of unfold error estimates in the unfold operator code

    NASA Astrophysics Data System (ADS)

    Fehl, D. L.; Biggs, F.

    1997-01-01

    Spectral unfolding is an inverse mathematical operation that attempts to obtain spectral source information from a set of response functions and data measurements. Several unfold algorithms have appeared over the past 30 years; among them is the unfold operator (UFO) code written at Sandia National Laboratories. In addition to an unfolded spectrum, the UFO code also estimates the unfold uncertainty (error) induced by estimated random uncertainties in the data. In UFO the unfold uncertainty is obtained from the error matrix. This built-in estimate has now been compared to error estimates obtained by running the code in a Monte Carlo fashion with prescribed data distributions (Gaussian deviates). In the test problem studied, data were simulated from an arbitrarily chosen blackbody spectrum (10 keV) and a set of overlapping response functions. The data were assumed to have an imprecision of 5% (standard deviation). One hundred random data sets were generated. The built-in estimate of unfold uncertainty agreed with the Monte Carlo estimate to within the statistical resolution of this relatively small sample size (95% confidence level). A possible 10% bias between the two methods was unresolved. The Monte Carlo technique is also useful in underdetermined problems, for which the error matrix method does not apply. UFO has been applied to the diagnosis of low energy x rays emitted by Z-pinch and ion-beam driven hohlraums.

  14. Verification of unfold error estimates in the unfold operator code

    SciTech Connect

    Fehl, D.L.; Biggs, F.

    1997-01-01

    Spectral unfolding is an inverse mathematical operation that attempts to obtain spectral source information from a set of response functions and data measurements. Several unfold algorithms have appeared over the past 30 years; among them is the unfold operator (UFO) code written at Sandia National Laboratories. In addition to an unfolded spectrum, the UFO code also estimates the unfold uncertainty (error) induced by estimated random uncertainties in the data. In UFO the unfold uncertainty is obtained from the error matrix. This built-in estimate has now been compared to error estimates obtained by running the code in a Monte Carlo fashion with prescribed data distributions (Gaussian deviates). In the test problem studied, data were simulated from an arbitrarily chosen blackbody spectrum (10 keV) and a set of overlapping response functions. The data were assumed to have an imprecision of 5{percent} (standard deviation). One hundred random data sets were generated. The built-in estimate of unfold uncertainty agreed with the Monte Carlo estimate to within the statistical resolution of this relatively small sample size (95{percent} confidence level). A possible 10{percent} bias between the two methods was unresolved. The Monte Carlo technique is also useful in underdetermined problems, for which the error matrix method does not apply. UFO has been applied to the diagnosis of low energy x rays emitted by Z-pinch and ion-beam driven hohlraums. {copyright} {ital 1997 American Institute of Physics.}

  15. Trapping a salt-dependent unfolding intermediate of the marginally stable protein Yfh1.

    PubMed

    Vilanova, Bartolomé; Sanfelice, Domenico; Martorell, Gabriel; Temussi, Piero A; Pastore, Annalisa

    2014-01-01

    Yfh1, the yeast ortholog of frataxin, is a protein of limited thermodynamic stability which undergoes cold denaturation at temperatures above the water freezing point. We have previously demonstrated that its stability is strongly dependent on ionic strength and that monovalent or divalent cations are able to considerably stabilize the fold. Here, we present a study of the folded state and of the structural determinants that lead to the strong salt dependence. We demonstrate by nuclear magnetic resonance that, at room temperature, Yfh1 exists as an equilibrium mixture of a folded species and a folding intermediate in slow exchange equilibrium. The equilibrium completely shifts in favor of the folded species by the addition of even small concentrations of salt. We demonstrate that Yfh1 is destabilized by a localized energetic frustration arising from an "electrostatic hinge" made of negatively charged residues mapped in the ?-sheet. Salt interactions at this site have a "frustration-relieving" effect. We discuss the consequences of our findings for the function of Yfh1 and for our understanding of protein folding stability. PMID:25988154

  16. Trapping a salt-dependent unfolding intermediate of the marginally stable protein Yfh1

    PubMed Central

    Vilanova, Bartolomé; Sanfelice, Domenico; Martorell, Gabriel; Temussi, Piero A.; Pastore, Annalisa

    2014-01-01

    Yfh1, the yeast ortholog of frataxin, is a protein of limited thermodynamic stability which undergoes cold denaturation at temperatures above the water freezing point. We have previously demonstrated that its stability is strongly dependent on ionic strength and that monovalent or divalent cations are able to considerably stabilize the fold. Here, we present a study of the folded state and of the structural determinants that lead to the strong salt dependence. We demonstrate by nuclear magnetic resonance that, at room temperature, Yfh1 exists as an equilibrium mixture of a folded species and a folding intermediate in slow exchange equilibrium. The equilibrium completely shifts in favor of the folded species by the addition of even small concentrations of salt. We demonstrate that Yfh1 is destabilized by a localized energetic frustration arising from an “electrostatic hinge” made of negatively charged residues mapped in the ?-sheet. Salt interactions at this site have a “frustration-relieving” effect. We discuss the consequences of our findings for the function of Yfh1 and for our understanding of protein folding stability. PMID:25988154

  17. Partially Unfolded Forms of the Prion Protein Populated under Misfolding-promoting Conditions: CHARACTERIZATION BY HYDROGEN EXCHANGE MASS SPECTROMETRY AND NMR.

    PubMed

    Moulick, Roumita; Das, Ranabir; Udgaonkar, Jayant B

    2015-10-16

    The susceptibility of the cellular prion protein (PrP(C)) to convert to an alternative misfolded conformation (PrP(Sc)), which is the key event in the pathogenesis of prion diseases, is indicative of a conformationally flexible native (N) state. In the present study, hydrogen-deuterium exchange (HDX) in conjunction with mass spectrometry and nuclear magnetic resonance spectroscopy were used for the structural and energetic characterization of the N state of the full-length mouse prion protein, moPrP(23-231), under conditions that favor misfolding. The kinetics of HDX of 34 backbone amide hydrogens in the N state were determined at pH 4. In contrast to the results of previous HDX studies on the human and Syrian hamster prion proteins at a higher pH, various segments of moPrP were found to undergo different extents of subglobal unfolding events at pH 4, a pH at which the protein is known to be primed to misfold to a ?-rich conformation. No residual structure around the disulfide bond was observed for the unfolded state at pH 4. The N state of the prion protein was observed to be at equilibrium with at least two partially unfolded forms (PUFs). These PUFs, which are accessed by stochastic fluctuations of the N state, have altered surface area exposure relative to the N state. One of these PUFs resembles a conformation previously implicated to be an initial intermediate in the conversion of monomeric protein into misfolded oligomer at pH 4. PMID:26306043

  18. Mechanics of collective unfolding

    E-print Network

    M Caruel; J. -M Allain; L Truskinovsky

    2015-01-07

    Mechanically induced unfolding of passive crosslinkers is a fundamental biological phenomenon encountered across the scales from individual macro-molecules to cytoskeletal actin networks. In this paper we study a conceptual model of athermal load-induced unfolding and use a minimalistic setting allowing one to emphasize the role of long-range interactions while maintaining full analytical transparency. Our model can be viewed as a description of a parallel bundle of N bistable units confined between two shared rigid backbones that are loaded through a series spring. We show that the ground states in this model correspond to synchronized, single phase configurations where all individual units are either folded or unfolded. We then study the fine structure of the wiggly energy landscape along the reaction coordinate linking the two coherent states and describing the optimal mechanism of cooperative unfolding. Quite remarkably, our study shows the fundamental difference in the size and structure of the folding-unfolding energy barriers in the hard (fixed displacements) and soft (fixed forces) loading devices which persists in the continuum limit. We argue that both, the synchronization and the non-equivalence of the mechanical responses in hard and soft devices, have their origin in the dominance of long-range interactions. We then apply our minimal model to skeletal muscles where the power-stroke in acto-myosin crossbridges can be interpreted as passive folding. A quantitative analysis of the muscle model shows that the relative rigidity of myosin backbone provides the long-range interaction mechanism allowing the system to effectively synchronize the power-stroke in individual crossbridges even in the presence of thermal fluctuations. In view of the prototypical nature of the proposed model, our general conclusions pertain to a variety of other biological systems where elastic interactions are mediated by effective backbones.

  19. Microscopic dynamics of water around unfolded structures of barstar at room temperature

    SciTech Connect

    Pal, Somedatta; Chakraborty, Kaushik; Khatua, Prabir; Bandyopadhyay, Sanjoy

    2015-02-07

    The breaking of the native structure of a protein and its influences on the dynamic response of the surrounding solvent is an important issue in protein folding. In this work, we have carried out atomistic molecular dynamics simulations to unfold the protein barstar at two different temperatures (400 K and 450 K). The two unfolded forms obtained at such high temperatures are further studied at room temperature to explore the effects of nonuniform unfolding of the protein secondary structures along two different pathways on the microscopic dynamical properties of the surface water molecules. It is demonstrated that though the structural transition of the protein in general results in less restricted water motions around its segments, but there are evidences of formation of new conformational motifs upon unfolding with increasingly confined environment around them, thereby resulting in further restricted water mobility in their hydration layers. Moreover, it is noticed that the effects of nonuniform unfolding of the protein segments on the relaxation times of the protein–water (PW) and the water–water (WW) hydrogen bonds are correlated with hindered hydration water motions. However, the kinetics of breaking and reformation of such hydrogen bonds are found to be influenced differently at the interface. It is observed that while the effects of unfolding on the PW hydrogen bond kinetics seem to be minimum, but the kinetics involving the WW hydrogen bonds around the protein segments exhibit noticeably heterogeneous characteristics. We believe that this is an important observation, which can provide valuable insights on the origin of heterogeneous influence of unfolding of a protein on the microscopic properties of its hydration water.

  20. Thermodynamic and fibril formation studies of full length immunoglobulin light chain AL-09 and its germline protein using scan rate dependent thermal unfolding.

    PubMed

    Blancas-Mejía, Luis M; Horn, Timothy J; Marin-Argany, Marta; Auton, Matthew; Tischer, Alexander; Ramirez-Alvarado, Marina

    2015-12-01

    Light chain (AL) amyloidosis is a fatal disease where monoclonal immunoglobulin light chains deposit as insoluble amyloid fibrils. For many years it has been considered that AL amyloid deposits are formed primarily by the variable domain, while its constant domain has been considered not to be amyloidogenic. However recent studies identify full length (FL) light chains as part of the amyloid deposits. In this report, we compare the stabilities and amyloidogenic properties of two light chains, an amyloid-associated protein AL-09 FL, and its germline protein ? I O18/O8 FL (IGKV 1-33). We demonstrate that the thermal unfolding for both proteins is irreversible and scan rate dependent, with similar stability parameters compared to their VL counterparts. In addition, the constant domain seems to modulate their amyloidogenic properties and affect the morphology of the amyloid fibrils. These results allow us to understand the role of the kappa constant domain in AL amyloidosis. PMID:26263488

  1. Quantification of Drive-Response Relationships Between Residues During Protein Folding

    PubMed Central

    Qi, Yifei; Im, Wonpil

    2013-01-01

    Mutual correlation and cooperativity are commonly used to describe residue-residue interactions in protein folding/function. However, these metrics do not provide any information on the causality relationships between residues. Such drive-response relationships are poorly studied in protein folding/function and difficult to measure experimentally due to technical limitations. In this study, using the information theory transfer entropy (TE) that provides a direct measurement of causality between two times series, we have quantified the drive-response relationships between residues in the folding/unfolding processes of four small proteins generated by molecular dynamics simulations. Instead of using a time-averaged single TE value, the time-dependent TE is measured with the Q-scores based on residue-residue contacts and with the statistical significance analysis along the folding/unfolding processes. The TE analysis is able to identify the driving and responding residues that are different from the highly correlated residues revealed by the mutual information analysis. In general, the driving residues have more regular secondary structures, are more buried, and show greater effects on the protein stability as well as folding and unfolding rates. In addition, the dominant driving and responding residues from the TE analysis on the whole trajectory agree with those on a single folding event, demonstrating that the drive-response relationships are preserved in the non-equilibrium process. Our study provides detailed insights into the protein folding process and has potential applications in protein engineering and interpretation of time-dependent residue-based experimental observables for protein function. PMID:24223527

  2. Predicting stabilizing mutations in proteins using Poisson-Boltzmann based models: study of unfolded state ensemble models and development of a successful binary classifier based on residue interaction energies.

    PubMed

    Estrada, Jorge; Echenique, Pablo; Sancho, Javier

    2015-11-18

    In many cases the stability of a protein has to be increased to permit its biotechnological use. Rational methods of protein stabilization based on optimizing electrostatic interactions have provided some fine successful predictions. However, the precise calculation of stabilization energies remains challenging, one reason being that the electrostatic effects on the unfolded state are often neglected. We have explored here the feasibility of incorporating Poisson-Boltzmann model electrostatic calculations performed on representations of the unfolded state as large ensembles of geometrically optimized conformations calculated using the ProtSA server. Using a data set of 80 electrostatic mutations experimentally tested in two-state proteins, the predictive performance of several such models has been compared to that of a simple one that considers an unfolded structure of non-interacting residues. The unfolded ensemble models, while showing correlation between the predicted stabilization values and the experimental ones, are worse than the simple model, suggesting that the ensembles do not capture well the energetics of the unfolded state. A more attainable goal is classifying potential mutations as either stabilizing or non-stabilizing, rather than accurately calculating their stabilization energies. To implement a fast classification method that can assist in selecting stabilizing mutations, we have used a much simpler electrostatic model based only on the native structure and have determined its precision using different stabilizing energy thresholds. The binary classifier developed finds 7 true stabilizing mutants out of every 10 proposed candidates and can be used as a robust tool to propose stabilizing mutations. PMID:26530878

  3. Individual Globular Domains and Domain Unfolding Visualized in Overstretched Titin Molecules with Atomic Force Microscopy

    PubMed Central

    Mártonfalvi, Zsolt; Kellermayer, Miklós

    2014-01-01

    Titin is a giant elastomeric protein responsible for the generation of passive muscle force. Mechanical force unfolds titin’s globular domains, but the exact structure of the overstretched titin molecule is not known. Here we analyzed, by using high-resolution atomic force microscopy, the structure of titin molecules overstretched with receding meniscus. The axial contour of the molecules was interrupted by topographical gaps with a mean width of 27.7 nm that corresponds well to the length of an unfolded globular (immunoglobulin and fibronectin) domain. The wide gap-width distribution suggests, however, that additional mechanisms such as partial domain unfolding and the unfolding of neighboring domain multimers may also be present. In the folded regions we resolved globules with an average spacing of 5.9 nm, which is consistent with a titin chain composed globular domains with extended interdomain linker regions. Topographical analysis allowed us to allocate the most distal unfolded titin region to the kinase domain, suggesting that this domain systematically unfolds when the molecule is exposed to overstretching forces. The observations support the prediction that upon the action of stretching forces the N-terminal ß-sheet of the titin kinase unfolds, thus exposing the enzyme’s ATP-binding site and hence contributing to the molecule’s mechanosensory function. PMID:24465745

  4. Wang-Landau density of states based study of the folding-unfolding transition in the mini-protein Trp-cage (TC5b)

    SciTech Connect

    Singh, Priya; Sarkar, Subir K.; Bandyopadhyay, Pradipta

    2014-07-07

    We present the results of a high-statistics equilibrium study of the folding/unfolding transition for the 20-residue mini-protein Trp-cage (TC5b) in water. The ECEPP/3 force field is used and the interaction with water is treated by a solvent-accessible surface area method. A Wang-Landau type simulation is used to calculate the density of states and the conditional probabilities for the various values of the radius of gyration and the number of native contacts at fixed values of energy—along with a systematic check on their convergence. All thermodynamic quantities of interest are calculated from this information. The folding-unfolding transition corresponds to a peak in the temperature dependence of the computed specific heat. This is corroborated further by the structural signatures of folding in the distributions for radius of gyration and the number of native contacts as a function of temperature. The potentials of mean force are also calculated for these variables, both separately and jointly. A local free energy minimum, in addition to the global minimum, is found in a temperature range substantially below the folding temperature. The free energy at this second minimum is approximately 5?k{sub B}T higher than the value at the global minimum.

  5. Unfolding-synthesis technique for digital pulse processing. Part 1: Unfolding

    NASA Astrophysics Data System (ADS)

    Jordanov, Valentin T.

    2016-01-01

    The unfolding-synthesis technique is used in the development of digital pulse processing systems used in radiation measurements. This technique is applied to digital signals obtained by digitization of analog signals that represent the combined response of the radiation detectors and the associated signal conditioning electronics. The salient features of the unfolding-synthesis technique are first the unfolding of the digital signals into unit impulses, followed by the synthesis of digital signal processing systems with unit impulse responses equivalent to the desired pulse shapes. Part 1 of this paper covers the unfolding part of this technique.

  6. Diverse role of conformational dynamics in carboxypeptidase A-driven peptide and ester hydrolyses: disclosing the "perfect induced fit" and "protein local unfolding" pathways by altering protein stability.

    PubMed

    Shushanyan, Mikhael; Khoshtariya, Dimitri E; Tretyakova, Tatyana; Makharadze, Maya; van Eldik, Rudi

    2011-12-01

    Catalytic mechanisms of carboxypeptidase A (CPA) are well known for their diversity and the relative inaccessibility for a decisive comprehension. Recent encouraging attempts through modern computational techniques promoted new challenges for the complementary experimental endeavors. In this work, we have applied the stopped-flow technique and the method of reaction progress curve fitting to extract kinetic parameters for the CPA-catalyzed hydrolyses of smaller (typical) peptide and ester substrates, known for their strong activating/inhibiting impact, thus to which the traditional method of "initial rates" is not applicable. Our approach that innately implies the overall constancy of the affecter (substrate plus "active" product) concentration, made it possible to rigorously determine the physically meaningful "effective" values for the catalytic and Michaelis constants under diverse experimental conditions including variable temperature and urea or trimethylamine N-oxide concentrations. Analysis of the obtained results allowed for: (i) the further substantiation of diverse mechanistic patterns for archetypal specific peptide and ester substrates, (ii) testing and disclosure of intrinsic links between the stabilizing/destabilizing and activating/inhibiting effects for the important model enzyme, CPA, and (iii) tentative explanation of a distinct activating/inhibiting impact of these substrates through the strong specific interaction of their benzyl (Bz) moiety with the substrate binding S(3) subsite of CPA. We have demonstrated that stabilization of CPA either through the interaction with an extra Bz moiety (belonging to another substrate or to the product) leads to the increase of its catalytic power with respect to the specific peptide substrate and to its decrease with respect to the counterpart ester substrate. We conjecture that the catalytic mechanisms operating in these two cases include: (a) the "promoted water" mechanism for the peptide substrate that, seemingly, provides the almost "perfect induced fit" (low-barrier conformational adaptation), and (b) presumably, the "anhydride intermediate" mechanism for the ester substrate that, anyway, requires substantial conformational rearrangement (in fact, "partial or local unfolding") of the protein environment in the course of the rate-determining step. PMID:21698595

  7. BUMS—Bonner sphere Unfolding Made Simple: an HTML based multisphere neutron spectrometer unfolding package

    NASA Astrophysics Data System (ADS)

    Sweezy, Jeremy; Hertel, Nolan; Veinot, Ken

    2002-01-01

    A new multisphere neutron spectrometer unfolding package, Bonner sphere Unfolding Made Simple (BUMS) has been developed that uses an HTML interface to simplify data input and code execution for the novice and the advanced user. This new unfolding package combines the unfolding algorithms contained in other popular unfolding codes under one easy to use interface. The interface makes use of web browsing software to provide a graphical user interface to the unfolding algorithms. BUMS integrates the SPUNIT, BON, MAXIET, and SAND-II unfolding algorithms into a single package. This package also includes a library of 14 response matrices, 58 starting spectra, and 24 dose and detector responses. BUMS has several improvements beyond the addition of unfolding algorithms. It has the ability to search for the most appropriate starting spectra. Also, plots of the unfolded neutron spectra are automatically generated. The BUMS package runs via a web server and may be accessed by any computer with access to the Internet at http://nukeisit.gatech.edu/bums.

  8. Towards improved membrane protein production in Pichia pastoris: general and specific transcriptional response to membrane protein overexpression.

    PubMed

    Vogl, Thomas; Thallinger, Gerhard G; Zellnig, Guenther; Drew, David; Cregg, James M; Glieder, Anton; Freigassner, Maria

    2014-12-25

    Membrane proteins are the largest group of human drug targets and are also used as biocatalysts. However, due to their complexity, efficient expression remains a bottleneck for high level production. In recent years, the methylotrophic yeast Pichia pastoris has emerged as one of the most commonly used expression systems for membrane protein production. Here, we have analysed the transcriptomes of P. pastoris strains producing different classes of membrane proteins (mitochondrial, ER/Golgi and plasma membrane localized) to understand the cellular response and to identify targets to engineer P. pastoris towards an improved chassis for membrane protein production. Microarray experiments revealed varying transcriptional responses depending on the enzymatic activity, subcellular localization and physiological role of the membrane proteins. While an alternative oxidase evoked primarily a response within the mitochondria, the overexpression of transporters entering the secretory pathway had a wide effect on lipid metabolism and induced the upregulation of the UPR (unfolded protein response) transcription factor Hac1p. Coexpression of P. pastoris endogenous HAC1 increased the levels of ER-resident membrane proteins 1.5- to 2.1-fold. Subsequent transcriptome analysis of HAC1 coexpression revealed an upregulation of the folding machinery correlating with an expansion of the ER membrane capacity, thus boosting membrane protein production. Hence, our study has helped to elucidate the cellular response of P. pastoris to the expression of different classes of membrane proteins and led specifically to new insights into the effect of PpHac1p on membrane proteins entering the secretory pathway. PMID:24594271

  9. Transient 2D IR spectroscopy of ubiquitin unfolding dynamics

    PubMed Central

    Chung, Hoi Sung; Ganim, Ziad; Jones, Kevin C.; Tokmakoff, Andrei

    2007-01-01

    Transient two-dimensional infrared (2D IR) spectroscopy is used as a probe of protein unfolding dynamics in a direct comparison of fast unfolding experiments with molecular dynamics simulations. In the experiments, the unfolding of ubiquitin is initiated by a laser temperature jump, and protein structural evolution from nanoseconds to milliseconds is probed using amide I 2D IR spectroscopy. The temperature jump prepares a subensemble near the unfolding transition state, leading to quasi-barrierless unfolding (the “burst phase”) before the millisecond activated unfolding kinetics. The burst phase unfolding of ubiquitin is characterized by a loss of the coupling between vibrations of the ?-sheet, a process that manifests itself in the 2D IR spectrum as a frequency blue-shift and intensity decrease of the diagonal and cross-peaks of the sheet's two IR active modes. As the sheet unfolds, increased fluctuations and solvent exposure of the ?-sheet amide groups are also characterized by increases in homogeneous linewidth. Experimental spectra are compared with 2D IR spectra calculated from the time-evolving structures in a molecular dynamics simulation of ubiquitin unfolding. Unfolding is described as a sequential unfolding of strands in ubiquitin's ?-sheet, using two collective coordinates of the sheet: (i) the native interstrand contacts between adjacent ?-strands I and II and (ii) the remaining ?-strand contacts within the sheet. The methods used illustrate the general principles by which 2D IR spectroscopy can be used for detailed dynamical comparisons of experiment and simulation. PMID:17551015

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

    E-print Network

    Nicchitta, Chris

    , Aurora, Colorado, United States of America, 2 Cell and Molecular Biology Program, Cancer Biology, College, United States of America, 4 Cancer Center Metabolomics Core, Anschutz Medical Center, University as well as increased phospholipid turnover. Elevated levels of amino acids, antioxidants, and cholesterol

  11. Lean forward: Genetic analysis of temperature-sensitive mutants unfolds the secrets of oligomeric protein complex assembly

    PubMed Central

    McMurray, Michael

    2014-01-01

    Multisubunit protein complexes are essential for cellular function. Genetic analysis of essential processes requires special tools, among which temperature-sensitive (Ts) mutants have historically been crucial. Many researchers assume that the effect of temperature on such mutants is to drive their proteolytic destruction. In fact, degradation-mediated elimination of mutant proteins likely explains only a fraction of the phenotypes associated with Ts mutants. Here I discuss insights gained from analysis of Ts mutants in oligomeric proteins, with particular focus on the study of septins, GTP-binding subunits of cytoskeletal filaments whose structures and functions are the subject of current investigation in my and many other labs. I argue that the kinds of unbiased forward genetic approaches that generate Ts mutants provide information that is largely inaccessible to modern reverse genetic methodologies, and will continue to drive our understanding of higher-order assembly by septins and other oligomeric proteins. PMID:25048147

  12. Canine pancreatic responses to intestinally perfused proteins and protein digests.

    PubMed

    Meyer, J H; Kelly, G A

    1976-09-01

    Pancreatic bicarbonate and protein secretory responses to intestinally perfused proteins or digests of proteins were measured in dogs with chronic gastric and pancreatic fistulas when luminalpancreatic protease concentrations were reduced to undetectable levels. Protein digests were analyzed for amino acid content, and various other indirect met-ods were used to assess the composition of the digest mixtures. Of five undigested proteins, none evoked more pancreatic secretion than a control perfusion with saline. Peptic digestion of these same proteins converted four of them to polypeptides that were poten stimuli of a pancreatic juice similar in HCO3-/protein ratios to that evoked by luminal amino acids. Dialyzed peptic digests of one of the proteins, bovine serum albumin (BSA), retained potency. Likewise, digestion of BSA with endogenous or exogenous pancreatic proteases converted the protein to a stimulus about equipotent with the peptic digest, though the composition of the pancreatic digests differed markedly from that of the peptic digests. We conclude that a) luminal peptides of four or more amino acids can stimulate the pancreas and b) during protein alimentation a wide array of luminal protein products may evoke pancreatic secretion. PMID:970450

  13. Osmolyte Effects on the Unfolding Pathway of ?-Lactoglobulin

    NASA Astrophysics Data System (ADS)

    Meng, Wei; Pan, Hai; Qin, Meng; Cao, Yi; Wang, Wei

    2013-10-01

    There are large amounts of osmolytes inside cells, which impact many physiological processes by complicated mechanisms. The osmolyte effects on the stability and folding of proteins have been studied in detail using simple two-state folding proteins. However, many important functional proteins fold in complex pathways involving various intermediates. Little is known about the osmolyte effects on the folding and unfolding of these proteins. It is noted that ?-lactoglobulin (BLG) is an example of such proteins, whose unfolding involves an obvious intermediate state. Using equilibrium chemical denaturation and stopped-flow kinetics, we investigate the unfolding of BLG in the presence of different osmolytes, e.g., glycerol, ethylene glycol (EG) and poly(ethylene glycol)400 (PEG400). It is found that all these osmolytes can stabilize the unfolding intermediate by modulating the relative unfolding kinetics of the native and the intermediate states. The stabilization effects are similar for EG and PEG400 but distinct for glycerol. Since the unfolding intermediates of many proteins are directly related to protein misfolding diseases, evaluation of the osmolyte effects for the unfolding of these proteins in vitro should be beneficial for the understanding of the occurrence of the related diseases in vivo.

  14. Probing Folded and Unfolded States of Outer Membrane Protein A using Steady-State and Time-Resolved Tryptophan Fluorescence

    PubMed Central

    Kim, Judy E.; Arjara, Gitrada; Richards, John H.; Gray, Harry B.; Winkler, Jay R.

    2008-01-01

    Steady-state and time-resolved fluorescence measurements on each of five native tryptophan residues in full-length and truncated variants of E. coli outer-membrane protein A (OmpA) have been made in folded and denatured states. Tryptophan singlet excited-state lifetimes are multiexponential and vary among the residues. In addition, substantial increases in excited-state lifetimes accompany OmpA folding, with longer lifetimes in micelles than in phospholipid bilayers. This finding suggests that the Trp environments of OmpA folded in micelles and phospholipid bilayers are different. Measurements of Trp fluorescence decay kinetics with full-length OmpA folded in brominated lipid vesicles reveal that W102 is the most distant fluorophore from the hydrocarbon core, while W7 is the closest. Steady-state and time-resolved polarized fluorescence measurements indicate reduced Trp mobility when OmpA is folded in a micelle, and even lower mobility when the protein is folded in a bilayer. The fluorescence properties of truncated OmpA, in which the soluble periplasmic domain is removed, only modestly differ from those of the full-length form, suggesting similar folded structures for the two forms under these conditions. PMID:16942111

  15. The arrest of secretion response in yeast: signaling from the secretory path to the nucleus via Wsc proteins and Pkc1p.

    PubMed

    Nanduri, J; Tartakoff, A M

    2001-08-01

    The arrest of secretion response (ASR) in sec mutants reversibly inhibits nuclear import and relocates nuclear proteins to the cytoplasm. sec mutants also relocate nucleoporins; however, endocytic and Golgi-to-vacuole transport mutants do not cause relocation. The ASR requires Wsc membrane proteins that are trapped along the secretory path, rather than those which are at the plasma membrane. The activity of the downstream kinase, Pkc1p, is also required; however, the Pkc1p MAP kinase cascade is not. sec mutants initiate compensatory transcriptional changes distinct from those of the unfolded protein response. PMID:11545731

  16. Unfolding single- and multilayers

    NASA Astrophysics Data System (ADS)

    Llorens, Maria-Gema; Bons, Paul D.; Griera, Albert; Gomez-Rivas, Enrique

    2014-05-01

    When planar structures (e.g. sedimentary layers, veins, dykes, cleavages, etc.) are subjected to deformation, they have about equal chances to be shortened or stretched. The most common shortening and stretching structures are folds and boudinage, respectively. However, boudinage requires additional deformation mechanisms apart from viscous flow, like formation of fractures or strain localization. When folded layers are subjected to extension, they could potentially unfold back to straight layers. Although probably not uncommon, this would be difficult to recognize. Open questions are whether folded layers can unfold, what determines their mechanical behaviour and how we can recognize them in the field. In order to approach these questions, we present a series of numerical experiments that simulate stretching of previously folded single- and multi-layers in simple shear, using the two dimensional numerical modelling platform ELLE, including the finite element module BASIL that calculates viscous deformation. We investigate the parameters that affect a fold train once it rotates into the extensional field. The results show that the unfolding process strongly depends on the viscosity contrast between the layer and matrix (Llorens et al., 2013). Layers do not completely unfold when they experience softening before or during the stretching process or when other neighbouring competent layers prevent them from unfolding. The foliation refraction patterns are the main indicators of unfolded folds. Additionally, intrafolial folds and cusp-like folds adjacent to straight layers, as well as variations in fold amplitudes and limb lengths of irregular folds can also be used as indicators of stretching of a layer after shortening and folding. References: Llorens, M-.G., Bons, P.D., Griera, A. and Gomez-Rivas, E. 2013. When do folds unfold during progressive shear?. Geology, 41, 563-566.

  17. Stable Intermediate States and High Energy Barriers in the Unfolding of GFP

    E-print Network

    Jackson, Sophie

    Stable Intermediate States and High Energy Barriers in the Unfolding of GFP Jie-rong Huang, Timothy of green fluorescent protein (GFP). Chemical denaturation is used to unfold the protein, with changes. The results show that the denaturation behaviour of GFP is complex compared to many small proteins

  18. Desiccation and zinc binding induce transition of tomato abscisic acid stress ripening 1, a water stress- and salt stress-regulated plant-specific protein, from unfolded to folded state.

    PubMed

    Goldgur, Yehuda; Rom, Slava; Ghirlando, Rodolfo; Shkolnik, Doron; Shadrin, Natalia; Konrad, Zvia; Bar-Zvi, Dudy

    2007-02-01

    Abscisic acid stress ripening 1 (ASR1) is a low molecular weight plant-specific protein encoded by an abiotic stress-regulated gene. Overexpression of ASR1 in transgenic plants increases their salt tolerance. The ASR1 protein possesses a zinc-dependent DNA-binding activity. The DNA-binding site was mapped to the central part of the polypeptide using truncated forms of the protein. Two additional zinc-binding sites were shown to be localized at the amino terminus of the polypeptide. ASR1 protein is presumed to be an intrinsically unstructured protein using a number of prediction algorithms. The degree of order of ASR1 was determined experimentally using nontagged recombinant protein expressed in Escherichia coli and purified to homogeneity. Purified ASR1 was shown to be unfolded using dynamic light scattering, gel filtration, microcalorimetry, circular dichroism, and Fourier transform infrared spectrometry. The protein was shown to be monomeric by analytical ultracentrifugation. Addition of zinc ions resulted in a global change in ASR1 structure from monomer to homodimer. Upon binding of zinc ions, the protein becomes ordered as shown by Fourier transform infrared spectrometry and microcalorimetry, concomitant with dimerization. Tomato (Solanum lycopersicum) leaf soluble ASR1 is unstructured in the absence of added zinc and gains structure upon binding of the metal ion. The effect of zinc binding on ASR1 folding and dimerization is discussed. PMID:17189335

  19. Protein response to ligation reactions in myoglobin

    SciTech Connect

    Causgrove, T.P.; Dyer, R.B.

    1992-12-31

    The protein response to the photodissociation, escape and subsequent rebinding of carbon monoxide in myoglobin is studied using time-resolved infrared (TRIR) spectroscopy. All phases of these reactions areinvestigated, from ultrafast phenomena (picoseconds) to relatively slow processes (milliseconds). Conformational changes in myoglobin (Mb) are detected by time-resolved absorption changes in the amide I band. On the hundreds of nanoseconds to milliseconds timescale, a ``real-time`` apparatus is used. This apparatus is based on a tunable diode laser operating in the region of 1650 cm{sup {minus}1} . The time course ofchanges in the amide I band are shown to follow the recombination of CO with photolyzed Mb. On the basis of the rise times of the amide I and FE-CO signals, it is concluded that protein motion is complete within 100 n. A time-resolved difference spectrum in the amide I region is generated from single wavelength transients taken throughout the amide I envelope. A static difference spectrum is also generated by subtracting FTIR spectra of carbonmonoxy and deoxy myoglobin. The two difference spectra are compared and are interpreted in terms of the three-dimensional structures of deoxy and carbonmonoxy Mb. Preliminary picosecond TRIR data are also given for the ultrafast response of the protein immediately following photodissociation of CO.

  20. Protein response to ligation reactions in myoglobin

    SciTech Connect

    Causgrove, T.P.; Dyer, R.B.

    1992-01-01

    The protein response to the photodissociation, escape and subsequent rebinding of carbon monoxide in myoglobin is studied using time-resolved infrared (TRIR) spectroscopy. All phases of these reactions areinvestigated, from ultrafast phenomena (picoseconds) to relatively slow processes (milliseconds). Conformational changes in myoglobin (Mb) are detected by time-resolved absorption changes in the amide I band. On the hundreds of nanoseconds to milliseconds timescale, a real-time'' apparatus is used. This apparatus is based on a tunable diode laser operating in the region of 1650 cm[sup [minus]1] . The time course ofchanges in the amide I band are shown to follow the recombination of CO with photolyzed Mb. On the basis of the rise times of the amide I and FE-CO signals, it is concluded that protein motion is complete within 100 n. A time-resolved difference spectrum in the amide I region is generated from single wavelength transients taken throughout the amide I envelope. A static difference spectrum is also generated by subtracting FTIR spectra of carbonmonoxy and deoxy myoglobin. The two difference spectra are compared and are interpreted in terms of the three-dimensional structures of deoxy and carbonmonoxy Mb. Preliminary picosecond TRIR data are also given for the ultrafast response of the protein immediately following photodissociation of CO.

  1. RESEARCH ARTICLE Pharmaceutical Biotechnology Role of Benzyl Alcohol in the Unfolding and Aggregation of

    E-print Network

    Mallela, Krishna M. G.

    RESEARCH ARTICLE ­ Pharmaceutical Biotechnology Role of Benzyl Alcohol in the Unfolding (wileyonlinelibrary.com). DOI 10.1002/jps.24105 ABSTRACT: Benzyl alcohol (BA) is the most widely used antimicrobial: protein aggregation; protein folding; protein formulation; excipients; stability; benzyl alcohol

  2. Thermal unfolding studies of a phytocyanin.

    PubMed

    Guzzi, Rita; Sportelli, Luigi; Sato, Katsuko; Cannistraro, Salvatore; Dennison, Christopher

    2008-12-01

    The thermal stability of umecyanin, a stellacyanin from horseradish roots, has been investigated by differential scanning calorimetry, optical absorption and fluorescence spectroscopy at neutral and alkaline pH. Above pH 9 the Cu(II) protein experiences a blue shift of the main visible absorption band at approximately 600 nm and changes colour from blue to violet. The thermal transition of the protein is irreversible and occurs between 61.4 and 68.8 degrees C at pH 7.5 and between 50.7 and 57.4 degrees C at pH 9.8. The calorimetric data indicates that at both pH values the thermally induced transition of the protein between the native and denaturated states can be described in terms of the classical Lumry-Eyring unfolding model Native<-->Unfolded-->Final. The analysis of the reversible step in the unfolding pathway demonstrates a significant reduction in conformational stability (DeltaG) of the alkaline form of the protein. Such a reduction is consistent with an enhanced flexibility of UMC at high pH and has mainly entropic character. PMID:18706533

  3. Exploring the minimally frustrated energy landscape of unfolded ACBP.

    PubMed

    Ozenne, Valéry; Noel, Jeffrey K; Heidarsson, Pétur O; Brander, Søren; Poulsen, Flemming M; Jensen, Malene Ringkjøbing; Kragelund, Birthe B; Blackledge, Martin; Danielsson, Jens

    2014-02-01

    The unfolded state of globular proteins is not well described by a simple statistical coil due to residual structural features, such as secondary structure or transiently formed long-range contacts. The principle of minimal frustration predicts that the unfolded ensemble is biased toward productive regions in the conformational space determined by the native structure. Transient long-range contacts, both native-like and non-native-like, have previously been shown to be present in the unfolded state of the four-helix-bundle protein acyl co-enzyme binding protein (ACBP) as seen from both perturbations in nuclear magnetic resonance (NMR) chemical shifts and structural ensembles generated from NMR paramagnetic relaxation data. To study the nature of the contacts in detail, we used paramagnetic NMR relaxation enhancements, in combination with single-point mutations, to obtain distance constraints for the acid-unfolded ensemble of ACBP. We show that, even in the acid-unfolded state, long-range contacts are specific in nature and single-point mutations affect the free-energy landscape of the unfolded protein. Using this approach, we were able to map out concerted, interconnected, and productive long-range contacts. The correlation between the native-state stability and compactness of the denatured state provides further evidence for native-like contact formation in the denatured state. Overall, these results imply that, even in the earliest stages of folding, ACBP dynamics are governed by native-like contacts on a minimally frustrated energy landscape. PMID:24211721

  4. Yeast Biological Networks Unfold the Interplay of Antioxidants, Genome and Phenotype, and Reveal a Novel Regulator of the Oxidative Stress Response

    PubMed Central

    Udatha, D. B. R. K. Gupta; Nielsen, Jens; Panagiotou, Gianni

    2010-01-01

    Background Identifying causative biological networks associated with relevant phenotypes is essential in the field of systems biology. We used ferulic acid (FA) as a model antioxidant to characterize the global expression programs triggered by this small molecule and decipher the transcriptional network controlling the phenotypic adaptation of the yeast Saccharomyces cerevisiae. Methodology/Principal Findings By employing a strict cut off value during gene expression data analysis, 106 genes were found to be involved in the cell response to FA, independent of aerobic or anaerobic conditions. Network analysis of the system guided us to a key target node, the FMP43 protein, that when deleted resulted in marked acceleration of cellular growth (?15% in both minimal and rich media). To extend our findings to human cells and identify proteins that could serve as drug targets, we replaced the yeast FMP43 protein with its human ortholog BRP44 in the genetic background of the yeast strain ?fmp43. The conservation of the two proteins was phenotypically evident, with BRP44 restoring the normal specific growth rate of the wild type. We also applied homology modeling to predict the 3D structure of the FMP43 and BRP44 proteins. The binding sites in the homology models of FMP43 and BRP44 were computationally predicted, and further docking studies were performed using FA as the ligand. The docking studies demonstrated the affinity of FA towards both FMP43 and BRP44. Conclusions This study proposes a hypothesis on the mechanisms yeast employs to respond to antioxidant molecules, while demonstrating how phenome and metabolome yeast data can serve as biomarkers for nutraceutical discovery and development. Additionally, we provide evidence for a putative therapeutic target, revealed by replacing the FMP43 protein with its human ortholog BRP44, a brain protein, and functionally characterizing the relevant mutant strain. PMID:21049050

  5. Role of conformational entropy in force-induced bio-polymer unfolding

    E-print Network

    Sanjay Kumar; Iwan Jensen; Jesper L. Jacobsen; Anthony J. Guttmann

    2007-02-19

    A statistical mechanical description of flexible and semi-flexible polymer chains in a poor solvent is developed in the constant force and constant distance ensembles. We predict the existence of many intermediate states at low temperatures stabilized by the force. A unified response to pulling and compressing forces has been obtained in the constant distance ensemble. We show the signature of a cross-over length which increases linearly with the chain length. Below this cross-over length, the critical force of unfolding decreases with temperature, while above, it increases with temperature. For stiff chains, we report for the first time "saw-tooth" like behavior in the force-extension curves which has been seen earlier in the case of protein unfolding.

  6. Acute phase proteins response in hunting dogs.

    PubMed

    Casella, Stefania; Fazio, Francesco; Russo, Carmelo; Giudice, Elisabetta; Piccione, Giuseppe

    2013-09-01

    Haptoglobin (Hp), serum amyloid A (SAA), C-reactive protein (CRP), and white blood cells (WBC) were assessed in 20 dogs divided into 2 groups. The dogs of group A were not subjected to hunting exercise (control group), while the dogs of group B were subjected to hunting exercise for 3 hr (experimental group). Blood samples were collected from each animal before hunting (T0), immediately after 3 hr of hunting (T1), and after 1 hr of recovery (T2). The general linear model (GLM) repeated measures procedure showed a significant difference between the 2 groups (P < 0.0001) and a significant rise (P < 0.0001) in concentration of Hp, SAA, and CRP after hunting exercise, with a consequent decline during recovery period in group B. These parameters could be considered valid and easily obtainable biomarkers in relation to hunting stress in dogs. Additional studies will continue to elucidate the magnitude and the time of response of other acute phase proteins. PMID:23864258

  7. Modeling force-induced bio-polymer unfolding

    E-print Network

    Anthony J. Guttmann; Jesper L. Jacobsen; Iwan Jensen; Sanjay Kumar

    2007-11-21

    We study the conformations of polymer chains in a poor solvent, with and without bending rigidity, by means of a simple statistical mechanics model. This model can be exactly solved for chains of length up to N=55 using exact enumeration techniques. We analyze in details the differences between the constant force and constant distance ensembles for large but finite N. At low temperatures, and in the constant force ensemble, the force-extension curve shows multiple plateaus (intermediate states), in contrast with the abrupt transition to an extended state prevailing in the $N \\to \\infty$ limit. In the constant distance ensemble, the same curve provides a unified response to pulling and compressing forces, and agrees qualitatively with recent experimental results. We identify a cross-over length, proportional to $N$, below which the critical force of unfolding decreases with temperature, while above, it increases wiyh temperature. Finally, the force-extension curve for stiff chains exhibits "saw-tooth" like behavior, as observed in protein unfolding experiments.

  8. Biology of the Heat Shock Response and Protein Chaperones: Budding Yeast (Saccharomyces cerevisiae) as a Model System

    PubMed Central

    Verghese, Jacob; Abrams, Jennifer; Wang, Yanyu

    2012-01-01

    Summary: The eukaryotic heat shock response is an ancient and highly conserved transcriptional program that results in the immediate synthesis of a battery of cytoprotective genes in the presence of thermal and other environmental stresses. Many of these genes encode molecular chaperones, powerful protein remodelers with the capacity to shield, fold, or unfold substrates in a context-dependent manner. The budding yeast Saccharomyces cerevisiae continues to be an invaluable model for driving the discovery of regulatory features of this fundamental stress response. In addition, budding yeast has been an outstanding model system to elucidate the cell biology of protein chaperones and their organization into functional networks. In this review, we evaluate our understanding of the multifaceted response to heat shock. In addition, the chaperone complement of the cytosol is compared to those of mitochondria and the endoplasmic reticulum, organelles with their own unique protein homeostasis milieus. Finally, we examine recent advances in the understanding of the roles of protein chaperones and the heat shock response in pathogenic fungi, which is being accelerated by the wealth of information gained for budding yeast. PMID:22688810

  9. The Application of an Unfolding Model of the PIRT Type to the Measurement of Attitude.

    ERIC Educational Resources Information Center

    Andrich, David

    1988-01-01

    A simple probabilistic model for unfolding data collected by a direct response design in which responses were scored dichotomously was applied to the measurement of attitudes toward capital punishment. Responses conformed to the unfolding mechanism. Scale values of the statements were statistically equivalent to those of Thurstone's methods. (SLD)

  10. Thermal unfolding mechanism of lipocalin-type prostaglandin D synthase.

    PubMed

    Iida, Tsukimi; Nishimura, Shigenori; Mochizuki, Maki; Uchiyama, Susumu; Ohkubo, Tadayasu; Urade, Yoshihiro; Tanaka, Akiyoshi; Inui, Takashi

    2008-01-01

    Lipocalin-type prostaglandin (PG) D synthase (L-PGDS) is a dual-functioning protein in the lipocalin family, acting as a PGD(2)-synthesizing enzyme and as an extracellular transporter for small lipophilic molecules. We earlier reported that denaturant-induced unfolding of L-PGDS follows a four-state pathway, including an activity-enhanced state and an inactive intermediate state. In this study, we investigated the thermal unfolding mechanism of L-PGDS by using differential scanning calorimetry (DSC) and CD spectroscopy. DSC measurements revealed that the thermal unfolding of L-PGDS was a completely reversible process at pH 4.0. The DSC curves showed no concentration dependency, demonstrating that the thermal unfolding of L-PGDS involved neither intermolecular interaction nor aggregation. On the basis of a simple two-state unfolding mechanism, the ratio of van't Hoff enthalpy (DeltaH(vH)) to calorimetric enthalpy (DeltaH(cal)) was below 1, indicating the presence of an intermediate state (I) between the native state (N) and unfolded state (U). Then, statistical thermodynamic analyses of a three-state unfolding process were performed. The heat capacity curves fit well with a three-state process; and the estimated transition temperature (T(m)) and enthalpy change (DeltaH(cal)) of the N<-->I and I<-->U transitions were 48.2 degrees C and 190 kJ.mol(-1), and 60.3 degrees C and 144 kJ.mol(-1), respectively. Correspondingly, the thermal unfolding monitored by CD spectroscopy at 200, 235 and 290 nm revealed that L-PGDS unfolded through the intermediate state, where its main chain retained the characteristic beta-sheet structure without side-chain interactions. PMID:18076651

  11. A stable intermediate in the thermal unfolding process of a chimeric 3-isopropylmalate dehydrogenase between a thermophilic and a mesophilic enzymes.

    PubMed Central

    Hayashi-Iwasaki, Y.; Numata, K.; Yamagishi, A.; Yutani, K.; Sakurai, M.; Tanaka, N.; Oshima, T.

    1996-01-01

    The thermal unfolding process of a chimeric 3-isopropylmalate dehydrogenase made of parts from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis, enzymes was studied by CD spectrophotometry and differential scanning calorimetry (DSC). The enzyme is a homodimer with a subunit containing two structural domains. The DSC melting profile of the chimeric enzyme in 20 mM NaHCO3, pH 10.4, showed two endothermic peaks, whereas that of the T. thermophilus wild-type enzyme had one peak. The CD melting profiles of the chimeric enzyme under the same conditions as the DSC measurement, also indicated biphasic unfolding transition. Concentration dependence of the unfolding profile revealed that the first phase was protein concentration-independent, whereas the second transition was protein concentration-dependent. When cooled after the first transition, the intermediate was isolated, which showed only the second transition upon heating. These results indicated the existence of a stable dimeric intermediate followed by the further unfolding and dissociation in the thermal unfolding of the chimeric enzyme at pH 10-11. Because the portion derived from the mesophilic isopropylmalate dehydrogenase in the chimeric enzyme is located in the hinge region between two domains of the enzyme, it is probably responsible for weakening of the interdomain interaction and causing the decooperativity of two domains. The dimeric form of the intermediate suggested that the first unfolding transition corresponds to the unfolding of domain 1 containing the N- and C-termini of the enzyme, and the second to that of domain 2 containing the subunit interface. PMID:8868488

  12. Glycaemic Response to Quality Protein Maize Grits

    PubMed Central

    Panlasigui, Leonora N.; Bayaga, Cecile L. T.; Barrios, Erniel B.; Cochon, Kim L.

    2010-01-01

    Background. Carbohydrates have varied rates of digestion and absorption that induces different hormonal and metabolic responses in the body. Given the abundance of carbohydrate sources in the Philippines, the determination of the glycaemic index (GI) of local foods may prove beneficial in promoting health and decreasing the risk of diabetes in the country. Methods. The GI of Quality Protein Maize (QPM) grits, milled rice, and the mixture of these two food items were determined in ten female subjects. Using a randomized crossover design, the control bread and three test foods were given on separate occasions after an overnight fast. Blood samples were collected through finger prick at time intervals of 0, 15, 30, 45, 60, 90, and 120 min and analyzed for glucose concentrations. Results. The computed incremental area under the glucose response curve (IAUC) varies significantly across test foods (P < .0379) with the pure QPM grits yielding the lowest IAUC relative to the control by 46.38. Resulting GI values of the test foods (bootstrapped) were 80.36 (SEM 14.24), 119.78 (SEM 18.81), and 93.17 (SEM 27.27) for pure QPM grits, milled rice, and rice-QPM grits mixture, respectively. Conclusion. Pure QPM corn grits has a lower glycaemic response compared to milled rice and the rice-corn grits mixture, which may be related in part to differences in their dietary fibre composition and physicochemical characteristics. Pure QPM corn grits may be a more health beneficial food for diabetic and hyperlipidemic individuals. PMID:20862364

  13. Unfolding the Sulcus

    NASA Astrophysics Data System (ADS)

    Hohlfeld, Evan; Mahadevan, L.

    2011-03-01

    Sulci are localized furrows on the surface of soft materials that form by a compression-induced instability. We unfold this instability by breaking its natural scale and translation invariance, and compute a limiting bifurcation diagram for sulcfication showing that it is a scale-free, subcritical nonlinear instability. In contrast with classical nucleation, sulcification is continuous, occurs in purely elastic continua and is structurally stable in the limit of vanishing surface energy. During loading, a sulcus nucleates at a point with an upper critical strain and an essential singularity in the linearized spectrum. On unloading, it quasistatically shrinks to a point with a lower critical strain, explained by breaking of scale symmetry. At intermediate strains the system is linearly stable but nonlinearly unstable with no energy barrier. Simple experiments confirm the existence of these two critical strains.

  14. Unfolding the Sulcus

    E-print Network

    Evan Hohlfeld; L. Mahadevan

    2011-03-11

    Sulci are localized furrows on the surface of soft materials that form by a compression-induced instability. We unfold this instability by breaking its natural scale and translation invariance, and compute a limiting bifurcation diagram for sulcfication showing that it is a scale-free, sub-critical {\\em nonlinear} instability. In contrast with classical nucleation, sulcification is {\\em continuous}, occurs in purely elastic continua and is structurally stable in the limit of vanishing surface energy. During loading, a sulcus nucleates at a point with an upper critical strain and an essential singularity in the linearized spectrum. On unloading, it quasi-statically shrinks to a point with a lower critical strain, explained by breaking of scale symmetry. At intermediate strains the system is linearly stable but nonlinearly unstable with {\\em no} energy barrier. Simple experiments confirm the existence of these two critical strains.

  15. Redox control of protein degradation.

    PubMed

    Pajares, Marta; Jiménez-Moreno, Natalia; Dias, Irundika H K; Debelec, Bilge; Vucetic, Milica; Fladmark, Kari E; Basaga, Huveyda; Ribaric, Samo; Milisav, Irina; Cuadrado, Antonio

    2015-12-01

    Intracellular proteolysis is critical to maintain timely degradation of altered proteins including oxidized proteins. This review attempts to summarize the most relevant findings about oxidant protein modification, as well as the impact of reactive oxygen species on the proteolytic systems that regulate cell response to an oxidant environment: the ubiquitin-proteasome system (UPS), autophagy and the unfolded protein response (UPR). In the presence of an oxidant environment, these systems are critical to ensure proteostasis and cell survival. An example of altered degradation of oxidized proteins in pathology is provided for neurodegenerative diseases. Future work will determine if protein oxidation is a valid target to combat proteinopathies. PMID:26381917

  16. Redox control of protein degradation

    PubMed Central

    Pajares, Marta; Jiménez-Moreno, Natalia; Dias, Irundika H.K.; Debelec, Bilge; Vucetic, Milica; Fladmark, Kari E.; Basaga, Huveyda; Ribaric, Samo; Milisav, Irina; Cuadrado, Antonio

    2015-01-01

    Intracellular proteolysis is critical to maintain timely degradation of altered proteins including oxidized proteins. This review attempts to summarize the most relevant findings about oxidant protein modification, as well as the impact of reactive oxygen species on the proteolytic systems that regulate cell response to an oxidant environment: the ubiquitin-proteasome system (UPS), autophagy and the unfolded protein response (UPR). In the presence of an oxidant environment, these systems are critical to ensure proteostasis and cell survival. An example of altered degradation of oxidized proteins in pathology is provided for neurodegenerative diseases. Future work will determine if protein oxidation is a valid target to combat proteinopathies. PMID:26381917

  17. Verification of unfold error estimates in the UFO code

    SciTech Connect

    Fehl, D.L.; Biggs, F.

    1996-07-01

    Spectral unfolding is an inverse mathematical operation which attempts to obtain spectral source information from a set of tabulated response functions and data measurements. Several unfold algorithms have appeared over the past 30 years; among them is the UFO (UnFold Operator) code. In addition to an unfolded spectrum, UFO also estimates the unfold uncertainty (error) induced by running the code in a Monte Carlo fashion with prescribed data distributions (Gaussian deviates). In the problem studied, data were simulated from an arbitrarily chosen blackbody spectrum (10 keV) and a set of overlapping response functions. The data were assumed to have an imprecision of 5% (standard deviation). 100 random data sets were generated. The built-in estimate of unfold uncertainty agreed with the Monte Carlo estimate to within the statistical resolution of this relatively small sample size (95% confidence level). A possible 10% bias between the two methods was unresolved. The Monte Carlo technique is also useful in underdetemined problems, for which the error matrix method does not apply. UFO has been applied to the diagnosis of low energy x rays emitted by Z-Pinch and ion-beam driven hohlraums.

  18. Pyrvinium Targets the Unfolded Protein Response to Hypoglycemia and Its Anti-Tumor Activity Is Enhanced by Combination Therapy

    PubMed Central

    Yu, De-Hua; Macdonald, James; Liu, Guohong; Lee, Amy S.; Ly, Mimi; Davis, Timothy; Ke, Ning; Zhou, Demin; Wong-Staal, Flossie; Li, Qi-Xiang

    2008-01-01

    We identified pyrvinium pamoate, an old anthelminthic medicine, which preferentially inhibits anchorage-independent growth of cancer cells over anchorage-dependent growth (?10 fold). It was also reported by others to have anti-tumor activity in vivo and selective toxicity against cancer cells under glucose starvation in vitro, but with unknown mechanism. Here, we provide evidence that pyrvinium suppresses the transcriptional activation of GRP78 and GRP94 induced by glucose deprivation or 2-deoxyglucose (2DG, a glycolysis inhibitor), but not by tunicamycin or A23187. Other UPR pathways induced by glucose starvation, e.g. XBP-1, ATF4, were also found suppressed by pyrvinium. Constitutive expression of GRP78 via transgene partially protected cells from pyrvinium induced cell death under glucose starvation, suggesting that suppression of the UPR is involved in pyrvinium mediated cytotoxicity under glucose starvation. Xenograft experiments showed rather marginal overall anti-tumor activity for pyrvinium as a monotherapy. However, the combination of pyrvinium and Doxorubicin demonstrated significantly enhanced efficacy in vivo, supporting a mechanistic treatment concept based on tumor hypoglycemia and UPR. PMID:19079611

  19. CSE IGERTCOMPUTATIONAL SCIENCE AND ENGINEERING IGERT QUARTERLy NEwSLETTER Mechanism of the Unfolded Protein Response. Theresa Yuraszeck

    E-print Network

    Bigelow, Stephen

    to model wildlife migration and gene flow across landscapes. Because of the high volume of data thesis, which aims to better understand and manipulate complex biological oscillators that give rise for the analysis of circadian performance, a control objective for phase re-synchronization, and a criterion

  20. Protein Contribution to Plant Salinity Response and Tolerance Acquisition

    PubMed Central

    Kosová, Klára; Prášil, Ilja T.; Vítámvás, Pavel

    2013-01-01

    The review is focused on plant proteome response to salinity with respect to physiological aspects of plant salt stress response. The attention is paid to both osmotic and ionic effects of salinity stress on plants with respect to several protein functional groups. Therefore, the role of individual proteins involved in signalling, changes in gene expression, protein biosynthesis and degradation and the resulting changes in protein relative abundance in proteins involved in energy metabolism, redox metabolism, stressand defence-related proteins, osmolyte metabolism, phytohormone, lipid and secondary metabolism, mechanical stress-related proteins as well as protein posttranslational modifications are discussed. Differences between salt-sensitive (glycophytes) and salt-tolerant (halophytes) plants are analysed with respect to differential salinity tolerance. In conclusion, contribution of proteomic studies to understanding plant salinity tolerance is summarised and discussed. PMID:23531537

  1. Piezotropic unfolding of lysozyme in pure D 2O at the outer edge of excess hydration

    NASA Astrophysics Data System (ADS)

    Pfeiffer, Helge; Heremans, Karel; Wevers, Martine

    2009-02-01

    This Letter reports a FTIR study on the pressure-induced unfolding of lysozyme in pure D2O close to the conditions of non excess hydration. An essential population of the proteins apparently refold into its native structure after pressure release. It could furthermore be shown that down to a hydration of h = 2 (h = mprot/mD2O ; m = mass), the unfolding pressure did not vary with hydration. Hydration dependent behaviour was found with respect to the change of the wavenumbers of the ?-helical structure during unfolding. This result is discussed with respect to the reversibility of unfolding in pure solvents and with the effects of hydrogen exchange.

  2. Temperature and Chemical Denaturant Dependence of Forced Unfolding of Titin I27 Eric Botello,

    E-print Network

    Kiang, Ching-Hwa

    Temperature and Chemical Denaturant Dependence of Forced Unfolding of Titin I27 Eric Botello, Nolan used force as a physical parameter under the traditional chemical and temperature denaturing and Li on protein mechanical unfolding in solutions containing chemical denaturants has demonstrated

  3. Unfolding pathway of CotA-laccase and the role of copper on the prevention of refolding through aggregation of the unfolded state

    SciTech Connect

    Fernandes, Andre T.; Lopes, Carlos; Martins, Ligia O.; Melo, Eduardo Pinho

    2012-06-08

    Highlights: Black-Right-Pointing-Pointer CotA-laccase unfolds with an intermediate state. Black-Right-Pointing-Pointer Copper stabilizes the native and the intermediate state. Black-Right-Pointing-Pointer Copper binding to the unfolded state prevents refolding through protein aggregation. Black-Right-Pointing-Pointer Copper incorporation in CotA-laccase occurs as a later step during folding. -- Abstract: Copper is a redox-active metal and the main player in electron transfer reactions occurring in multicopper oxidases. The role of copper in the unfolding pathway and refolding of the multicopper oxidase CotA laccase in vitro was solved using double-jump stopped-flow experiments. Unfolding of apo- and holo-CotA was described as a three-state process with accumulation of an intermediate in between the native and unfolded state. Copper stabilizes the native holo-CotA but also the intermediate state showing that copper is still bound to this state. Also, copper binds to unfolded holo-CotA in a non-native coordination promoting CotA aggregation and preventing refolding to the native structure. These results gather information on unfolding/folding pathways of multicopper oxidases and show that copper incorporation in vivo should be a tight controlled process as copper binding to the unfolded state under native conditions promotes protein aggregation.

  4. Mechanical unfolding pathway of a model ?-peptide foldamer

    NASA Astrophysics Data System (ADS)

    Uribe, Lalita; Jaschonek, Stefan; Gauss, Jürgen; Diezemann, Gregor

    2015-05-01

    Foldamers constructed from oligomers of ?-peptides form stable secondary helix structures already for small chain lengths, which makes them ideal candidates for the investigation of the (un)folding of polypeptides. Here, the results of molecular simulations of the mechanical unfolding of a ?-heptapeptide in methanol solvent revealing the detailed unfolding pathway are reported. The unfolding process is shown to proceed via a stable intermediate even for such a small system. This result is arrived at performing non-equilibrium force ramp simulations employing different pulling velocities and also using standard calculations of the potential of mean force, i.e., the free energy as a function of the helix elongation. It is thus demonstrated that even with the rather large pulling velocities employed in the force ramp simulations relevant information about the equilibrium kinetics can be obtained. The smallness of the system allows a detailed analysis of the unfolding pathway, which is characterized by an opening of the terminal loops followed by the unfolding of the center. This sequence is in accord with the configurational preferences of the system that also are responsible for the stability of the 314-helix. From an analysis of the distributions of rupture forces and the force spectra, the kinetic rates for both transitions were determined and common models were used to extract geometric quantities describing the free energy landscape of the system.

  5. Conformational Heterogeneity Determined by Folding and Oligomer Assembly Routes of the Interferon Response Inhibitor NS1 Protein, Unique to Human Respiratory Syncytial Virus.

    PubMed

    Pretel, Esteban; Sánchez, Ignacio E; Fassolari, Marisol; Chemes, Lucía B; de Prat-Gay, Gonzalo

    2015-08-25

    The nonstructural NS1 protein is an essential virulence factor of the human respiratory syncytial virus, with a predominant role in the inhibition of the host antiviral innate immune response. This inhibition is mediated by multiple protein-protein interactions and involves the formation of large oligomeric complexes. There is neither a structure nor sequence or functional homologues of this protein, which points to a distinctive mechanism for blocking the interferon response among viruses. The NS1 native monomer follows a simple unfolding kinetics via a nativelike transition state ensemble, with a half-life of 45 min, in agreement with a highly stable core structure at equilibrium. Refolding is a complex process that involves several slowly interconverting species compatible with proline isomerization. However, an ultrafast folding event with a half-life of 0.2 ms is indicative of a highly folding compatible species within the unfolded state ensemble. On the other hand, the oligomeric assembly route from the native monomer, which does not involve unfolding, shows a monodisperse and irreversible end-point species triggered by a mild temperature change, with half-lives of 160 and 26 min at 37 and 47 °C, respectively, and at a low protein concentration (10 ?M). A large secondary structure change into ?-sheet structure and the formation of a dimeric nucleus precede polymerization by the sequential addition of monomers at the surprisingly low rate of one monomer every 34 s. The polymerization phase is followed by the binding to thioflavin-T indicative of amyloid-like, albeit soluble, repetitive ?-sheet quaternary structure. The overall process is reversible only up until ~8 min, a time window in which most of the secondary structure change takes place. NS1's multiple binding activities must be accommodated in a few binding interfaces at most, something to be considered remarkable given its small size (15 kDa). Thus, conformational heterogeneity, and in particular oligomer formation, may provide a means of expand its binding repertoire. These equilibria will be determined by variables such as macromolecular crowding, protein-protein interactions, expression levels, turnover, or specific subcellular localization. The irreversible and quasi-spontaneous nature of the oligomer assembly, together with the fact that NS1 is the most abundant viral protein in infected cells, makes its accumulation highly conceivable under conditions compatible with the cellular milieu. The implications of NS1 oligomers in the viral life cycle and the inhibition of host innate immune response remain to be determined. PMID:26237467

  6. Bioinformatics and variability in drug response: a protein structural perspective

    PubMed Central

    Lahti, Jennifer L.; Tang, Grace W.; Capriotti, Emidio; Liu, Tianyun; Altman, Russ B.

    2012-01-01

    Marketed drugs frequently perform worse in clinical practice than in the clinical trials on which their approval is based. Many therapeutic compounds are ineffective for a large subpopulation of patients to whom they are prescribed; worse, a significant fraction of patients experience adverse effects more severe than anticipated. The unacceptable risk–benefit profile for many drugs mandates a paradigm shift towards personalized medicine. However, prior to adoption of patient-specific approaches, it is useful to understand the molecular details underlying variable drug response among diverse patient populations. Over the past decade, progress in structural genomics led to an explosion of available three-dimensional structures of drug target proteins while efforts in pharmacogenetics offered insights into polymorphisms correlated with differential therapeutic outcomes. Together these advances provide the opportunity to examine how altered protein structures arising from genetic differences affect protein–drug interactions and, ultimately, drug response. In this review, we first summarize structural characteristics of protein targets and common mechanisms of drug interactions. Next, we describe the impact of coding mutations on protein structures and drug response. Finally, we highlight tools for analysing protein structures and protein–drug interactions and discuss their application for understanding altered drug responses associated with protein structural variants. PMID:22552919

  7. Genetically engineered protein in hydrogels tailors stimuli-responsive characteristics

    NASA Astrophysics Data System (ADS)

    Ehrick, Jason D.; Deo, Sapna K.; Browning, Tyler W.; Bachas, Leonidas G.; Madou, Marc J.; Daunert, Sylvia

    2005-04-01

    Certain proteins undergo a substantial conformational change in response to a given stimulus. This conformational change can manifest in different manners and result in an actuation, that is, catalytic or signalling event, movement, interaction with other proteins, and so on. In all cases, the sensing-actuation process of proteins is initiated by a recognition event that translates into a mechanical action. Thus, proteins are ideal components for designing new nanomaterials that are intelligent and can perform desired mechanical actions in response to target stimuli. A number of approaches have been undertaken to mimic nature's sensing-actuating process. We now report a new hybrid material that integrates genetically engineered proteins within hydrogels capable of producing a stimulus-responsive action mechanism. The mechanical effect is a result of an induced conformational change and binding affinities of the protein in response to a stimulus. The stimuli-responsive hydrogel exhibits three specific swelling stages in response to various ligands offering additional fine-tuned control over a conventional two-stage swelling hydrogel. The newly prepared material was used in the sensing, and subsequent gating and transport of biomolecules across a polymer network, demonstrating its potential application in microfluidics and miniaturized drug-delivery systems.

  8. Genetically engineered protein in hydrogels tailors stimuli-responsive characteristics.

    PubMed

    Ehrick, Jason D; Deo, Sapna K; Browning, Tyler W; Bachas, Leonidas G; Madou, Marc J; Daunert, Sylvia

    2005-04-01

    Certain proteins undergo a substantial conformational change in response to a given stimulus. This conformational change can manifest in different manners and result in an actuation, that is, catalytic or signalling event, movement, interaction with other proteins, and so on. In all cases, the sensing-actuation process of proteins is initiated by a recognition event that translates into a mechanical action. Thus, proteins are ideal components for designing new nanomaterials that are intelligent and can perform desired mechanical actions in response to target stimuli. A number of approaches have been undertaken to mimic nature's sensing-actuating process. We now report a new hybrid material that integrates genetically engineered proteins within hydrogels capable of producing a stimulus-responsive action mechanism. The mechanical effect is a result of an induced conformational change and binding affinities of the protein in response to a stimulus. The stimuli-responsive hydrogel exhibits three specific swelling stages in response to various ligands offering additional fine-tuned control over a conventional two-stage swelling hydrogel. The newly prepared material was used in the sensing, and subsequent gating and transport of biomolecules across a polymer network, demonstrating its potential application in microfluidics and miniaturized drug-delivery systems. PMID:15765106

  9. Residual dipolar couplings measured in unfolded proteins are sensitive to amino-acid-specific geometries as well as local conformational sampling.

    PubMed

    Huang, Jie-rong; Gentner, Martin; Vajpai, Navratna; Grzesiek, Stephan; Blackledge, Martin

    2012-10-01

    Many functional proteins do not have well defined folded structures. In recent years, both experimental and computational approaches have been developed to study the conformational behaviour of this type of protein. It has been shown previously that experimental RDCs (residual dipolar couplings) can be used to study the backbone sampling of disordered proteins in some detail. In these studies, the backbone structure was modelled using a common geometry for all amino acids. In the present paper, we demonstrate that experimental RDCs are also sensitive to the specific geometry of each amino acid as defined by energy-minimized internal co-ordinates. We have modified the FM (flexible-Meccano) algorithm that constructs conformational ensembles on the basis of a statistical coil model, to account for these differences. The modified algorithm inherits the advantages of the FM algorithm to efficiently sample the potential energy landscape for coil conformations. The specific geometries incorporated in the new algorithm result in a better reproduction of experimental RDCs and are generally applicable for further studies to characterize the conformational properties of intrinsically disordered proteins. In addition, the internal-co-ordinate-based algorithm is an order of magnitude more efficient, and facilitates side-chain construction, surface osmolyte simulation, spin-label distribution sampling and proline cis/trans isomer simulation. PMID:22988852

  10. Structure of the transition state for the folding/unfolding of the barley chymotrypsin inhibitor 2 and its implications for mechanisms of protein folding.

    PubMed Central

    Otzen, D E; Itzhaki, L S; elMasry, N F; Jackson, S E; Fersht, A R

    1994-01-01

    The equilibrium and kinetics of folding of the single-domain protein chymotrypsin inhibitor 2 conform to the simple two-state model. The structure of the rate-determining transition state has been mapped out at the resolution of individual side chains by using the protein engineering method on 74 mutants that have been constructed at 37 of the 64 residues. The structure contains no elements of secondary structure that are fully formed. The majority of interactions are weakened by > 50% in the transition state, although most regions do have some very weak structure. The structure of the transition state appears to be an expanded form of the native state in which secondary and tertiary elements have been partly formed concurrently. This is consistent with a "global collapse" model of folding rather than a framework model in which folding is initiated from fully preformed local secondary structural elements. This may be a general feature for the folding of proteins lacking a folding intermediate and is perhaps representative of the early stages of folding for multidomain or multimodule proteins. The major transition state for the folding of barnase, for example, has some fully formed secondary and tertiary structural elements in the major transition state, and barnase appears to form by a framework process. However, the fully formed framework may be preceded by a global collapse, and a unified folding scheme is presented. Images PMID:7937967

  11. Characterization of the unfolding, refolding, and aggregation pathways of two protein implicated in cataractogenesis : human gamma D and human gamma S crystallin

    E-print Network

    Kosinski-Collins, Melissa Sue, 1978-

    2005-01-01

    Human [gamma]D crystallin (H[gamma]D-Crys) and human [gamma]S crystallin (H[gamma]S-Crys), are major proteins of the human eye lens and are components of cataracts. H[gamma]D-Crys is expressed early in life in the lens ...

  12. Genes that encode different stress-response proteins --proteins that

    E-print Network

    Bedwell, David M.

    to oil or chemical dispersant as a result of the 2010 BP Deepwater Horizon oil spill. Stephen Watts, Ph minnows show an inflammatory response to Corexit®, the dispersant used to clean the 2010 oil spill. Watts the effect the dispersant used in the cleanup of 2010 Gulf oil spill has on bull minnows. Watts' team

  13. Application of Wavelet Unfolding Technique in Neutron Spectroscopic Analysis

    NASA Astrophysics Data System (ADS)

    Hartman, Jessica; Barzilov, Alexander

    Nonproliferation of nuclear materials is important in nuclear power industry and fuel cycle facilities. It requires technologies capable of measuring and assessing the radiation signatures of fission events. Neutrons produced in spontaneous or induced fission reactions are mainly fast. The neutron energy information allows characterization of nuclear materials and neutron sources. It can also be applied in remote sensing and source search tasks. The plastic scintillator EJ-299-33A was studied as a fast neutron detector. The detector response to a polyenergetic flux was unfolded usingthe multiple linear regression method. It yields the intensities of neutron flux of particular energy, hence, enabling the spectroscopic analysis. The wavelet technique was evaluated for the unfolding of neutron spectrum using the scintillator's response functions between 1 MeV and 14 MeV computed with the MCNPX code. This paperpresents the computational results of the wavelet-based spectrum unfolding applied to a scintillator detector with neutron / photon pulse shape discrimination properties.

  14. Stimuli-Responsive Nanomaterials for Therapeutic Protein Delivery

    PubMed Central

    Lu, Yue; Sun, Wujin; Gu, Zhen

    2014-01-01

    Protein therapeutics have emerged as a significant role in treatment of a broad spectrum of diseases, including cancer, metabolic disorders and autoimmune diseases. The efficacy of protein therapeutics, however, is limited by their instability, immunogenicity and short half-life. In order to overcome these barriers, tremendous efforts have recently been made in developing controlled protein delivery systems. Stimuli-triggered release is an appealing and promising approach for protein delivery and has made protein delivery with both spatiotemporal- and dosage-controlled manners possible. This review surveys recent advances in controlled protein delivery of proteins or peptides using stimuli-responsive nanomaterials. Strategies utilizing both physiological and external stimuli are introduced and discussed. PMID:25151983

  15. Stress proteins are induced by space environment

    NASA Astrophysics Data System (ADS)

    Takahashi, Akihisa; Ohnishi, Takeo

    The space environment contains two major biologically significant influences such as space radiations and microgravity. Almost all organisms possess essential recognition and response systems for environmental changes. The famous one of cellular stress responses is the gene induction of heat shock protein (HSP). HSP expression is increased under elevated temperatures, and also increased by other sources of cellular stress, including ionizing radiation, oxidative injury, osmotic stress and the unfolded protein response. HSPs assist in the folding and maintenance of newly translated proteins, the refolding of denatured proteins and the further unfolding of misfolded or destabilized proteins to protect the cell from crisis. Based on our space experiment, we report the results and discussion from the viewpoint of HSP expression after exposure to space environment.

  16. Modified Likelihood-Based Item Fit Statistics for the Generalized Graded Unfolding Model

    ERIC Educational Resources Information Center

    Roberts, James S.

    2008-01-01

    Orlando and Thissen (2000) developed an item fit statistic for binary item response theory (IRT) models known as S-X[superscript 2]. This article generalizes their statistic to polytomous unfolding models. Four alternative formulations of S-X[superscript 2] are developed for the generalized graded unfolding model (GGUM). The GGUM is a…

  17. Proteome response at the edge of protein aggregation

    PubMed Central

    Sanchez de Groot, Natalia; Gomes, Ricardo A.; Villar-Pique, Anna; Babu, M. Madan; Coelho, Ana Varela; Ventura, Salvador

    2015-01-01

    Proteins adopt defined structures and are crucial to most cellular functions. Their misfolding and aggregation is associated with numerous degenerative human disorders such as type II diabetes, Huntington's or Alzheimer's diseases. Here, we aim to understand why cells promote the formation of protein foci. Comparison of two amyloid-?-peptide variants, mostly insoluble but differently recruited by the cell (inclusion body versus diffused), reveals small differences in cell fitness and proteome response. We suggest that the levels of oxidative stress act as a sensor to trigger protein recruitment into foci. Our data support a common cytoplasmic response being able to discern and react to the specific properties of polypeptides. PMID:25673330

  18. RNA-Processing Protein TDP-43 Regulates FOXO-Dependent Protein Quality Control in Stress Response

    PubMed Central

    Zhang, Tao; Baldie, Gerard; Periz, Goran; Wang, Jiou

    2014-01-01

    Protein homeostasis is critical for cell survival and functions during stress and is regulated at both RNA and protein levels. However, how the cell integrates RNA-processing programs with post-translational protein quality control systems is unknown. Transactive response DNA-binding protein (TARDBP/TDP-43) is an RNA-processing protein that is involved in the pathogenesis of major neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here, we report a conserved role for TDP-43, from C. elegans to mammals, in the regulation of protein clearance via activation of FOXO transcription factors. In response to proteotoxic insults, TDP-43 redistributes from the nucleus to the cytoplasm, promoting nuclear translocation of FOXOs and relieving an inhibition of FOXO activity in the nucleus. The interaction between TDP-43 and the FOXO pathway in mammalian cells is mediated by their competitive binding to 14-3-3 proteins. Consistent with FOXO-dependent protein quality control, TDP-43 regulates the levels of misfolded proteins. Therefore, TDP-43 mediates stress responses and couples the regulation of RNA metabolism and protein quality control in a FOXO-dependent manner. The results suggest that compromising the function of TDP-43 in regulating protein homeostasis may contribute to the pathogenesis of related neurodegenerative diseases. PMID:25329970

  19. Evidences for the unfolding mechanism of three-dimensional domain swapping

    PubMed Central

    Liu, Zhirong; Huang, Yongqi

    2013-01-01

    The full or partial unfolding of proteins is widely believed to play an essential role in three-dimensional domain swapping. However, there is little research that has rigorously evaluated the association between domain swapping and protein folding/unfolding. Here, we examined a kinetic model in which domain swapping occurred via the denatured state produced by the complete unfolding of proteins. The relationships between swapping kinetics and folding/unfolding thermodynamics were established, which were further adopted as criteria to show that the proposed mechanism dominates in three representative proteins: Cyanovirin-N (CV-N), the C-terminal domain of SARS-CoV main protease (Mpro-C), and a single mutant of oxidized thioredoxin (Trx_W28Aox). PMID:23238853

  20. Distinguishing Unfolding and Functional Conformational Transitions of Calmodulin Using Ultraviolet Resonance Raman Spectroscopy

    SciTech Connect

    Jones, Eric M.; Balakrishnan, G.; Squier, Thomas C.; Spiro, Thomas

    2014-06-14

    Calmodulin (CaM) is a ubiquitous moderator protein for calcium signaling in all eukaryotic cells. This small calcium-binding protein exhibits a broad range of structural transitions, including domain opening and folding-unfolding, that allow it to recognize a wide variety of binding partners in vivo. While the static structures of CaM associated with its various binding activities are fairly well known, it has been challenging to examine the dynamics of transition between these structures in real-time, due to a lack of suitable spectroscopic probes of CaM structure. In this paper, we examine the potential of ultraviolet resonance Raman (UVRR) spectroscopy for clarifying the nature of structural transitions in CaM. We find that the UVRR spectral change (with 229 nm excitation) due to thermal unfolding of CaM is qualitatively different from that associated with opening of the C-terminal domain in response to Ca2+ binding. This spectral difference is entirely due to differences in teritary contacts at the inter-domain tyrosine residue Tyr138, toward which other spectroscopic methods are not sensitive. We conclude that UVRR is ideally suited to identifying the different types of structural transitions in CaM and other proteins with conformation-sensitive tyrosine residues, opening a path to time-resolved studies of CaM dynamics using Raman spectroscopy.

  1. Proteomic profiling of k-11706 responsive proteins.

    PubMed

    Horie, M; Kawashima, Y; Naka, A; Matsumoto, K; Kodera, Y; Maeda, T; Iida, K

    2011-07-01

    Erythropoietin promotes the production of red blood cells. Recombinant human erythropoietin is illicitly used to improve performance in endurance sports. Expression of the ERYTHROPOIETIN gene is negatively controlled by the transcription factor GATA-binding protein (GATA). Specific GATA inhibitors have recently been developed as novel drugs for the management of anemia. These drugs could, therefore, be illicitly used like recombinant human erythropoietin to improve performance in sports. To examine alterations in levels of plasma protein after administration of GATA inhibitors, proteomic analyses were conducted on mouse plasma samples treated with the potent GATA inhibitor K-11706. The analysis based on gel electrophoresis identified 41 protein spots differentially expressed when compared with normal plasma. Each spot was identified with liquid chromatography coupled to tandem mass spectrometry and 2 of them, fetuin-B and prothrombin, were verified by Western blotting. The results showed that the expression of fetuin-B in mice plasma was increased by K-11706, but not by recombinant human erythropoietin or hypoxia. These results suggest the potential of proteomic-based approaches as tools to identify biomarkers for the illegal use of novel drugs (e.g., GATA inhibitors). Also, fetuin-B could be a sensitive marker for the detection of abuse of GATA inhibitors. PMID:21567354

  2. Aging Is Accompanied by a Blunted Muscle Protein Synthetic Response to Protein Ingestion

    PubMed Central

    Wall, Benjamin Toby; Gorissen, Stefan H.; Pennings, Bart; Koopman, René; Groen, Bart B. L.; Verdijk, Lex B.; van Loon, Luc J. C.

    2015-01-01

    Purpose Progressive loss of skeletal muscle mass with aging (sarcopenia) forms a global health concern. It has been suggested that an impaired capacity to increase muscle protein synthesis rates in response to protein intake is a key contributor to sarcopenia. We assessed whether differences in post-absorptive and/or post-prandial muscle protein synthesis rates exist between large cohorts of healthy young and older men. Procedures We performed a cross-sectional, retrospective study comparing in vivo post-absorptive muscle protein synthesis rates determined with stable isotope methodologies between 34 healthy young (22±1 y) and 72 older (75±1 y) men, and post-prandial muscle protein synthesis rates between 35 healthy young (22±1 y) and 40 older (74±1 y) men. Findings Post-absorptive muscle protein synthesis rates did not differ significantly between the young and older group. Post-prandial muscle protein synthesis rates were 16% lower in the older subjects when compared with the young. Muscle protein synthesis rates were >3 fold more responsive to dietary protein ingestion in the young. Irrespective of age, there was a strong negative correlation between post-absorptive muscle protein synthesis rates and the increase in muscle protein synthesis rate following protein ingestion. Conclusions Aging is associated with the development of muscle anabolic inflexibility which represents a key physiological mechanism underpinning sarcopenia. PMID:26536130

  3. Expanded Monomeric Intermediate upon Cold and Heat Unfolding of Phosphofructokinase-2 from Escherichia coli

    PubMed Central

    Baez, Mauricio; Wilson, Christian A.M.; Ramírez-Sarmiento, César A.; Guixé, Victoria; Babul, Jorge

    2012-01-01

    Folding studies have been focused mainly on small, single-domain proteins or isolated single domains of larger proteins. However, most of the proteins present in biological systems are composed of multiple domains, and to date, the principles that underlie its folding remain elusive. The unfolding of Pfk-2 induced by GdnHCl has been described by highly cooperative three-state equilibrium (N2?2I?2U). This is characterized by a strong coupling between the subunits’ tertiary structure and the integrity of the dimer interface because “I” represents an unstructured and expanded monomeric intermediate. Here we report that cold and heat unfolding of Pfk-2 resembles the N2?2I step of chemically induced unfolding. Moreover, cold unfolding appears to be as cooperative as that induced chemically and even more so than its heat-unfolding counterpart. Because Pfk-2 is a large homodimer of 66 kDa with a complex topology consisting of well-defined domains, these results are somewhat unexpected considering that cold unfolding has been described as a special kind of perturbation that decouples the cooperative unfolding of several proteins. PMID:23200052

  4. Unfolding the fast neutron spectra of a BC501A liquid scintillation detector using GRAVEL method

    NASA Astrophysics Data System (ADS)

    Chen, YongHao; Chen, XiMeng; Lei, JiaRong; An, Li; Zhang, XiaoDong; Shao, JianXiong; Zheng, Pu; Wang, XinHua

    2014-10-01

    Accurate knowledge of the neutron energy spectra is useful in basic research and applications. The overall procedure of measuring and unfolding the fast neutron energy spectra with BC501A liquid scintillation detector is described. The recoil proton spectrum of 241Am-Be neutrons was obtained experimentally. With the NRESP7 code, the response matrix of detector was simulated. Combining the recoil proton spectrum and response matrix, the unfolding of neutron spectra was performed by GRAVEL iterative algorithm. A MatLab program based on the GRAVEL method was developed. The continuous neutron spectrum of 241Am-Be source and monoenergetic neutron spectrum of D-T source have been unfolded successfully and are in good agreement with their standard reference spectra. The unfolded 241Am-Be spectrum are more accurate than the spectra unfolded by artificial neural networks in recent years.

  5. Folding and Unfolding in Computational Geometry

    E-print Network

    O'Rourke, Joseph

    from sheet metal leads directly to unfolding issues. A 3D part isFolding and Unfolding in Computational Geometry Joseph O'Rourke Department of Computer Science. Abstract. Three open problems on folding/unfolding are discussed: (1) Can every convex polyhedron be cut

  6. Computerized Classification Testing under the Generalized Graded Unfolding Model

    ERIC Educational Resources Information Center

    Wang, Wen-Chung; Liu, Chen-Wei

    2011-01-01

    The generalized graded unfolding model (GGUM) has been recently developed to describe item responses to Likert items (agree-disagree) in attitude measurement. In this study, the authors (a) developed two item selection methods in computerized classification testing under the GGUM, the current estimate/ability confidence interval method and the cut…

  7. A role for SR proteins in plant stress responses

    PubMed Central

    2011-01-01

    Members of the SR (serine/arginine-rich) protein gene family are key players in the regulation of alternative splicing, an important means of generating proteome diversity and regulating gene expression. In plants, marked changes in alternative splicing are induced by a wide variety of abiotic stresses, suggesting a role for this highly versatile gene regulation mechanism in the response to environmental cues. In support of this notion, the expression of plant SR proteins is stress-regulated at multiple levels, with environmental signals controlling their own alternative splicing patterns, phosphorylation status and subcellular distribution. Most importantly, functional links between these RNA-binding proteins and plant stress tolerance are beginning to emerge, including a role in the regulation of abscisic acid (ABA) signaling. Future identification of the physiological mRNA targets of plant SR proteins holds much promise for the elucidation of the molecular mechanisms underlying their role in the response to abiotic stress. PMID:21258207

  8. The stress response system of proteins: Implications for bioreactor scaleup

    NASA Technical Reports Server (NTRS)

    Goochee, Charles F.

    1988-01-01

    Animal cells face a variety of environmental stresses in large scale bioreactors, including periodic variations in shear stress and dissolved oxygen concentration. Diagnostic techniques were developed for identifying the particular sources of environmental stresses for animal cells in a given bioreactor configuration. The mechanisms by which cells cope with such stresses was examined. The individual concentrations and synthesis rates of hundreds of intracellular proteins are affected by the extracellular environment (medium composition, dissolved oxygen concentration, ph, and level of surface shear stress). Techniques are currently being developed for quantifying the synthesis rates and concentrations of the intracellular proteins which are most sensitive to environmental stress. Previous research has demonstrated that a particular set of stress response proteins are synthesized by mammalian cells in response to temperature fluctuations, dissolved oxygen deprivation, and glucose deprivation. Recently, it was demonstrated that exposure of human kidney cells to high shear stress results in expression of a completely distinct set of intracellular proteins.

  9. Probing the interface in a human co-chaperonin heptamer: residues disrupting oligomeric unfolded state identified

    PubMed Central

    Guidry, Jesse J; Shewmaker, Frank; Maskos, Karol; Landry, Samuel; Wittung-Stafshede, Pernilla

    2003-01-01

    Background The co-chaperonin protein 10 (cpn10) assists cpn60 in the folding of nonnative polypeptides in a wide range of organisms. All known cpn10 molecules are heptamers of seven identical subunits that are linked together by ?-strand interactions at a large and flexible interface. Unfolding of human mitochondrial cpn10 in urea results in an unfolded heptameric state whereas GuHCl additions result in unfolded monomers. To address the role of specific interface residues in the assembly of cpn10 we prepared two point-mutated variants, in each case removing a hydrophobic residue positioned at the subunit-subunit interface. Results Replacing valine-100 with a glycine (Val100Gly cpn10) results in a wild-type-like protein with seven-fold symmetry although the thermodynamic stability is decreased and the unfolding processes in urea and GuHCl both result in unfolded monomers. In sharp contrast, replacing phenylalanine-8 with a glycine (Phe8Gly cpn10) results in a protein that has lost the ability to assemble. Instead, this protein exists mostly as unfolded monomers. Conclusions We conclude that valine-100 is a residue important to adopt an oligomeric unfolded state but it does not affect the ability to assemble in the folded state. In contrast, phenylalanine-8 is required for both heptamer assembly and monomer folding and therefore this mutation results in unfolded monomers at physiological conditions. Despite the plasticity and large size of the cpn10 interface, our observations show that isolated interface residues can be crucial for both the retention of a heptameric unfolded structure and for subunit folding. PMID:14525625

  10. The early steps in the unfolding of azurin.

    PubMed

    Rizzuti, Bruno; Daggett, Valerie; Guzzi, Rita; Sportelli, Luigi

    2004-12-14

    High-temperature molecular dynamics simulations were used to gain insight into the early steps in the unfolding pathway of azurin, a blue copper protein with a beta-barrel structure formed by two sheets arranged in a Greek key folding topology. The results reveal that unfolding of the beta-barrel in azurin is associated with dislocation of its unique alpha-helix with respect to the protein scaffold. Exposure of the hydrophobic core to solvent precedes complete disruption of secondary and tertiary structure, and modifications in the region around the active site are directly connected with this event. Denaturation of the protein initiates from the sheet coordinating the copper ion, and the other sheet maintains its topology. Results derived from the simulation were compared with experimental data obtained with different techniques, showing excellent agreement and providing a framework to understand the process of disruption and formation of the beta-barrel in azurin. PMID:15581373

  11. Carbohydrate Responsive Proteins in the Roots of Pennisetum americanum

    PubMed Central

    Baysdorfer, Chris; VanDerWoude, William J.

    1988-01-01

    The effect of changes in carbohydrate status on the synthesis of specific proteins was investigated in millet (Pennisetum americanum L., Leeke, Tift 23B1E1) seedlings grown in sterile solution culture. Carbohydrate status was altered by extended darkness and sucrose feeding. Root proteins from intact seedlings were labeled with [35S]methionine, phenol-extracted, separated by two-dimensional gel electrophoresis, and visualized by autoradiography. In four separate experiments, two proteins showed a consistent change in labeling when root carbohydrate levels were varied between 200 and 1000 micromole hexose per gram residual dry weight. Labeling of the first protein (P47, Mr 47 kD) increased as the carbohydrate levels rose above 500 micromole hexose per gram residual dry weight. Labeling of the second protein (P34, Mr 34 kD) increased as carbohydrate levels declined from 500 to 200 micromole hexose per gram residual dry weight. Under extreme conditions, when carbohydrate levels fell below 100 micromole hexose per gram residual dry weight, the labeling pattern of most proteins was drastically altered. It is suggested that P47 and P34 are `carbohydrate responsive proteins,' i.e. proteins whose concentrations are controlled either directly or indirectly by tissue carbohydrate status. In contrast, the changes in protein labeling that occur once carbohydrate pools are depleted may be involved in adaptation to periods of prolonged starvation. Images Fig. 3 Fig. 4 Fig. 5 Fig. 6 PMID:16666186

  12. Export is the default pathway for soluble unfolded polypeptides that accumulate during expression in Escherichia coli

    SciTech Connect

    Scotto-Lavino, E.; Freimuth, P.; Bai, M.; Zhang, Y.-B.

    2011-09-01

    Several E. coli endogenous, cytoplasmic proteins that are known clients of the chaperonin GroEL were overexpressed to examine the fate of accumulated unfolded polypeptides. Substantial fractions of about half of the proteins formed insoluble aggregates, consistent with the hypothesis that these proteins were produced at rates or in amounts that exceeded the protein-folding capacity of GroEL. In addition, large fractions of three overexpressed GroEL client proteins were localized in an extra-cytoplasmic, osmotically-sensitive compartment, suggesting they had initially accumulated in the cytoplasm as soluble unfolded polypeptides and thus were able to access a protein export pathway. Consistent with this model, an intrinsically unfoldable, hydrophilic, non-secretory polypeptide was quantitatively exported from the E. coli cytoplasm into an osmotically-sensitive compartment. Our results support the conclusion that a soluble, unfolded conformation alone may be sufficient to direct non-secretory polypeptides into a protein export pathway for signal peptide-independent translocation across the inner membrane, and that export rather than degradation by cytoplasmic proteases is the preferred fate for newly-synthesized, soluble, unfolded polypeptides that accumulate in the cytoplasm. The stable folded conformation of exported GroEL client proteins further suggests that the requirement for GroEL may be conditional on protein folding in the molecularly-crowded environment of the cytoplasm.

  13. The Metabolic Response to Hypocaloric Protein Diets in Obese Man

    PubMed Central

    Marliss, Errol B.; Murray, Frederick T.; Nakhooda, Azima F.

    1978-01-01

    Exogenous protein in the absence of other calories can cause protein-sparing, but the mechanisms involved are controversial. It has been postulated that low insulin and high fat-derived substrate levels are necessary and sufficient conditions for such protein-sparing. We therefore established such conditions with differing protocols of protein input to define the role of protein input in mediating the response. Three groups of obese, nondiabetic subjects received the following diets: (1) 82.5±1.0 g protein/day (400 cal/day) for 21 days, n = 7; (2) the same, but as a refeeding diet for 7 days after 21-28 days of total fasts, n = 7; and (3) commencing with the same input, but with daily stepwise decrements over 14 days to 19.4±2.2 g/day, then maintained an additional 7 days, n = 4. Diet 3 gave approximately the amount and pattern of protein lost during total fasting. The circulating hormone and substrate responses of diets 1 and 3 were comparable and resembled those of total fasts, in that plasma glucose and insulin fell and free fatty acids rose. Blood levels of alanine, pyruvate, and other glucogenic amino acids fell and blood levels of branched-chain amino acids rose transiently. Blood 3-hydroxybutyrate levels and urinary excretion were greater in diet 3 than diet 1, but less than in total fasting. Nitrogen balance in diet 1 was transiently negative, but in equilibrium from 12 to 21 days. In diet 3, it was constantly negative at ?6 g/day, the values also observed at 21 days of fasting. Mean 3-methylhistidine excretion decreased by 170 ?mol/day in diet 1 and 107 ?mol/day in diet 3, reflecting decreased muscle protein catabolism. The refed, protein-depleted subjects, diet 2, showed an increase in plasma glucose without alteration in insulin levels. Free fatty acid and ketone body levels decreased to those of the steady state observed in diet 1. Glucogenic and branched-chain amino acids decreased transiently. Nitrogen balance became positive, and the low 3-methylhistidine excretion increased by 152 ?mol/day. The differing responses of nitrogen balance could not be accounted for on the basis of levels of insulin or of fat-derived substrates. The primary determinants of the protein-sparing observed appeared to be the protein supply itself, and the magnitude of the decrease in endogenous protein catabolism. The positive balance on refeeding after prior depletion of protein stores was likely due to the exogenous supply, combined with decreased catabolism and considerably increased reutilization. PMID:670403

  14. Therapeutic Protein Aggregation: Mechanisms, Design, and Control

    PubMed Central

    Roberts, Christopher J.

    2014-01-01

    While it is well known that proteins are only marginally stable in their folded states, it is often less well appreciated that most proteins are inherently aggregation-prone in their unfolded or partially unfolded states, and the resulting aggregates can be extremely stable and long-lived. For therapeutic proteins, aggregates are a significant risk factor for deleterious immune responses in patients, and can form via a variety of mechanisms. Controlling aggregation using a mechanistic approach may allow improved design of therapeutic protein stability, as a complement to existing design strategies that target desired protein structures and function. Recent results highlight the importance of balancing protein environment with the inherent aggregation propensities of polypeptide chains. PMID:24908382

  15. Identifying Kinetic Barriers to Mechanical Unfolding of the T. thermophila Ribozyme

    NASA Astrophysics Data System (ADS)

    Onoa, Bibiana; Dumont, Sophie; Liphardt, Jan; Smith, Steven B.; Tinoco, Ignacio; Bustamante, Carlos

    2003-03-01

    Mechanical unfolding trajectories for single molecules of the Tetrahymena thermophila ribozyme display eight intermediates corresponding to discrete kinetic barriers that oppose mechanical unfolding with lifetimes of seconds and rupture forces between 10 and 30 piconewtons. Barriers are magnesium dependent and correspond to known intra- and interdomain interactions. Several barrier structures are ``brittle,'' breakage requiring high forces but small (1 to 3 nanometers) deformations. Barrier crossing is stochastic, leading to variable unfolding paths. The response of complex RNA structures to locally applied mechanical forces may be analogous to the responses of RNA during translation, messenger RNA export from the nucleus, and viral replication.

  16. Empirical Spacings of Unfolded Eigenvalues

    E-print Network

    Kristina Schubert; Martin Venker

    2015-08-23

    We study random points on the real line generated by the eigenvalues in unitary invariant random matrix ensembles or by more general repulsive particle systems. As the number of points tends to infinity, we prove convergence of the empirical distribution of nearest neighbor spacings. We extend existing results for the spacing distribution in two ways. On the one hand, we believe the empirical distribution to be of more practical relevance than the so far considered expected distribution. On the other hand, we use the unfolding, a non-linear rescaling, which transforms the ensemble such that the density of particles is asymptotically constant. This allows to consider all empirical spacings, where previous results were restricted to a tiny fraction of the particles. Moreover, we prove bounds on the rates of convergence. The main ingredient for the proof, a strong bulk universality result for correlation functions in the unfolded setting including optimal rates, should be of independent interest.

  17. Induction of Antioxidant and Heat Shock Protein Responses During Torpor in the Gray Mouse Lemur, Microcebus murinus

    PubMed Central

    Wu, Cheng-Wei; Biggar, Kyle K.; Zhang, Jing; Tessier, Shannon N.; Pifferi, Fabien; Perret, Martine; Storey, Kenneth B.

    2015-01-01

    A natural tolerance of various environmental stresses is typically supported by various cytoprotective mechanisms that protect macromolecules and promote extended viability. Among these are antioxidant defenses that help to limit damage from reactive oxygen species and chaperones that help to minimize protein misfolding or unfolding under stress conditions. To understand the molecular mechanisms that act to protect cells during primate torpor, the present study characterizes antioxidant and heat shock protein (HSP) responses in various organs of control (aroused) and torpid gray mouse lemurs, Microcebus murinus. Protein expression of HSP70 and HSP90? was elevated to 1.26 and 1.49 fold, respectively, in brown adipose tissue during torpor as compared with control animals, whereas HSP60 in liver of torpid animals was 1.15 fold of that in control (P < 0.05). Among antioxidant enzymes, protein levels of thioredoxin 1 were elevated to 2.19 fold in white adipose tissue during torpor, whereas Cu–Zn superoxide dismutase 1 levels rose to 1.1 fold in skeletal muscle (P < 0.05). Additionally, total antioxidant capacity was increased to 1.6 fold in liver during torpor (P < 0.05), while remaining unchanged in the five other tissues. Overall, our data suggest that antioxidant and HSP responses are modified in a tissue-specific manner during daily torpor in gray mouse lemurs. Furthermore, our data also show that cytoprotective strategies employed during primate torpor are distinct from the strategies in rodent hibernation as reported in previous studies. PMID:26092183

  18. Induction of Antioxidant and Heat Shock Protein Responses During Torpor in the Gray Mouse Lemur, Microcebus murinus.

    PubMed

    Wu, Cheng-Wei; Biggar, Kyle K; Zhang, Jing; Tessier, Shannon N; Pifferi, Fabien; Perret, Martine; Storey, Kenneth B

    2015-04-01

    A natural tolerance of various environmental stresses is typically supported by various cytoprotective mechanisms that protect macromolecules and promote extended viability. Among these are antioxidant defenses that help to limit damage from reactive oxygen species and chaperones that help to minimize protein misfolding or unfolding under stress conditions. To understand the molecular mechanisms that act to protect cells during primate torpor, the present study characterizes antioxidant and heat shock protein (HSP) responses in various organs of control (aroused) and torpid gray mouse lemurs, Microcebus murinus. Protein expression of HSP70 and HSP90? was elevated to 1.26 and 1.49 fold, respectively, in brown adipose tissue during torpor as compared with control animals, whereas HSP60 in liver of torpid animals was 1.15 fold of that in control (P<0.05). Among antioxidant enzymes, protein levels of thioredoxin 1 were elevated to 2.19 fold in white adipose tissue during torpor, whereas Cu-Zn superoxide dismutase 1 levels rose to 1.1 fold in skeletal muscle (P<0.05). Additionally, total antioxidant capacity was increased to 1.6 fold in liver during torpor (P<0.05), while remaining unchanged in the five other tissues. Overall, our data suggest that antioxidant and HSP responses are modified in a tissue-specific manner during daily torpor in gray mouse lemurs. Furthermore, our data also show that cytoprotective strategies employed during primate torpor are distinct from the strategies in rodent hibernation as reported in previous studies. PMID:26092183

  19. High temperature unfolding of a truncated hemoglobin by molecular dynamics simulation.

    PubMed

    Sharma, Ravi Datta; Kanwal, Rajnee; Lynn, Andrew M; Singh, Prerna; Pasha, Syed Tazeen; Fatma, Tasneem; Jawaid, Safdar

    2013-09-01

    Heme containing proteins are associated with peroxidase activity. The proteins like hemoglobin, myoglobins, cytochrome c and micro-peroxidase other than peroxidases have been shown to exhibit weak peroxidase-like activity. This weak peroxidase-like activity in hemoglobin-like molecules is due to heme moiety. We conducted molecular dynamics (MD) studies to decipher the unfolding path of Ba-Glb (a truncated hemoglobin from Bacillus anthracis) and the role of heme moiety to its unfolding path. The similar unfolding path is also observed in vitro by UV/VIS spectroscopy. The data confirmed that the unfolding of Ba-Glb follows a three state process with a meta-stable (intermediate) state between the native and unfolded conformations. The present study is supported by several unfolding parameters like root-mean-square-deviation (RMSD), dictionary of protein secondary structure (DSSP), and free energy landscape. Understanding the structure of hemoglobin like proteins in unicellular dreaded pathogens like B. anthracis will pave way for newer drug discovery targets and in the disease management of anthrax. PMID:23839248

  20. Correlation Analysis of Trp-Cage Dynamics in Folded and Unfolded States.

    PubMed

    Palese, Luigi L

    2015-12-24

    A fundamental and still debated problem is how folded structures of proteins are related to their unfolded state. Besides the classical view, in which a large number of conformations characterize the unfolded state while the folded one is dominated by a single structure, recently a reassessment of the denatured state has been suggested. A growing amount of evidence indicates that not only the folded but also the unfolded state is at least partially organized. Here, we try to answer the question of how different protein dynamics is in folded and unfolded states by performing all-atom molecular dynamics simulations on the model protein Trp-cage. Random matrix theory inspired analysis of the correlation matrices has been carried out. The spectra of these correlation matrices show that the low rank modes of Trp-cage dynamics are outside of the limit expected for a random system both in folded and in unfolded conditions. These findings shed light on the nature of the unfolded state of the proteins, suggesting that it is much less random than previously thought. PMID:26619349

  1. Mechanical Unfolding of Acylphosphatase Studied by Single-Molecule Force Spectroscopy and MD Simulations

    PubMed Central

    Arad-Haase, Gali; Chuartzman, Silvia G.; Dagan, Shlomi; Nevo, Reinat; Kouza, Maksim; Mai, Binh Khanh; Nguyen, Hung Tien; Li, Mai Suan; Reich, Ziv

    2010-01-01

    Abstract Single-molecule manipulation methods provide a powerful means to study protein transitions. Here we combined single-molecule force spectroscopy and steered molecular-dynamics simulations to study the mechanical properties and unfolding behavior of the small enzyme acylphosphatase (AcP). We find that mechanical unfolding of AcP occurs at relatively low forces in an all-or-none fashion and is decelerated in the presence of a ligand, as observed in solution measurements. The prominent energy barrier for the transition is separated from the native state by a distance that is unusually long for ?/? proteins. Unfolding is initiated at the C-terminal strand (?T) that lies at one edge of the ?-sheet of AcP, followed by unraveling of the strand located at the other. The central strand of the sheet and the two helices in the protein unfold last. Ligand binding counteracts unfolding by stabilizing contacts between an arginine residue (Arg-23) and the catalytic loop, as well as with ?T of AcP, which renders the force-bearing units of the protein resistant to force. This stabilizing effect may also account for the decelerated unfolding of ligand-bound AcP in the absence of force. PMID:20655852

  2. Simulations of the folding/unfolding of biomolecules under solvent, and pressure perturbations

    NASA Astrophysics Data System (ADS)

    Garcia, Angel

    2012-02-01

    Proteins exhibit marginal stability, determined by the balance of many competing effects. This stability can be perturbed by changes in temperature, pH, pressure, and other solvent conditions. Osmolytes are small organic compounds that modulate the conformational equilibrium, folded (F) and unfolded (U), of proteins as cosolvents. Protecting osmolytes such as trimethylamine N-oxide (TMAO), glycerol, and sugars that push the equilibrium toward F play a crucial role in maintaining the function of intracellular proteins in extreme environmental conditions. Urea is a denaturing osmolyte that shifts the equilibrium toward U. We will describe calculations of the reversible folding/unfolding equilibrium, under various solution conditions that include urea, high pressure, and different charge states of the Trp-cage miniprotein. The folding/unfolding equilibrium is studied using all-atom Replica exchange MD simulations. For urea, the simulations capture the experimentally observed linear dependence of unfolding free energy on urea concentration. We find that the denaturation is driven by favorable direct interaction of urea with the protein through both electrostatic and van der Waals forces and quantify their contribution. Though the magnitude of direct electrostatic interaction of urea is larger than van der Waals, the difference between unfolded and folded ensembles is dominated by the van der Waals interaction. We also find that hydrogen bonding of urea to the peptide backbone does not play a dominant role in denaturation. The unfolded ensemble sampled depends on urea concentration, with greater urea concentration favoring conformations with greater solvent exposure.

  3. Molecular Dynamics Simulations of the Temperature Induced Unfolding of Crambin Follow the Arrhenius Equation.

    PubMed Central

    Dalby, Andrew; Shamsir, Mohd Shahir

    2015-01-01

    Molecular dynamics simulations have been used extensively to model the folding and unfolding of proteins. The rates of folding and unfolding should follow the Arrhenius equation over a limited range of temperatures. This study shows that molecular dynamic simulations of the unfolding of crambin between 500K and 560K do follow the Arrhenius equation. They also show that while there is a large amount of variation between the simulations the average values for the rate show a very high degree of correlation. PMID:26539292

  4. Unfolding mechanism of lysozyme in various urea solutions: Insights from fluorescence spectroscopy

    NASA Astrophysics Data System (ADS)

    Chen, Bang; Zhang, Hongjia; Xi, Wenying; Zhao, Liqing; Liang, Li; Chen, Yantao

    2014-11-01

    Fluorescence spectroscopic technique is very popular in exploring the folding/unfolding process of proteins. In this paper, unfolding process of hen egg-white lysozyme was investigated in various denaturing solutions. Firstly, polymer solution theory was employed to comprehend the dependence of fluorescence quenching effect on protein concentration, and dynamic contact concentration was suggested as a critical value for related fluorescence experiment. Secondly, it was found that urea alone could not completely unfold lysozyme but did when together with DTT or HCl. Lysozyme was destabilized in concentrated urea solution, but still could maintain its spatial structure. Phase diagram of fluorescence intensities revealed that HCl could enhance the denaturing capacity of urea, resulting in the emergence of intermediate state in the thermodynamic unfolding process of lysozyme.

  5. Induced Dipole-Dipole Interactions Influence the Unfolding Pathways of Wild-Type and Mutant Amyloid ?-Peptides.

    PubMed

    Lemkul, Justin A; Huang, Jing; MacKerell, Alexander D

    2015-12-24

    Amyloid-forming proteins undergo a structural transition from ?-helical to disordered conformations and, ultimately, cross-? fibrils. The unfolding and aggregation of the amyloid ?-peptide (A?) have been implicated in the development and progression of Alzheimer's disease (AD) and cerebral amyloid angiopathy (CAA). However, the events underlying the initial structural transition leading to the disease state remain unclear. Although most cases are sporadic, several genetic variants exist that alter the electrostatic properties of A? and lead to more rapid unfolding and more severe phenotypes. In the present study, the enhanced unfolding is shown to be due to the mutated side chains altering the local peptide-bond dipole moments leading to local destabilization of the ?-helix, as determined from polarizable molecular dynamics (MD) simulations of wild-type (WT) A? fragments and several common mutations. The local perturbation of the helix then leads to progressive unwinding of the ?-helix in a cooperative fashion due to decreases in adjacent (i ± 1) and hydrogen-bonded (i + 4) peptide-bond dipole moments. Side-chain dynamics, subsequent variations in dipole moments, and ultimately the response in the peptide-bond dipole moments are all modulated by solvent dielectric properties based on simulations in water versus ethanol. The polarizable simulation results, along with simulations using the additive CHARMM36 force field, further indicate that cooperativity due to the alignment of peptide bonds leading to enhanced dipole moments is a fundamental force in stabilizing ?-helices. PMID:26629591

  6. Induced Dipole–Dipole Interactions Influence the Unfolding Pathways of Wild-Type and Mutant Amyloid ?-Peptides

    PubMed Central

    2015-01-01

    Amyloid-forming proteins undergo a structural transition from ?-helical to disordered conformations and, ultimately, cross-? fibrils. The unfolding and aggregation of the amyloid ?-peptide (A?) have been implicated in the development and progression of Alzheimer’s disease (AD) and cerebral amyloid angiopathy (CAA). However, the events underlying the initial structural transition leading to the disease state remain unclear. Although most cases are sporadic, several genetic variants exist that alter the electrostatic properties of A? and lead to more rapid unfolding and more severe phenotypes. In the present study, the enhanced unfolding is shown to be due to the mutated side chains altering the local peptide-bond dipole moments leading to local destabilization of the ?-helix, as determined from polarizable molecular dynamics (MD) simulations of wild-type (WT) A? fragments and several common mutations. The local perturbation of the helix then leads to progressive unwinding of the ?-helix in a cooperative fashion due to decreases in adjacent (i ± 1) and hydrogen-bonded (i + 4) peptide-bond dipole moments. Side-chain dynamics, subsequent variations in dipole moments, and ultimately the response in the peptide-bond dipole moments are all modulated by solvent dielectric properties based on simulations in water versus ethanol. The polarizable simulation results, along with simulations using the additive CHARMM36 force field, further indicate that cooperativity due to the alignment of peptide bonds leading to enhanced dipole moments is a fundamental force in stabilizing ?-helices. PMID:26629591

  7. A Euclidean Perspective on the Unfolding of Azurin: Spatial Correlations

    PubMed Central

    Warren, Jeffrey J.; Gray, Harry B.; Winkler, Jay R.; Kozak, John J.

    2013-01-01

    We investigate the stability to structural perturbation of Pseudomonas aeruginosa azurin using a previously developed geometric model. Our analysis considers Ru(2,2?,6?,2?-terpyridine)(1,10-phenanthroline)(His83)-labeled wild-type azurin and five variants with mutations to Cu-ligating residues. We find that in the early stages of unfolding, the ?-strands exhibit the most structural stability. The conserved residues comprising the hydrophobic core are dislocated only after nearly complete unfolding of the ?-barrel. Attachment of the Ru-complex at His83 does not destabilize the protein fold, despite causing some degree of structural rearrangement. Notably, replacing the Cys112 and/or Met121 Cu ligands does not affect the conformational integrity of the protein. Notably, these results are in accord with experimental evidence, as well as molecular dynamics simulations of the denaturation of azurin. PMID:23853392

  8. Chemical Theory and Computation Special Feature: Mechanical unfolding of RNA hairpins

    NASA Astrophysics Data System (ADS)

    Hyeon, Changbong; Thirumalai, D.

    2005-05-01

    Mechanical unfolding trajectories, generated by applying constant force in optical-tweezer experiments, show that RNA hairpins and the P5abc subdomain of the group I intron unfold reversibly. We use coarse-grained Go-like models for RNA hairpins to explore forced unfolding over a broad range of temperatures. A number of predictions that are amenable to experimental tests are made. At the critical force, the hairpin jumps between folded and unfolded conformations without populating any discernible intermediates. The phase diagram in the force-temperature (f, T) plane shows that the hairpin unfolds by an all-or-none process. The cooperativity of the unfolding transition increases dramatically at low temperatures. Free energy of stability, obtained from time averages of mechanical unfolding trajectories, coincides with ensemble averages, which establishes ergodicity. The hopping time between the native basin of attraction (NBA) and the unfolded basin increases dramatically along the phase boundary. Thermal unfolding is stochastic, whereas mechanical unfolding occurs in "quantized steps" with great variations in the step lengths. Refolding times, upon force quench, from stretched states to the NBA are at least an order of magnitude greater than folding times by temperature quench. Upon force quench from stretched states, the NBA is reached in at least three stages. In the initial stages, the mean end-to-end distance decreases nearly continuously, and there is a sudden transition to the NBA only in the last stage. Because of the generality of the results, we propose that similar behavior should be observed in force quench refolding of proteins.

  9. Investigation of the kinetics of protein folding and the ensemble of conformations in non-native states of proteins by liquid NMR spectroscopy

    E-print Network

    Wirmer, Julia

    2005-01-01

    For a complete description of protein folding dynamics and the structure of the folded state, of unfolded and of non-native states of proteins and the kinetics of protein folding from the unfolded state to the folded state ...

  10. The Intrinsic Dynamics and Unfolding Process of an Antibody Fab Fragment Revealed by Elastic Network Model

    PubMed Central

    Su, Ji-Guo; Zhang, Xiao; Han, Xiao-Ming; Zhao, Shu-Xin; Li, Chun-Hua

    2015-01-01

    Antibodies have been increasingly used as pharmaceuticals in clinical treatment. Thermal stability and unfolding process are important properties that must be considered in antibody design. In this paper, the structure-encoded dynamical properties and the unfolding process of the Fab fragment of the phosphocholine-binding antibody McPC603 are investigated by use of the normal mode analysis of Gaussian network model (GNM). Firstly, the temperature factors for the residues of the protein were calculated with GNM and then compared with the experimental measurements. A good result was obtained, which provides the validity for the use of GNM to study the dynamical properties of the protein. Then, with this approach, the mean-square fluctuation (MSF) of the residues, as well as the MSF in the internal distance (MSFID) between all pairwise residues, was calculated to investigate the mobility and flexibility of the protein, respectively. It is found that the mobility and flexibility of the constant regions are higher than those of the variable regions, and the six complementarity-determining regions (CDRs) in the variable regions also exhibit relative large mobility and flexibility. The large amplitude motions of the CDRs are considered to be associated with the immune function of the antibody. In addition, the unfolding process of the protein was simulated by iterative use of the GNM. In our method, only the topology of protein native structure is taken into account, and the protein unfolding process is simulated through breaking the native contacts one by one according to the MSFID values between the residues. It is found that the flexible regions tend to unfold earlier. The sequence of the unfolding events obtained by our method is consistent with the hydrogen-deuterium exchange experimental results. Our studies imply that the unfolding behavior of the Fab fragment of antibody McPc603 is largely determined by the intrinsic dynamics of the protein. PMID:26690429

  11. Intranuclear localization and UV response of ERCC5/XPG protein

    SciTech Connect

    Park, M.S.; Marrone, B.L.; MacInnes, M.A.

    1995-11-01

    The human ERCC5/XPG protein is defective in the hereditary genetic disorder xeroderma pigmentosum, group-G. The XPG gene encodes a single-strand DNA endonuclease which is essential for the incision step of nucleotide excision repair for a wide variety of DNA damages. We have shown previously by indirect immunofluorescence and biochemical fractionation that the XPG protein is localized in the nucleus, in discrete foci, and probably associated with the nuclear matrix. However, the intranuclear localization of XPG is markedly altered for a short time after UV irradiation. Here, we report the identification of XPG protein regions involved in the UV response, and its putative nuclear localization signals (NLS) using a B-galactosidase (B-gal) reporter gene system. Control and fusion reporter genes were expressed in Hela S3 cells after CaPO{sub 4} transfection. B-gal protein was detected by indirect immuno-fluorescence using an anti B-gal monoclonal antibody and FITC-labeled goat anti-mouse antiserum. Two NLS peptides of the XPG carboxy-terminal region (AA 1029-1069 and 1146-1186 term) were shown to independently localize B-gal fusion proteins to the nucleus (>90%). The C-terminus peptide was observed to further localize B-gal into nuclear foci and the perinucleolar regions. When B-gal was fused with two copies of the C-terminal NLS, in tandem, B-gal was extensively sublocalized to the perinucleolar regions. Shortly after cell UV irradiation (5 J/m{sup 2}) this B-gal fusion protein became dissociated from the perinucleolar regions whereupon it was distributed throughout the nucleus. Within 6 hours post-irradiation, the fusion protein reassociated again with the perinucleolar regions. These observations confirm and extend a similar UV response of endogenous XPG protein in UV-irradiation human cells. The involvement of XPG protein and its UV responses will be discussed in context of models nuclear matrix and preferential DNA repair in actively transcribed genes.

  12. The identification of unfolding facial expressions.

    PubMed

    Fiorentini, Chiara; Schmidt, Susanna; Viviani, Paolo

    2012-01-01

    We asked whether the identification of emotional facial expressions (FEs) involves the simultaneous perception of the facial configuration or the detection of emotion-specific diagnostic cues. We recorded at high speed (500 frames s-1) the unfolding of the FE in five actors, each expressing six emotions (anger, surprise, happiness, disgust, fear, sadness). Recordings were coded every 10 frames (20 ms of real time) with the Facial Action Coding System (FACS, Ekman et al 2002, Salt Lake City, UT: Research Nexus eBook) to identify the facial actions contributing to each expression, and their intensity changes over time. Recordings were shown in slow motion (1/20 of recording speed) to one hundred observers in a forced-choice identification task. Participants were asked to identify the emotion during the presentation as soon as they felt confident to do so. Responses were recorded along with the associated response times (RTs). The RT probability density functions for both correct and incorrect responses were correlated with the facial activity during the presentation. There were systematic correlations between facial activities, response probabilities, and RT peaks, and significant differences in RT distributions for correct and incorrect answers. The results show that a reliable response is possible long before the full FE configuration is reached. This suggests that identification is reached by integrating in time individual diagnostic facial actions, and does not require perceiving the full apex configuration. PMID:23025158

  13. Characterization of the protein apparently responsible for the elevated tyrosine protein kinase activity in LSTRA cells.

    PubMed Central

    Voronova, A F; Buss, J E; Patschinsky, T; Hunter, T; Sefton, B M

    1984-01-01

    The LSTRA murine thymoma cell line contains an elevated level of tyrosine protein kinase activity. When a microsomal preparation from these cells is incubated in vitro with ATP, the principal tyrosine protein kinase substrate is a 56,000-dalton protein, p56. We have found that an activity phosphorylating p56 on tyrosine can also be detected at low levels in microsomes from most, but not all, T lymphoma cell lines and from normal thymic tissue. Only 1 of 30 other lymphoma cell lines was found to contain an elevated level of such a tyrosine protein kinase. An activity that phosphorylated p56 in vitro was not detectable in the cells of other hematopoietic lineages. Anti-peptide antibodies reactive with the site of in vitro tyrosine phosphorylation of p56 allowed us to determine that the apparent abundance of the p56 polypeptide parallels closely the level of the tyrosine protein kinase activity in the cell lines examined. This suggests that p56 is the protein kinase responsible for the elevated tyrosine protein kinase activity in LSTRA cells and that the phosphorylation of p56 observed in vitro results from autophosphorylation. Two-dimensional tryptic peptide mapping revealed that p56 is distinct from the proteins encoded by the cellular genes which are the progenitors of retroviral tyrosine protein kinases, src, yes, fgr, abl, fes, and ros. Additionally, none of these proto-oncogenes was found to be transcribed at elevated levels in LSTRA or Thy19 cells. Like the catalytic subunit of the cyclic AMP-dependent protein kinase, the cellular and viral forms of p60src, and the protein phosphatase calcineurin B, p56 contains covalently bound fatty acid. Images PMID:6543243

  14. Structure of the Periplasmic Stress Response Protein CpxP?†

    PubMed Central

    Thede, Gina L.; Arthur, David C.; Edwards, Ross A.; Buelow, Daelynn R.; Wong, Julia L.; Raivio, Tracy L.; Glover, J. N. Mark

    2011-01-01

    CpxP is a novel bacterial periplasmic protein with no homologues of known function. In Gram-negative enteric bacteria, CpxP is thought to interact with the two-component sensor kinase, CpxA, to inhibit induction of the Cpx envelope stress response in the absence of protein misfolding. CpxP has also been shown to facilitate DegP-mediated proteolysis of misfolded proteins. Six mutations that negate the ability of CpxP to function as a signaling protein are localized in or near two conserved LTXXQ motifs that define a class of proteins with similarity to CpxP, Pfam PF07813. To gain insight into how these mutations might affect CpxP signaling and/or proteolytic adaptor functions, the crystal structure of CpxP from Escherichia coli was determined to 2.85-Å resolution. The structure revealed an antiparallel dimer of intertwined ?-helices with a highly basic concave surface. Each protomer consists of a long, hooked and bent hairpin fold, with the conserved LTXXQ motifs forming two diverging turns at one end. Biochemical studies demonstrated that CpxP maintains a dimeric state but may undergo a slight structural adjustment in response to the inducing cue, alkaline pH. Three of the six previously characterized cpxP loss-of-function mutations, M59T, Q55P, and Q128H, likely result from a destabilization of the protein fold, whereas the R60Q, D61E, and D61V mutations may alter intermolecular interactions. PMID:21317318

  15. Multidimensional free energy surface of unfolding of HP-36: Microscopic origin of ruggedness

    NASA Astrophysics Data System (ADS)

    Ghosh, Rikhia; Roy, Susmita; Bagchi, Biman

    2014-10-01

    The protein folding funnel paradigm suggests that folding and unfolding proceed as directed diffusion in a multidimensional free energy surface where a multitude of pathways can be traversed during the protein's sojourn from initial to final state. However, finding even a single pathway, with the detail chronicling of intermediates, is an arduous task. In this work we explore the free energy surface of unfolding pathway through umbrella sampling, for a small globular ?-helical protein chicken-villin headpiece (HP-36) when the melting of secondary structures is induced by adding DMSO in aqueous solution. We find that the unfolding proceeds through the initial separation or melting of aggregated hydrophobic core that comprises of three phenylalanine residues (Phe7, Phe11, and Phe18). This separation is accompanied by simultaneous melting of the second helix. Unfolding is found to be a multistage process involving crossing of three consecutive minima and two barriers at the initial stage. At a molecular level, Phe18 is observed to reorient itself towards other hydrophobic grooves to stabilize the intermediate states. We identify the configuration of the intermediates and correlate the intermediates with those obtained in our previous works. We also give an estimate of the barriers for different transition states and observe the softening of the barriers with increasing DMSO concentration. We show that higher concentration of DMSO tunes the unfolding pathway by destabilizing the third minimum and stabilizing the second one, indicating the development of a solvent modified, less rugged pathway. The prime outcome of this work is the demonstration that mixed solvents can profoundly transform the nature of the energy landscape and induce unfolding via a modified route. A successful application of Kramer's rate equation correlating the free energy simulation results shows faster rate of unfolding with increasing DMSO concentration. This work perhaps presents the first systematic theoretical study of the effect of a chemical denaturant on the microscopic free energy surface and rates of unfolding of HP-36.

  16. A stress-responsive system for mitochondrial protein degradation

    PubMed Central

    Heo, Jin-Mi; Livnat-Levanon, Nurit; Taylor, Eric B.; Jones, Kevin T.; Dephoure, Noah; Ring, Julia; Xie, Jianxin; Brodsky, Jeffrey L.; Madeo, Frank; Gygi, Steven P.; Ashrafi, Kaveh; Glickman, Michael H.; Rutter, Jared

    2010-01-01

    We show that Ydr049 (renamed VCP/ Cdc48-associated Mitochondrial Stress-responsive—Vms1), a member of an unstudied pan-eukaryotic protein family, translocates from the cytosol to mitochondria upon mitochondrial stress. Cells lacking Vms1 show progressive mitochondrial failure, hypersensitivity to oxidative stress and decreased chronological lifespan. Both yeast and mammalian Vms1 stably interact with Cdc48/ VCP/ p97, a component of the ubiquitin/ proteasome system with a well-defined role in endoplasmic reticulum-associated protein degradation (ERAD), wherein misfolded ER proteins are degraded in the cytosol. We show that oxidative stress triggers mitochondrial localization of Cdc48 and this is dependent on Vms1. When this system is impaired by mutation of Vms1, ubiquitin-dependent mitochondrial protein degradation, mitochondrial respiratory function and cell viability are compromised. We demonstrate that Vms1 is a required component of an evolutionarily conserved system for mitochondrial protein degradation, which is necessary to maintain mitochondrial, cellular and organismal viability. PMID:21070972

  17. A Generalization of the Source Unfolding of Convex Polyhedra

    E-print Network

    Demaine, Erik D.

    2012-01-01

    We present a new method for unfolding a convex polyhedron into one piece without overlap, based on shortest paths to a convex curve on the polyhedron. Our “sun unfoldings” encompass source unfolding from a point, source ...

  18. LON is the master protease that protects against protein aggregation in human mitochondria through direct degradation of misfolded proteins.

    PubMed

    Bezawork-Geleta, Ayenachew; Brodie, Erica J; Dougan, David A; Truscott, Kaye N

    2015-01-01

    Maintenance of mitochondrial protein homeostasis is critical for proper cellular function. Under normal conditions resident molecular chaperones and proteases maintain protein homeostasis within the organelle. Under conditions of stress however, misfolded proteins accumulate leading to the activation of the mitochondrial unfolded protein response (UPR(mt)). While molecular chaperone assisted refolding of proteins in mammalian mitochondria has been well documented, the contribution of AAA+ proteases to the maintenance of protein homeostasis in this organelle remains unclear. To address this gap in knowledge we examined the contribution of human mitochondrial matrix proteases, LONM and CLPXP, to the turnover of OTC-?, a folding incompetent mutant of ornithine transcarbamylase, known to activate UPR(mt). Contrary to a model whereby CLPXP is believed to degrade misfolded proteins, we found that LONM, and not CLPXP is responsible for the turnover of OTC-? in human mitochondria. To analyse the conformational state of proteins that are recognised by LONM, we examined the turnover of unfolded and aggregated forms of malate dehydrogenase (MDH) and OTC. This analysis revealed that LONM specifically recognises and degrades unfolded, but not aggregated proteins. Since LONM is not upregulated by UPR(mt), this pathway may preferentially act to promote chaperone mediated refolding of proteins. PMID:26627475

  19. LON is the master protease that protects against protein aggregation in human mitochondria through direct degradation of misfolded proteins

    PubMed Central

    Bezawork-Geleta, Ayenachew; Brodie, Erica J.; Dougan, David A.; Truscott, Kaye N.

    2015-01-01

    Maintenance of mitochondrial protein homeostasis is critical for proper cellular function. Under normal conditions resident molecular chaperones and proteases maintain protein homeostasis within the organelle. Under conditions of stress however, misfolded proteins accumulate leading to the activation of the mitochondrial unfolded protein response (UPRmt). While molecular chaperone assisted refolding of proteins in mammalian mitochondria has been well documented, the contribution of AAA+ proteases to the maintenance of protein homeostasis in this organelle remains unclear. To address this gap in knowledge we examined the contribution of human mitochondrial matrix proteases, LONM and CLPXP, to the turnover of OTC-?, a folding incompetent mutant of ornithine transcarbamylase, known to activate UPRmt. Contrary to a model whereby CLPXP is believed to degrade misfolded proteins, we found that LONM, and not CLPXP is responsible for the turnover of OTC-? in human mitochondria. To analyse the conformational state of proteins that are recognised by LONM, we examined the turnover of unfolded and aggregated forms of malate dehydrogenase (MDH) and OTC. This analysis revealed that LONM specifically recognises and degrades unfolded, but not aggregated proteins. Since LONM is not upregulated by UPRmt, this pathway may preferentially act to promote chaperone mediated refolding of proteins. PMID:26627475

  20. RecQ-core of BLM unfolds telomeric G-quadruplex in the absence of ATP

    PubMed Central

    Budhathoki, Jagat B.; Ray, Sujay; Urban, Vaclav; Janscak, Pavel; Yodh, Jaya G.; Balci, Hamza

    2014-01-01

    Various helicases and single-stranded DNA (ssDNA) binding proteins are known to destabilize G-quadruplex (GQ) structures, which otherwise result in genomic instability. Bulk biochemical studies have shown that Bloom helicase (BLM) unfolds both intermolecular and intramolecular GQ in the presence of ATP. Using single molecule FRET, we show that binding of RecQ-core of BLM (will be referred to as BLM) to ssDNA in the vicinity of an intramolecular GQ leads to destabilization and unfolding of the GQ in the absence of ATP. We show that the efficiency of BLM-mediated GQ unfolding correlates with the binding stability of BLM to ssDNA overhang, as modulated by the nucleotide state, ionic conditions, overhang length and overhang directionality. In particular, we observed enhanced GQ unfolding by BLM in the presence of non-hydrolysable ATP analogs, which has implications for the underlying mechanism. We also show that increasing GQ stability, via shorter loops or higher ionic strength, reduces BLM-mediated GQ unfolding. Finally, we show that while WRN has similar activity as BLM, RecQ and RECQ5 helicases do not unfold GQ in the absence of ATP at physiological ionic strength. In summary, our study points to a novel and potentially very common mechanism of GQ destabilization mediated by proteins binding to the vicinity of these structures. PMID:25245947

  1. Declining global warming effects on the phenology of spring leaf unfolding

    NASA Astrophysics Data System (ADS)

    Fu, Yongshuo H.; Zhao, Hongfang; Piao, Shilong; Peaucelle, Marc; Peng, Shushi; Zhou, Guiyun; Ciais, Philippe; Huang, Mengtian; Menzel, Annette; Peñuelas, Josep; Song, Yang; Vitasse, Yann; Zeng, Zhenzhong; Janssens, Ivan A.

    2015-10-01

    Earlier spring leaf unfolding is a frequently observed response of plants to climate warming. Many deciduous tree species require chilling for dormancy release, and warming-related reductions in chilling may counteract the advance of leaf unfolding in response to warming. Empirical evidence for this, however, is limited to saplings or twigs in climate-controlled chambers. Using long-term in situ observations of leaf unfolding for seven dominant European tree species at 1,245 sites, here we show that the apparent response of leaf unfolding to climate warming (ST, expressed in days advance of leaf unfolding per °C warming) has significantly decreased from 1980 to 2013 in all monitored tree species. Averaged across all species and sites, ST decreased by 40% from 4.0 +/- 1.8 days °C-1 during 1980-1994 to 2.3 +/- 1.6 days °C-1 during 1999-2013. The declining ST was also simulated by chilling-based phenology models, albeit with a weaker decline (24-30%) than observed in situ. The reduction in ST is likely to be partly attributable to reduced chilling. Nonetheless, other mechanisms may also have a role, such as `photoperiod limitation' mechanisms that may become ultimately limiting when leaf unfolding dates occur too early in the season. Our results provide empirical evidence for a declining ST, but also suggest that the predicted strong winter warming in the future may further reduce ST and therefore result in a slowdown in the advance of tree spring phenology.

  2. Protein-responsive ribozyme switches in eukaryotic cells

    PubMed Central

    Kennedy, Andrew B.; Vowles, James V.; d'Espaux, Leo; Smolke, Christina D.

    2014-01-01

    Genetic devices that directly detect and respond to intracellular concentrations of proteins are important synthetic biology tools, supporting the design of biological systems that target, respond to or alter specific cellular states. Here, we develop ribozyme-based devices that respond to protein ligands in two eukaryotic hosts, yeast and mammalian cells, to regulate the expression of a gene of interest. Our devices allow for both gene-ON and gene-OFF response upon sensing the protein ligand. As part of our design process, we describe an in vitro characterization pipeline for prescreening device designs to identify promising candidates for in vivo testing. The in vivo gene-regulatory activities in the two types of eukaryotic cells correlate with in vitro cleavage activities determined at different physiologically relevant magnesium concentrations. Finally, localization studies with the ligand demonstrate that ribozyme switches respond to ligands present in the nucleus and/or cytoplasm, providing new insight into their mechanism of action. By extending the sensing capabilities of this important class of gene-regulatory device, our work supports the implementation of ribozyme-based devices in applications requiring the detection of protein biomarkers. PMID:25274734

  3. Response and function of skeletal muscle heat shock protein 70.

    PubMed

    Liu, Yuefei; Gampert, Larissa; Nething, Katja; Steinacker, Jürgen M

    2006-01-01

    In response to stress, cells produce a series of heat shock proteins (Hsps). One of the most prominent Hsps, is the 70 kDa Hsp (Hsp70). Hsp70 is a highly conserved and essential protein against stress. The skeletal muscle responds to a diverse group of stress signals namely, muscle contraction linked energy and milieu challenges, ischemia and exercise by producing Hsp70. The extent of this Hsp70 response in skeletal muscle depends on the type and intensity of the signal, and is characterized in a muscle fiber specific manner by a special time course. Hsp70 in the skeletal muscle is regulated at transcriptional, translational and posttranslational levels. Hsp70 serves as an indicator for cellular stress as a molecular chaperone, plays pivotal role in maintaining cellular homeostasis by preventing apoptosis, influences energy metabolism, facilitates cellular processes in terms of muscular adaptation and interacts with other signalling pathways. This review summarizes our current knowledge on the skeletal muscle Hsp70 response. PMID:16720354

  4. What are proteins? Proteins are important; e.g. for catalyzing and regulating biochemical reactions,

    E-print Network

    Gerstein, Mark

    (or lack thereof) ·Proteins unfold or denature with increasing temperature or chemical denaturants reactions, transporting molecules, ... ·Linear polymer chain composed of tens (peptides) to thousands interactions, ... ·Unfolding: increase in conformational entropy, electric charge... H (hydrophobic) P (polar

  5. Rational design of a photo-responsive UVR8-derived protein and a self-assembling peptide-protein conjugate for responsive hydrogel formation

    NASA Astrophysics Data System (ADS)

    Zhang, Xiaoli; Dong, Chunming; Huang, Weiyun; Wang, Huaimin; Wang, Ling; Ding, Dan; Zhou, Hao; Long, Jiafu; Wang, Tingliang; Yang, Zhimou

    2015-10-01

    Responsive hydrogels hold great potential in controllable drug delivery, regenerative medicine, sensing, etc. We introduced in this study the first example of a photo-responsive protein for hydrogel formation. Based on the first example of the crystal structure of a photo-responsive protein, Arabidopsis thaliana protein UVR8, we designed and expressed its derived protein UVR8-1 with a hexa-peptide WRESAI. We also prepared supramolecular nanofibers with a TIP-1 protein at their surface. The simple mixing of these two components resulted in rapid hydrogel formation through the specific interactions between the protein TIP-1 and the peptide WRESAI. Since the protein could show a reversible dimer-monomer transformation, the resulting gels also showed a reversible gel-sol phase transition which was controlled by photo-irradiation. The photo-controllable gel-sol phase transition could be applied for protein delivery and cell separation.Responsive hydrogels hold great potential in controllable drug delivery, regenerative medicine, sensing, etc. We introduced in this study the first example of a photo-responsive protein for hydrogel formation. Based on the first example of the crystal structure of a photo-responsive protein, Arabidopsis thaliana protein UVR8, we designed and expressed its derived protein UVR8-1 with a hexa-peptide WRESAI. We also prepared supramolecular nanofibers with a TIP-1 protein at their surface. The simple mixing of these two components resulted in rapid hydrogel formation through the specific interactions between the protein TIP-1 and the peptide WRESAI. Since the protein could show a reversible dimer-monomer transformation, the resulting gels also showed a reversible gel-sol phase transition which was controlled by photo-irradiation. The photo-controllable gel-sol phase transition could be applied for protein delivery and cell separation. Electronic supplementary information (ESI) available. See DOI: 10.1039/c5nr05213k

  6. Mechanical unfolding of RNA: From hairpins to structures with internal multiloops

    E-print Network

    Changbong Hyeon; D. Thirumalai

    2006-09-20

    Mechanical unfolding of RNA structures, ranging from hairpins to ribozymes, using laser optical tweezer (LOT) experiments have begun to reveal the features of the energy landscape that cannot be easily explored using conventional experiments. Upon application of constant force ($f$), RNA hairpins undergo cooperative transitions from folded to unfolded states whereas subdomains of ribozymes unravel one at a time. Here, we use a self-organized polymer (SOP) model and Brownian dynamics simulations to probe mechanical unfolding at constant force and constant-loading rate of four RNA structures of varying complexity. Our work shows (i) the response of RNA to force is largely determined by the native structure; (ii) only by probing mechanical unfolding over a wide range of forces can the underlying energy landscape be fully explored.

  7. An approach to investigate intracellular protein network responses.

    PubMed

    Currie, Holly N; Vrana, Julie A; Han, Alice A; Scardoni, Giovanni; Boggs, Nate; Boyd, Jonathan W

    2014-01-21

    Modern toxicological evaluations have evolved to consider toxicity as a perturbation of biological pathways or networks. As such, toxicity testing approaches are shifting from common end point evaluations to pathway based approaches, where the degree of perturbation of select biological pathways is monitored. These new approaches are greatly increasing the data available to toxicologists, but methods of analyses to determine the inter-relationships between potentially affected pathways are needed to fully understand the consequences of exposure. An approach to construct dose-response curves that use graph theory to describe network perturbations among three disparate mitogen-activated protein kinase (MAPK) pathways is presented. Mitochondrial stress was induced in human hepatocytes (HepG2) by exposing the cells to increasing doses of the complex I inhibitor, deguelin. The relative phosphorylation responses of proteins involved in the regulation of the stress response were measured. Graph theory was applied to the phosphorylation data to obtain parameters describing the network perturbations at each individual dose tested. The graph theory results depicted the dynamic nature of the relationship between p38, JNK, and ERK1/2 under conditions of mitochondrial stress and revealed shifts in the relationships between these MAPK pathways at low doses. The inter-relationship, or crosstalk, among these 3 traditionally linear MAPK cascades was further probed by coexposing cells to deguelin plus SB202190 (JNK and p38 inhibitor) or deguelin plus SB202474 (JNK inhibitor). The cells exposed to deguelin plus SB202474 resulted in significantly decreased viability, which could be visualized and attributed to the decrease of ERK1/2 network centrality. The approach presented here allows for the construction and visualization of dose-response curves that describe network perturbations induced by chemical stress, which provides an informative and sensitive means of assessing toxicological effects on biological systems. PMID:24359296

  8. An Approach to Investigate Intracellular Protein Network Responses

    PubMed Central

    Currie, Holly N.; Vrana, Julie A.; Han, Alice A.; Scardoni, Giovanni; Boggs, Nate; Boyd, Jonathan W.

    2014-01-01

    Modern toxicological evaluations have evolved to consider toxicity as a perturbation of biological pathways or networks. As such, toxicity testing approaches are shifting from common endpoint evaluations to pathway based approaches, where the degree of perturbation of select biological pathways is monitored. These new approaches are greatly increasing the data available to toxicologists, but methods of analyses to determine the inter-relationships between potentially affected pathways are needed to fully understand the consequences of exposure. An approach to construct dose-response curves that use graph theory to describe network perturbations amongst three disparate mitogen-activated protein kinase (MAPK) pathways is presented. Mitochondrial stress was induced in human hepatocytes (HepG2) by exposing the cells to increasing doses of the complex I inhibitor, deguelin. The relative phosphorylation responses of proteins involved in the regulation of the stress response were measured. Graph theory was applied to the phosphorylation data to obtain parameters describing the network perturbations at each individual dose tested. The graph theory results depicted the dynamic nature of the relationship between p38, JNK, and ERK1/2 under conditions of mitochondrial stress, and revealed shifts in the relationships between these MAPK pathways at low doses. The inter-relationship, or crosstalk, amongst these 3 traditionally linear MAPK cascades was further probed by co-exposing cells to deguelin plus SB202190 (JNK and p38 inhibitor) or deguelin plus SB202474 (JNK inhibitor). The cells exposed to deguelin plus SB202474 resulted in significantly decreased viability, which could be visualized and attributed to the decrease of ERK1/2 network centrality. The approach presented here allows for the construction and visualization of dose-response curves that describe network perturbations induced by chemical stress, which provides an informative and sensitive means of assessing toxicological effects on biological systems. PMID:24359296

  9. Unfolding large-scale online collaborative human dynamics

    E-print Network

    Zha, Yilong; Zhou, Changsong

    2015-01-01

    Large-scale interacting human activities underlie all social and economic phenomena, but quantitative understanding of regular patterns and mechanism is very challenging and still rare. Self-organized online collaborative activities with precise record of event timing provide unprecedented opportunity. Our empirical analysis of the history of millions of updates in Wikipedia shows a universal double power-law distribution of time intervals between consecutive updates of an article. We then propose a generic model to unfold collaborative human activities into three modules: (i) individual behavior characterized by Poissonian initiation of an action, (ii) human interaction captured by a cascading response to others with a power-law waiting time, and (iii) population growth due to increasing number of interacting individuals. This unfolding allows us to obtain analytical formula that is fully supported by the universal patterns in empirical data. Our modeling approaches reveal "simplicity" beyond complex interac...

  10. Protein-protein interactions between histidine kinases and response regulators of Mycobacterium tuberculosis H37Rv.

    PubMed

    Lee, Ha-Na; Jung, Kwang-Eun; Ko, In-Jeong; Baik, Hyung Suk; Oh, Jeong-Il

    2012-04-01

    Using yeast two-hybrid assay, we investigated protein-protein interactions between all orthologous histidine kinase (HK)/response regulator (RR) pairs of M. tuberculosis H37Rv and identified potential protein-protein interactions between a noncognate HK/RR pair, DosT/NarL. The protein interaction between DosT and NarL was verified by phosphotransfer reaction from DosT to NarL. Furthermore, we found that the DosT and DosS HKs, which share considerable sequence similarities to each other and form a two-component system with the DosR RR, have different cross-interaction capabilities with NarL: DosT interacted with NarL, while DosS did not. The dimerization domains of DosT and DosS were shown to be sufficient to confer specificity for DosR, and the different cross-interaction abilities of DosS and DosT with NarL were demonstrated to be attributable to variations in the amino acid sequences of the ?2-helices of their dimerization domains. PMID:22538656

  11. Conformational Properties of the Unfolded State of Im7 in Nondenaturing Conditions

    PubMed Central

    Pashley, Clare L.; Morgan, Gareth J.; Kalverda, Arnout P.; Thompson, Gary S.; Kleanthous, Colin; Radford, Sheena E.

    2012-01-01

    The unfolded ensemble in aqueous solution represents the starting point of protein folding. Characterisation of this species is often difficult since the native state is usually predominantly populated at equilibrium. Previous work has shown that the four-helix protein, Im7 (immunity protein 7), folds via an on-pathway intermediate. While the transition states and folding intermediate have been characterised in atomistic detail, knowledge of the unfolded ensemble under the same ambient conditions remained sparse. Here, we introduce destabilising amino acid substitutions into the sequence of Im7, such that the unfolded state becomes predominantly populated at equilibrium in the absence of denaturant. Using far- and near-UV CD, fluorescence, urea titration and heteronuclear NMR experiments, we show that three amino acid substitutions (L18A–L19A–L37A) are sufficient to prevent Im7 folding, such that the unfolded state is predominantly populated at equilibrium. Using measurement of chemical shifts, 15N transverse relaxation rates and sedimentation coefficients, we show that the unfolded species of L18A–L19A–L37A deviates significantly from random-coil behaviour. Specifically, we demonstrate that this unfolded species is compact (Rh = 25 Å) relative to the urea-denatured state (Rh ? 30 Å) and contains local clusters of hydrophobic residues in regions that correspond to the four helices in the native state. Despite these interactions, there is no evidence for long-range stabilising tertiary interactions or persistent helical structure. The results reveal an unfolded ensemble that is conformationally restricted in regions of the polypeptide chain that ultimately form helices I, II and IV in the native state. PMID:22226836

  12. Biological responses to spider silk-antibiotic fusion protein

    PubMed Central

    Gomes, Sílvia; Gallego-Llamas, Jabier; Leonor, Isabel B.; Mano, João F.; Reis, Rui L.; Kaplan, David L.

    2011-01-01

    The development of a new generation of multifunctional biomaterials is a continual goal for the field of materials science. The in vivo functional behaviour of a new fusion protein that combines the mechanical properties of spider silk with the antimicrobial properties of hepcidin was addressed in this study. This new chimeric protein, termed 6mer+hepcidin, fuses spider dragline consensus sequences (6mer) and the antimicrobial peptide hepcidin as we have recently described, with retention of bactericidal activity and low cytotoxicity. In the present study mice subcutaneous implants were studied to access the in vivo biological response to the 6mer+hepcidin, which were compared with controls of silk alone (6mer), poly-lactic-glycolic-acid (PLGA) films and empty defects. Along with visual observations, flow cytometry and histology analyses were used to determine the number and type of inflammatory cells at the implantation site. The results show a mild to low inflammatory reaction to the implanted materials and no apparent differences between the 6mer+hepcidin films and the other experimental controls, demonstrating that the new fusion protein has good in vivo biocompatibility, while maintaining antibiotic function. PMID:22514077

  13. Stress Responses from the Endoplasmic Reticulum in Cancer

    PubMed Central

    Kato, Hironori; Nishitoh, Hideki

    2015-01-01

    The endoplasmic reticulum (ER) is a dynamic organelle that is essential for multiple cellular functions. During cellular stress conditions, including nutrient deprivation and dysregulation of protein synthesis, unfolded/misfolded proteins accumulate in the ER lumen, resulting in activation of the unfolded protein response (UPR). The UPR also contributes to the regulation of various intracellular signaling pathways such as calcium signaling and lipid signaling. More recently, the mitochondria-associated ER membrane (MAM), which is a site of close contact between the ER and mitochondria, has been shown to function as a platform for various intracellular stress responses including apoptotic signaling, inflammatory signaling, the autophagic response, and the UPR. Interestingly, in cancer, these signaling pathways from the ER are often dysregulated, contributing to cancer cell metabolism. Thus, the signaling pathway from the ER may be a novel therapeutic target for various cancers. In this review, we discuss recent research on the roles of stress responses from the ER, including the MAM. PMID:25941664

  14. Integrated biophysical studies implicate partial unfolding of NBD1 of CFTR in the molecular pathogenesis of F508del cystic fibrosis

    PubMed Central

    Wang, Chi; Protasevich, Irina; Yang, Zhengrong; Seehausen, Derek; Skalak, Timothy; Zhao, Xun; Atwell, Shane; Spencer Emtage, J; Wetmore, Diana R; Brouillette, Christie G; Hunt, John F

    2010-01-01

    The lethal genetic disease cystic fibrosis is caused predominantly by in-frame deletion of phenylalanine 508 in the cystic fibrosis transmembrane conductance regulator (CFTR). F508 is located in the first nucleotide-binding domain (NBD1) of CFTR, which functions as an ATP-gated chloride channel on the cell surface. The F508del mutation blocks CFTR export to the surface due to aberrant retention in the endoplasmic reticulum. While it was assumed that F508del interferes with NBD1 folding, biophysical studies of purified NBD1 have given conflicting results concerning the mutation's influence on domain folding and stability. We have conducted isothermal (this paper) and thermal (accompanying paper) denaturation studies of human NBD1 using a variety of biophysical techniques, including simultaneous circular dichroism, intrinsic fluorescence, and static light-scattering measurements. These studies show that, in the absence of ATP, NBD1 unfolds via two sequential conformational transitions. The first, which is strongly influenced by F508del, involves partial unfolding and leads to aggregation accompanied by an increase in tryptophan fluorescence. The second, which is not significantly influenced by F508del, involves full unfolding of NBD1. Mg-ATP binding delays the first transition, thereby offsetting the effect of F508del on domain stability. Evidence suggests that the initial partial unfolding transition is partially responsible for the poor in vitro solubility of human NBD1. Second-site mutations that increase the solubility of isolated F508del-NBD1 in vitro and suppress the trafficking defect of intact F508del-CFTR in vivo also stabilize the protein against this transition, supporting the hypothesize that it is responsible for the pathological trafficking of F508del-CFTR. PMID:20687163

  15. Consistent Picture of the Reversible Thermal Unfolding of Hen Egg-White Lysozyme from Experiment and Molecular Dynamics

    PubMed Central

    Meersman, Filip; Atilgan, Canan; Miles, Andrew J.; Bader, Reto; Shang, Weifeng; Matagne, André; Wallace, B.A.; Koch, Michel H.J.

    2010-01-01

    Synchrotron radiation circular dichroism, Fourier transform infrared, and nuclear magnetic resonance spectroscopies, and small-angle x-ray scattering were used to monitor the reversible thermal unfolding of hen egg white lysozyme. The results were compared with crystal structures and high- and low-temperature structures derived from molecular-dynamics calculations. The results of both experimental and computational methods indicate that the unfolding process starts with the loss of ?-structures followed by the reversible loss of helix content from ?40% at 20°C to 27% at 70°C and ?20% at 77°C, beyond which unfolding becomes irreversible. Concomitantly there is a reversible increase in the radius of gyration of the protein from 15 Å to 18 Å. The reversible decrease in forward x-ray scattering demonstrates a lack of aggregation upon unfolding, suggesting the change is due to a larger dilation of hydration water than of bulk water. Molecular-dynamics simulations suggest a similar sequence of events and are in good agreement with the 1HN chemical shift differences in nuclear magnetic resonance. This study demonstrates the power of complementary methods for elucidating unfolding/refolding processes and the nature of both the unfolded structure, for which there is no crystallographic data, and the partially unfolded forms of the protein that can lead to fibril formation and disease. PMID:20923660

  16. Thermo-responsive adsorbent for size-selective protein adsorption.

    PubMed

    Lee, Micky Fu Xiang; Chan, Eng Seng; Tam, Kam Chiu; Tey, Beng Ti

    2015-05-15

    A thermo-responsive random copolymer, POEGMA (poly(oligoethylene glycol) methacrylate) grafted on cationized agarose adsorbent was used for size selective protein adsorption. The effects of OEGMA300 ((oligoethylene glycol) methyl ether methacrylate, Mn=300g/mol) content and temperature on the adsorption of bovine serum albumin (BSA) were evaluated. Increasing the content of OEGMA300 resulted a reduction in BSA adsorption due to the enhanced shielding effect of OEGMA300 chains. Grafting of POEGMA chains onto cationized agarose adsorbent reduced the BSA adsorption by more than 95% at 26.5°C, which is below the LCST (lower critical solution temperature) of POEGMA. The BSA adsorption capacities for adsorbents grafted with 10 and 20mol% of OEGMA300 decreased by 48% and 46% respectively at 38°C, a temperature higher than their LCSTs. The temperature-dependent adsorption of BSA on the adsorbents was attributed to changes in the polymer conformation. The thermal transition of grafted POEGMA conformation exposed the ligand when the temperature was increased. Myoglobin (Myo), which was smaller than BSA, its adsorption behavior was less dependent on the polymer conformation. The adsorption of myoglobin onto the adsorbent with and without POEGMA showed similar percentage of reduction whereas the adsorption of BSA onto the adsorbent with POEGMA decreased by 7.6 times compared to the one without POEGMA. The packed bed of POEGMA grafted adsorbent was used for flow through separation of a protein mixture consisted of virus-like particle, Hepatitis B virus-like particle (HBVLP), BSA and insulin aspart. The recovery of HBVLP in 20mol% of OEGMA300 grafted adsorbent was increased by 19% compared to ungrafted adsorbent. The flow through of BSA can be reduced by increasing the operating temperature above LCST of 20mol% of OEGMA300 while the smaller protein, insulin aspart, remained adsorbed onto the cationized surface. Hence, this thermo-responsive adsorbent has a potential for size-selective separation of protein especially for the recovery of large biomolecule. PMID:25836051

  17. Surface-enhanced unfolding of collapsed polymers in shear flow

    NASA Astrophysics Data System (ADS)

    Alexander-Katz, A.; Netz, R. R.

    2007-10-01

    Using hydrodynamic simulations we study the shear-induced unfolding of a collapsed polymer near a planar wall. Above a well-defined threshold shear rate \\dot{\\gamma}^* , the globule becomes unstable and displays stretching-refolding events. With decreasing distance from the surface, the critical shear rate \\dot{\\gamma}^* goes down, which is rationalized within a scaling analysis in terms of increased hydrodynamic stress due to a surface-induced slowing-down of globule rotation and translation. Our results are relevant for protein-assisted blood clotting in capillary vessels.

  18. AMP-activated protein kinase, stress responses and cardiovascular diseases

    PubMed Central

    WANG, Shaobin; SONG, Ping; ZOU, Ming-Hui

    2012-01-01

    AMPK (AMP-activated protein kinase) is one of the key players in maintaining intracellular homoeostasis. AMPK is well known as an energy sensor and can be activated by increased intracellular AMP levels. Generally, the activation of AMPK turns on catabolic pathways that generate ATP, while inhibiting cell proliferation and biosynthetic processes that consume ATP. In recent years, intensive investigations on the regulation and the function of AMPK indicates that AMPK not only functions as an intracellular energy sensor and regulator, but is also a general stress sensor that is important in maintaining intracellular homoeostasis during many kinds of stress challenges. In the present paper, we will review recent literature showing that AMPK functions far beyond its proposed energy sensor and regulator function. AMPK regulates ROS (reactive oxygen species)/redox balance, autophagy, cell proliferation, cell apoptosis, cellular polarity, mitochondrial function and genotoxic response, either directly or indirectly via numerous downstream pathways under physiological and pathological conditions. PMID:22390198

  19. 14-3-3 Proteins, FHA Domains and BRCT Domains in the DNA Damage Response

    E-print Network

    Mohammad, Duaa H.

    The DNA damage response depends on the concerted activity of protein serine/threonine kinases and modular phosphoserine/threonine-binding domains to relay the damage signal and recruit repair proteins. The PIKK family of ...

  20. Detection of Innate Immune Response Modulating Impurities in Therapeutic Proteins

    PubMed Central

    Haile, Lydia Asrat; Puig, Montserrat; Kelley-Baker, Logan; Verthelyi, Daniela

    2015-01-01

    Therapeutic proteins can contain multiple impurities, some of which are variants of the product, while others are derived from the cell substrate and the manufacturing process. Such impurities, even when present at trace levels, have the potential to activate innate immune cells in peripheral blood or embedded in tissues causing expression of cytokines and chemokines, increasing antigen uptake, facilitating processing and presentation by antigen presenting cells, and fostering product immunogenicity. Currently, while products are tested for host cell protein content, assays to control innate immune response modulating impurities (IIRMIs) in products are focused mainly on endotoxin and nucleic acids, however, depending on the cell substrate and the manufacturing process, numerous other IIRMI could be present. In these studies we assess two approaches that allow for the detection of a broader subset of IIRMIs. In the first, we use commercial cell lines transfected with Toll like receptors (TLR) to detect receptor-specific agonists. This method is sensitive to trace levels of IIRMI and provides information of the type of IIRMIs present but is limited by the availability of stably transfected cell lines and requires pre-existing knowledge of the IIRMIs likely to be present in the product. Alternatively, the use of a combination of macrophage cell lines of human and mouse origin allows for the detection of a broader spectrum of impurities, but does not identify the source of the activation. Importantly, for either system the lower limit of detection (LLOD) of impurities was similar to that of PBMC and it was not modified by the therapeutic protein tested, even in settings where the product had inherent immune modulatory properties. Together these data indicate that a cell-based assay approach could be used to screen products for the presence of IIRMIs and inform immunogenicity risk assessments, particularly in the context of comparability exercises. PMID:25901912

  1. Ligand-Induced Protein Responses and Mechanical Signal Propagation Described by Linear Response Theories

    PubMed Central

    Yang, Lee-Wei; Kitao, Akio; Huang, Bang-Chieh; G?, Nobuhiro

    2014-01-01

    In this study, a general linear response theory (LRT) is formulated to describe time-dependent and -independent protein conformational changes upon CO binding with myoglobin. Using the theory, we are able to monitor protein relaxation in two stages. The slower relaxation is found to occur from 4.4 to 81.2 picoseconds and the time constants characterized for a couple of aromatic residues agree with those observed by UV Resonance Raman (UVRR) spectrometry and time resolved x-ray crystallography. The faster “early responses”, triggered as early as 400 femtoseconds, can be best described by the theory when impulse forces are used. The newly formulated theory describes the mechanical propagation following ligand-binding as a function of time, space and types of the perturbation forces. The “disseminators”, defined as the residues that propagate signals throughout the molecule the fastest among all the residues in protein when perturbed, are found evolutionarily conserved and the mutations of which have been shown to largely change the CO rebinding kinetics in myoglobin. PMID:25229149

  2. Muscle protein synthesis in response to nutrition and exercise.

    PubMed

    Atherton, P J; Smith, K

    2012-03-01

    Muscle protein synthesis (MPS) is the driving force behind adaptive responses to exercise and represents a widely adopted proxy for gauging chronic efficacy of acute interventions, (i.e. exercise/nutrition). Recent findings in this arena have been progressive. Nutrient-driven increases in MPS are of finite duration (?1.5 h), switching off thereafter despite sustained amino acid availability and intramuscular anabolic signalling. Intriguingly, this 'muscle-full set-point' is delayed by resistance exercise (RE) (i.e. the feeding × exercise combination is 'more anabolic' than nutrition alone) even 24 h beyond a single exercise bout, casting doubt on the importance of nutrient timing vs. sufficiency per se. Studies manipulating exercise intensity/workload have shown that increases in MPS are negligible with RE at 20-40% but maximal at 70-90% of one-repetition maximum when workload is matched (according to load × repetition number). However, low-intensity exercise performed to failure equalises this response. Analysing distinct subcellular fractions (e.g. myofibrillar, sarcoplasmic, mitochondrial) may provide a readout of chronic exercise efficacy in addition to effect size in MPS per se, i.e. while 'mixed' MPS increases similarly with endurance and RE, increases in myofibrillar MPS are specific to RE, prophetic of adaptation (i.e. hypertrophy). Finally, the molecular regulation of MPS by exercise and its regulation via 'anabolic' hormones (e.g. IGF-1) has been questioned, leading to discovery of alternative mechanosensing-signalling to MPS. PMID:22289911

  3. Fibronectin unfolded by adherent but not suspended platelets: An in vitro explanation for its dual role in haemostasis.

    PubMed

    Huynh, Khon; Gyenes, Marianna; Hollenberg, Cornelis P; Nguyen, Thi-Hiep; Van Vo, Toi; Stoldt, Volker R

    2015-10-01

    Fibronectin (FN), a dimeric adhesive glycoprotein, which is present both in plasma and the extracellular matrix can interact with platelets and thus contribute to platelet adhesion and aggregation. It has been shown that FN can decrease platelet aggregation but enhance platelet adhesion, suggesting a dual role of FN in haemostasis. The prevalent function(s) of FN may be determined by its fibril form. To explore the suggested dual role of this adhesive protein for haemostasis in further detail, we now tested for any differences of adherent and suspended platelets with regard to their effect to unfold and assemble FN upon interaction. Platelet aggregation and adhesion assays were performed using washed platelets in the presence of exogenous FN. Addition of plasma FN reduced platelet aggregation in response to collagen or PMA by 50% or 25% but enhanced platelet adhesion onto immobilized collagen, as compared to control experiments. Analyses by fluorescence resonance energy transfer (FRET) demonstrated that adherent platelets but not suspended platelets were capable of unfolding FN during 3h incubation. Fluorescence microscopy and deoxycholate (DOC) solubility assays demonstrated that FN fibrils formed only on the surfaces of adherent platelets. In addition, platelets adherent onto FN revealed a significantly higher activity of specific Src phosphorylation (pY418) than platelets in suspension. These data suggest (1) that the function of FN in haemostasis is prevalent to its assembly, unfolding and subsequent fibril formation on the surface of adherent platelets and (2) that outside-in signaling contributes to the interaction of platelets and FN. PMID:26277681

  4. Unfolding and Refolding of Aspergillus Niger PhyB Phytase: Role of Disulfide Bridges

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Role of disulfide bridges in folding of Aspergillus niger phytase pH 2.5-optimum (PhyB) was investigated using dynamic light scattering (DLS). Guanidinium chloride (GuCl) at 1.0 M unfolded phytase; however, its removal by dialysis refolded the protein. Thiol reagent, tris (2-carboxyethyl) phosphin...

  5. Quenching of Tryptophan Fluorescence in Unfolded Cytochrome "c": A Biophysics Experiment for Physical Chemistry Students

    ERIC Educational Resources Information Center

    Schlamadinger, Diana E.; Kats, Dina I.; Kim, Judy E.

    2010-01-01

    Laboratory experiments that focus on protein folding provide excellent opportunities for undergraduate students to learn important topics in the expanding interdisciplinary field of biophysics. Here, we describe the use of Stern-Volmer plots to determine the extent of solvent accessibility of the single tryptophan residue (trp-59) in unfolded and…

  6. Structureenergy relations in hen egg white lysozyme observed during refolding from a quenched unfolded statew

    E-print Network

    Structure­energy relations in hen egg white lysozyme observed during refolding from a quenched by rapidly quenching thermally unfolded hen egg white lysozyme in a glassy matrix, followed by reheating possible tech- nique to quench fast-folding proteins.9 Hen egg white lysozyme was chosen as the model

  7. RESEARCH ARTICLE Pharmaceutical Biotechnology Role of Benzyl Alcohol in the Unfolding and Aggregation of

    E-print Network

    Mallela, Krishna M. G.

    RESEARCH ARTICLE ­ Pharmaceutical Biotechnology Role of Benzyl Alcohol in the Unfolding (wileyonlinelibrary.com). DOI 10.1002/jps.24105 ABSTRACT: Benzyl alcohol (BA) is the most widely used antimicrobial preservative; BA, benzyl alcohol; Cm, midpoint denaturant concentration in protein denaturant melt; Cyt c

  8. Unfolding collapsed polyelectrolytes in alternating-current electric fields Pai-Yi Hsiao,*a

    E-print Network

    Chang, Hsueh-Chia

    f We investigate the unfolding of single polyelectrolyte (PE) chains collapsed by trivalent salt fluctuation time, suggesting that the dissociation of condensed polyvalent counterion on the collapsed PE. Introduction To develop new ability to separate and stretch biomolecules, such as nucleic acids and proteins

  9. User's manual for the FERDO and FERD unfolding codes

    SciTech Connect

    Rust, B.W.; Ingersoll, D.T.; Burrus, W.R.

    1983-09-01

    FERDO and FERD are unfolding codes which can be used to correct observed pulse-height distributions for the nonideal response of a pulse-height spectrometer or to solve poorly conditioned linear equations. It is assumed that the response of the spectrometer is given by Ax = b, where A is the spectrometer response function matrix, x is the unknown spectrum, and b is the pulse-height distribution. FERDO does not solve directly for x, but instead solves for p = Wx, where W is a window function matrix. Typically, W is the resolution function of an ideal spectrometer which has a single Gaussian response. The effective resolution of the unfolding solution may be varied by choice of W. Confidence intervals (p/sup 10/, p/sup up/) are found for each element of the solution p. FERDO and FERD are written in IBM 360/370 Fortran (Level H). This manual describes and derives the mathematical procedures used by the codes, tells how to write input data for them, and gives solutions to some sample problems to illustrate the output.

  10. A neutron spectrum unfolding computer code based on artificial neural networks

    NASA Astrophysics Data System (ADS)

    Ortiz-Rodríguez, J. M.; Reyes Alfaro, A.; Reyes Haro, A.; Cervantes Viramontes, J. M.; Vega-Carrillo, H. R.

    2014-02-01

    The Bonner Spheres Spectrometer consists of a thermal neutron sensor placed at the center of a number of moderating polyethylene spheres of different diameters. From the measured readings, information can be derived about the spectrum of the neutron field where measurements were made. Disadvantages of the Bonner system are the weight associated with each sphere and the need to sequentially irradiate the spheres, requiring long exposure periods. Provided a well-established response matrix and adequate irradiation conditions, the most delicate part of neutron spectrometry, is the unfolding process. The derivation of the spectral information is not simple because the unknown is not given directly as a result of the measurements. The drawbacks associated with traditional unfolding procedures have motivated the need of complementary approaches. Novel methods based on Artificial Intelligence, mainly Artificial Neural Networks, have been widely investigated. In this work, a neutron spectrum unfolding code based on neural nets technology is presented. This code is called Neutron Spectrometry and Dosimetry with Artificial Neural networks unfolding code that was designed in a graphical interface. The core of the code is an embedded neural network architecture previously optimized using the robust design of artificial neural networks methodology. The main features of the code are: easy to use, friendly and intuitive to the user. This code was designed for a Bonner Sphere System based on a 6LiI(Eu) neutron detector and a response matrix expressed in 60 energy bins taken from an International Atomic Energy Agency compilation. The main feature of the code is that as entrance data, for unfolding the neutron spectrum, only seven rate counts measured with seven Bonner spheres are required; simultaneously the code calculates 15 dosimetric quantities as well as the total flux for radiation protection purposes. This code generates a full report with all information of the unfolding in the HTML format. NSDann unfolding code is freely available, upon request to the authors.

  11. OsHrd3 is necessary for maintaining the quality of endoplasmic reticulum-derived protein bodies in rice endosperm.

    PubMed

    Ohta, Masaru; Takaiwa, Fumio

    2015-08-01

    Large amounts of seed storage proteins (SSPs) are produced in the maturing endosperm of rice seeds. Rice SSPs are synthesized as secretory proteins on the rough endoplasmic reticulum (ER), and are transported and deposited into protein complexes called protein bodies (PB-I and PB-II). Due to the high production of SSPs, unfolded SSPs may be generated during this process. However, it was previously unclear how such unfolded proteins are selected among synthesized products and removed from the ER to maintain protein quality in the endosperm. Since Hrd3/SEL1L recognizes unfolded proteins in yeast and mammalian protein quality control systems, the role of OsHrd3 in protein quality control in rice endosperm was investigated. Co-immunoprecipitation experiments demonstrated that OsHrd3 interacts with components of the Hrd1 ubiquitin ligase complex such as OsOS-9 and OsHrd1 in rice protoplasts. Endosperm-specific suppression of OsHrd3 in transgenic rice reduced the levels of polyubiquitinated proteins and resulted in unfolded protein responses (UPRs) in the endosperm, suggesting that OsHrd3-mediated polyubiquitination plays an important role in ER quality control. It was found that a cysteine-rich 13kDa prolamin, RM1, was polyubiquitinated in wild-type (WT) seeds but not in OsHrd3 knockdown (KD) seeds. RM1 formed aberrant aggregates that were deposited abnormally in OsHrd3 KD seeds, resulting in deformed PB-I. Therefore, the quality of protein bodies is maintained by polyubiquitination of unfolded SSPs through the Hrd1 ubiquitin ligase system in rice endosperm. PMID:25977235

  12. OsHrd3 is necessary for maintaining the quality of endoplasmic reticulum-derived protein bodies in rice endosperm

    PubMed Central

    Ohta, Masaru; Takaiwa, Fumio

    2015-01-01

    Large amounts of seed storage proteins (SSPs) are produced in the maturing endosperm of rice seeds. Rice SSPs are synthesized as secretory proteins on the rough endoplasmic reticulum (ER), and are transported and deposited into protein complexes called protein bodies (PB-I and PB-II). Due to the high production of SSPs, unfolded SSPs may be generated during this process. However, it was previously unclear how such unfolded proteins are selected among synthesized products and removed from the ER to maintain protein quality in the endosperm. Since Hrd3/SEL1L recognizes unfolded proteins in yeast and mammalian protein quality control systems, the role of OsHrd3 in protein quality control in rice endosperm was investigated. Co-immunoprecipitation experiments demonstrated that OsHrd3 interacts with components of the Hrd1 ubiquitin ligase complex such as OsOS-9 and OsHrd1 in rice protoplasts. Endosperm-specific suppression of OsHrd3 in transgenic rice reduced the levels of polyubiquitinated proteins and resulted in unfolded protein responses (UPRs) in the endosperm, suggesting that OsHrd3-mediated polyubiquitination plays an important role in ER quality control. It was found that a cysteine-rich 13kDa prolamin, RM1, was polyubiquitinated in wild-type (WT) seeds but not in OsHrd3 knockdown (KD) seeds. RM1 formed aberrant aggregates that were deposited abnormally in OsHrd3 KD seeds, resulting in deformed PB-I. Therefore, the quality of protein bodies is maintained by polyubiquitination of unfolded SSPs through the Hrd1 ubiquitin ligase system in rice endosperm. PMID:25977235

  13. Increased dietary protein attenuates C-reactive protein and creatine kinase responses to exercise-induced energy deficit

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We determined if dietary protein (P) modulates responses of C-reactive protein (CRP) and creatine kinase (CK), biomarkers of inflammation and muscle damage, during exercise-induced energy deficit (DEF). Thirteen healthy men (22 +/- 1 y, VO2peak 60 +/- 2 ml.kg-1.min-1) balanced energy expenditure (EE...

  14. Calcineurin inhibitors recruit protein kinases JAK2 and JNK, TLR signaling and the UPR to activate NF-?B-mediated inflammatory responses in kidney tubular cells

    SciTech Connect

    González-Guerrero, Cristian; Ocaña-Salceda, Carlos; Berzal, Sergio; Carrasco, Susana; Fernández-Fernández, Beatriz; and others

    2013-11-01

    The calcineurin inhibitors (CNIs) cyclosporine (CsA) and tacrolimus are key drugs in current immunosuppressive regimes for solid organ transplantation. However, they are nephrotoxic and promote death and profibrotic responses in tubular cells. Moreover, renal inflammation is observed in CNI nephrotoxicity but the mechanisms are poorly understood. We have now studied molecular pathways leading to inflammation elicited by the CNIs in cultured and kidney tubular cells. Both CsA and tacrolimus elicited a proinflammatory response in tubular cells as evidenced by a transcriptomics approach. Transcriptomics also suggested several potential pathways leading to expression of proinflammatory genes. Validation and functional studies disclosed that in tubular cells, CNIs activated protein kinases such as the JAK2/STAT3 and TAK1/JNK/AP-1 pathways, TLR4/Myd88/IRAK signaling and the Unfolded Protein Response (UPR) to promote NF-?B activation and proinflammatory gene expression. CNIs also activated an Nrf2/HO-1-dependent compensatory response and the Nrf2 activator sulforaphane inhibited JAK2 and JNK activation and inflammation. A murine model of CsA nephrotoxicity corroborated activation of the proinflammatory pathways identified in cell cultures. Human CNIs nephrotoxicity was also associated with NF-?B, STAT3 and IRE1? activation. In conclusion, CNIs recruit several intracellular pathways leading to previously non-described proinflammatory actions in renal tubular cells. Identification of these pathways provides novel clues for therapeutic intervention to limit CNIs nephrotoxicity. - Highlights: • Molecular mechanisms modulating CNI renal inflammation were investigated. • Kinases, immune receptors and ER stress mediate the inflammatory response to CNIs. • Several intracellular pathways activate NF-?B in CNIs-treated tubular cells. • A NF-?B-dependent cytokine profile characterizes CNIs-induced inflammation. • CNI nephrotoxicity was associated to inflammatory events in mice and human.

  15. Development, Characterization, and Optimization of Protein Level in Date Bars Using Response Surface Methodology

    PubMed Central

    Nadeem, Muhammad; Salim-ur-Rehman; Muhammad Anjum, Faqir; Murtaza, Mian Anjum; Mueen-ud-Din, Ghulam

    2012-01-01

    This project was designed to produce a nourishing date bar with commercial value especially for school going children to meet their body development requirements. Protein level of date bars was optimized using response surface methodology (RSM). Economical and underutilized sources, that is, whey protein concentrate and vetch protein isolates, were explored for protein supplementation. Fourteen date bar treatments were produced using a central composite design (CCD) with 2 variables and 3 levels for each variable. Date bars were then analyzed for nutritional profile. Proximate composition revealed that addition of whey protein concentrate and vetch protein isolates improved the nutritional profile of date bars. Protein level, texture, and taste were considerably improved by incorporating 6.05% whey protein concentrate and 4.35% vetch protein isolates in date bar without affecting any sensory characteristics during storage. Response surface methodology was observed as an economical and effective tool to optimize the ingredient level and to discriminate the interactive effects of independent variables. PMID:22792044

  16. Proteomic analysis of rice plasma membrane reveals proteins involved in early defense response to bacterial blight.

    PubMed

    Chen, Fang; Yuan, Yuexing; Li, Qun; He, Zuhua

    2007-05-01

    Plant plasma membrane (PM) proteins play important roles in signal transduction during defense response to an attacking pathogen. By using an improved method of PM protein preparation and PM-bound green fluorescent protein fusion protein as a visible marker, we conducted PM proteomic analysis of the rice suspension cells expressing the disease resistance gene Xa21, to identify PM components involved in the early defense response to bacterial blight (Xanthomonas oryzae pv. oryzae). A total of 20 regulated protein spots were observed on 2-D gels of PM fractions at 12 and 24 h after pathogen inoculation, of which some were differentially regulated between the incompatible and compatible interactions mediated by Xa21, with good correlation between biological repeats. Eleven protein spots with predicted functions in plant defense were identified by MS/MS, including nine putative PM-associated proteins H+-ATPase, protein phosphatase, hypersensitive-induced response protein (OsHIR1), prohibitin (OsPHB2), zinc finger and C2 domain protein, universal stress protein (USP), and heat shock protein. OsHIR1 was modified by the microbial challenge, leading to two differentially accumulated protein spots. Transcript analysis showed that most of the genes were also regulated at transcriptional levels. Our study would provide a starting point for functionality of PM proteins in the rice defense. PMID:17407182

  17. Insight into bacterial virulence mechanisms against host immune response via the Yersinia pestis-human protein-protein interaction network.

    PubMed

    Yang, Huiying; Ke, Yuehua; Wang, Jian; Tan, Yafang; Myeni, Sebenzile K; Li, Dong; Shi, Qinghai; Yan, Yanfeng; Chen, Hui; Guo, Zhaobiao; Yuan, Yanzhi; Yang, Xiaoming; Yang, Ruifu; Du, Zongmin

    2011-11-01

    A Yersinia pestis-human protein interaction network is reported here to improve our understanding of its pathogenesis. Up to 204 interactions between 66 Y. pestis bait proteins and 109 human proteins were identified by yeast two-hybrid assay and then combined with 23 previously published interactions to construct a protein-protein interaction network. Topological analysis of the interaction network revealed that human proteins targeted by Y. pestis were significantly enriched in the proteins that are central in the human protein-protein interaction network. Analysis of this network showed that signaling pathways important for host immune responses were preferentially targeted by Y. pestis, including the pathways involved in focal adhesion, regulation of cytoskeleton, leukocyte transendoepithelial migration, and Toll-like receptor (TLR) and mitogen-activated protein kinase (MAPK) signaling. Cellular pathways targeted by Y. pestis are highly relevant to its pathogenesis. Interactions with host proteins involved in focal adhesion and cytoskeketon regulation pathways could account for resistance of Y. pestis to phagocytosis. Interference with TLR and MAPK signaling pathways by Y. pestis reflects common characteristics of pathogen-host interaction that bacterial pathogens have evolved to evade host innate immune response by interacting with proteins in those signaling pathways. Interestingly, a large portion of human proteins interacting with Y. pestis (16/109) also interacted with viral proteins (Epstein-Barr virus [EBV] and hepatitis C virus [HCV]), suggesting that viral and bacterial pathogens attack common cellular functions to facilitate infections. In addition, we identified vasodilator-stimulated phosphoprotein (VASP) as a novel interaction partner of YpkA and showed that YpkA could inhibit in vitro actin assembly mediated by VASP. PMID:21911467

  18. The non-uniform early structural response of globular proteins to cold denaturing conditions: A case study with Yfh1

    NASA Astrophysics Data System (ADS)

    Chatterjee, Prathit; Bagchi, Sayan; Sengupta, Neelanjana

    2014-11-01

    The mechanism of cold denaturation in proteins is often incompletely understood due to limitations in accessing the denatured states at extremely low temperatures. Using atomistic molecular dynamics simulations, we have compared early (nanosecond timescale) structural and solvation properties of yeast frataxin (Yfh1) at its temperature of maximum stability, 292 K (Ts), and the experimentally observed temperature of complete unfolding, 268 K (Tc). Within the simulated timescales, discernible "global" level structural loss at Tc is correlated with a distinct increase in surface hydration. However, the hydration and the unfolding events do not occur uniformly over the entire protein surface, but are sensitive to local structural propensity and hydrophobicity. Calculated infrared absorption spectra in the amide-I region of the whole protein show a distinct red shift at Tc in comparison to Ts. Domain specific calculations of IR spectra indicate that the red shift primarily arises from the beta strands. This is commensurate with a marked increase in solvent accessible surface area per residue for the beta-sheets at Tc. Detailed analyses of structure and dynamics of hydration water around the hydrophobic residues of the beta-sheets show a more bulk water like behavior at Tc due to preferential disruption of the hydrophobic effects around these domains. Our results indicate that in this protein, the surface exposed beta-sheet domains are more susceptible to cold denaturing conditions, in qualitative agreement with solution NMR experimental results.

  19. Regulating the ethylene response of a plant by modulation of F-box proteins

    DOEpatents

    Guo, Hongwei [Beijing, CN; Ecker, Joseph R [Carlsbad, CA

    2014-01-07

    The relationship between F-box proteins and proteins invovled in the ethylene response in plants is described. In particular, F-box proteins may bind to proteins involved in the ethylene response and target them for degradation by the ubiquitin/proteasome pathway. The transcription factor EIN3 is a key transcription factor mediating ethylne-regulated gene expression and morphological responses. EIN3 is degraded through a ubiquitin/proteasome pathway mediated by F-box proteins EBF1 and EBF2. The link between F-box proteins and the ethylene response is a key step in modulating or regulating the response of a plant to ethylene. Described herein are transgenic plants having an altered sensitivity to ethylene, and methods for making transgenic plant haing an althered sensitivity to ethylene by modulating the level of activity of F-box proteins. Methods of altering the ethylene response in a plant by modulating the activity or expression of an F-box protein are described. Also described are methods of identifying compounds that modulate the ethylene response in plants by modulating the level of F-box protein expression or activity.

  20. Oxidatively Responsive Chain Extension to Entangle Engineered Protein Hydrogels.

    PubMed

    Tang, Shengchang; Glassman, Matthew J; Li, Shuaili; Socrate, Simona; Olsen, Bradley D

    2014-01-28

    Engineering artificial protein hydrogels for medical applications requires precise control over their mechanical properties, including stiffness, toughness, extensibility and stability in the physiological environment. Here we demonstrate topological entanglement as an effective strategy to robustly increase the mechanical tunability of a transient hydrogel network based on coiled-coil interactions. Chain extension and entanglement are achieved by coupling the cysteine residues near the N- and C- termini, and the resulting chain distribution is found to agree with the Jacobson-Stockmayer theory. By exploiting the reversible nature of the disulfide bonds, the entanglement effect can be switched on and off by redox stimuli. With the presence of entanglements, hydrogels exhibit a 7.2-fold enhanced creep resistance and a suppressed erosion rate by a factor of 5.8, making the gels more mechanically stable in a physiologically relevant open system. While hardly affecting material stiffness (only resulting in a 1.5-fold increase in the plateau modulus), the entanglements remarkably lead to hydrogels with a toughness of 65,000 J m(-3) and extensibility to approximately 3,000% engineering strain, which enables the preparation of tough yet soft tissue simulants. This improvement in mechanical properties resembles that from double-network hydrogels, but is achieved with the use of a single associating network and topological entanglement. Therefore, redox-triggered chain entanglement offers an effective approach for constructing mechanically enhanced and responsive injectable hydrogels. PMID:24910474

  1. Oxidatively Responsive Chain Extension to Entangle Engineered Protein Hydrogels

    PubMed Central

    Tang, Shengchang; Glassman, Matthew J.; Li, Shuaili; Socrate, Simona; Olsen, Bradley D.

    2014-01-01

    Engineering artificial protein hydrogels for medical applications requires precise control over their mechanical properties, including stiffness, toughness, extensibility and stability in the physiological environment. Here we demonstrate topological entanglement as an effective strategy to robustly increase the mechanical tunability of a transient hydrogel network based on coiled-coil interactions. Chain extension and entanglement are achieved by coupling the cysteine residues near the N- and C- termini, and the resulting chain distribution is found to agree with the Jacobson-Stockmayer theory. By exploiting the reversible nature of the disulfide bonds, the entanglement effect can be switched on and off by redox stimuli. With the presence of entanglements, hydrogels exhibit a 7.2-fold enhanced creep resistance and a suppressed erosion rate by a factor of 5.8, making the gels more mechanically stable in a physiologically relevant open system. While hardly affecting material stiffness (only resulting in a 1.5-fold increase in the plateau modulus), the entanglements remarkably lead to hydrogels with a toughness of 65,000 J m-3 and extensibility to approximately 3,000% engineering strain, which enables the preparation of tough yet soft tissue simulants. This improvement in mechanical properties resembles that from double-network hydrogels, but is achieved with the use of a single associating network and topological entanglement. Therefore, redox-triggered chain entanglement offers an effective approach for constructing mechanically enhanced and responsive injectable hydrogels. PMID:24910474

  2. Molecular dynamics simulation studies of dielectric response and vibrational energy relaxation in photoactive yellow protein and green fluorescent protein

    NASA Astrophysics Data System (ADS)

    Xu, Yao; Gnanasekaran, Ramachandran; Leitner, David

    2012-02-01

    The first step in the photocycle of many proteins involves conformational change of a chromophore or a charge transfer reaction following photoexcitation. To explore the response of the protein and solvent environment to photoexcitation of the chromophore in photoactive yellow protein (PYP) and green fluorescent protein (GFP) we carried out molecular dynamics simulations of the dielectric response and vibrational energy relaxation (VER) from the chromophore to the protein and solvent. In PYP the time scale of the protein response, mainly contributed by Tyr42 and Glu46, to photoexcitation appears prominently between 0.1 and 0.3 picoseconds. The frequency-dependent VER rate also reveals dynamic coupling between the chromophore and residues that hydrogen bond to it. Resonances in the VER rate appear at frequencies comparable to the oscillations observed in recent fluorescence decay studies. In GFP, which undergoes excited state proton transfer about 10 ps following photoexcitation that may be assisted by specific chromophore vibrations, both the protein and water molecules inside the ?-barrel surrounding the chromophore mediate the dielectric response.

  3. Author's personal copy Defective responses to oxidative stress in protein L-isoaspartyl repair-deficient

    E-print Network

    Clarke, Steven

    Author's personal copy Defective responses to oxidative stress in protein L-isoaspartyl repair type of protein damage during aging occurs by the non- oxidative deamidation and isomerization'Connor and Yutzey, 1988) and membrane proteins (Ingrosso et al., 2000) exposed to oxidative stress, as well

  4. Collapsin response mediator protein-2 is a calmodulin-binding protein.

    PubMed

    Zhang, Z; Majava, V; Greffier, A; Hayes, R L; Kursula, P; Wang, K K W

    2009-02-01

    Collapsin response mediator protein-2 (CRMP-2) plays a crucial role in axonal guidance and neurite outgrowth during neural development and regeneration. We have studied the interaction between calmodulin (CaM) and CRMP-2 and how Ca(2+)/CaM binding modulates the biological functions of CRMP-2. We have shown that CRMP-2 binds to CaM directly in a Ca(2+)-dependent manner. The CaM binding site of CRMP-2 is proposed to reside in the last helix of the folded domain, and in line with this, a synthesized peptide representing this helix bound to CaM. In addition, CaM binding inhibits a homotetrameric assembly of CRMP-2 and attenuates calpainmediated CRMP-2 proteolysis. Furthermore, a CaM antagonist reduces the number and length of process induced by CRMP-2 overexpression in HEK293 cells. Take together, our data suggest that CRMP-2 is a novel CaM-binding protein and that CaM binding may play an important role in regulating CRMP-2 functions. PMID:19151921

  5. Collapsin response mediator protein-2 is a calmodulin-binding protein

    PubMed Central

    Majava, V.; Greffier, A.; Hayes, R. L.

    2009-01-01

    Collapsin response mediator protein-2 (CRMP-2) plays a crucial role in axonal guidance and neurite outgrowth during neural development and regeneration. We have studied the interaction between calmodulin (CaM) and CRMP-2 and how Ca2+/CaM binding modulates the biological functions of CRMP-2. We have shown that CRMP-2 binds to CaM directly in a Ca2+-dependent manner. The CaM-binding site of CRMP-2 is proposed to reside in the last helix of the folded domain, and in line with this, a synthesized peptide representing this helix bound to CaM. In addition, CaM binding inhibits a homotetrameric assembly of CRMP-2 and attenuates calpain-mediated CRMP-2 proteolysis. Furthermore, a CaM antagonist reduces the number and length of process induced by CRMP-2 overexpression in HEK293 cells. Take together, our data suggest that CRMP-2 is a novel CaM-binding protein and that CaM binding may play an important role in regulating CRMP-2 functions. PMID:19151921

  6. First Characterization of Avian Memory T Lymphocyte Responses to Avian Influenza Virus Proteins 

    E-print Network

    Singh, Shailbala

    2011-02-22

    + T lymphocyte responses in chickens to individual AIV proteins have not been defined, the modulation of responses of the memory CD8+ T lymphocytes to H5N9 AIV hemagglutinin (HA) and nucleocapsid (NP) proteins over a time course were evaluated. CD8+ T lymphocyte...

  7. Antagonism of Host Antiviral Responses by Kaposi's Sarcoma-Associated Herpesvirus Tegument Protein

    E-print Network

    Ronquist, Fredrik

    Antagonism of Host Antiviral Responses by Kaposi's Sarcoma-Associated Herpesvirus Tegument Protein responses. Kaposi's sarcoma-associated herpesvirus (KSHV) is demonstrated in this report to be able by Kaposi's Sarcoma-Associated Herpesvirus Tegument Protein ORF45. PLoS ONE 5(5): e10573. doi:10

  8. A superior drug carrier – aponeocarzinostatin in partially unfolded state fully protects the labile antitumor enediyne

    PubMed Central

    Shanmuganathan, Aranganathan; Kumar, Thallapuranam Krishnaswamy Suresh; Huang, Chiy-Mey; Yu, Chin; Chin, Der-Hang

    2009-01-01

    Background Neocarzinostatin is a potent antitumor drug consisting of an enediyne chromophore and a protein carrier. Methods We characterized an intermediate in the equilibrium unfolding pathway of aponeocarzinostatin, using a variety of biophysical techniques including 1-anilino-8-napthalene sulfonate binding studies, size-exclusion fast protein liquid chromatography, intrinsic tryptophan fluorescence, circular dichroism, and 1H-15N heteronuclear single quantum coherence spectroscopy. Results The partially unfolded protein is in molten globule-like state, in which ~60% and ~20% tertiary and secondary structure is disrupted respectively. Despite lacking a fully coordinated tertiary structure for assembling a functional binding cleft, the protein in molten globule-like state is still able to fully protect the labile chromophore. Titration of chromophore leads the partially denatured apoprotein to fold into its native state. Conclusion These findings bring insight into conserving mechanism of neocarzinostatin under harsh environment, where even the partially denatured apoprotein exhibits protective effect, confirming the superiority of the drug carrier. PMID:19463188

  9. The Skeletal Muscle Anabolic Response to Plant- versus Animal-Based Protein Consumption.

    PubMed

    van Vliet, Stephan; Burd, Nicholas A; van Loon, Luc J C

    2015-09-01

    Clinical and consumer market interest is increasingly directed toward the use of plant-based proteins as dietary components aimed at preserving or increasing skeletal muscle mass. However, recent evidence suggests that the ingestion of the plant-based proteins in soy and wheat results in a lower muscle protein synthetic response when compared with several animal-based proteins. The possible lower anabolic properties of plant-based protein sources may be attributed to the lower digestibility of plant-based sources, in addition to greater splanchnic extraction and subsequent urea synthesis of plant protein-derived amino acids compared with animal-based proteins. The latter may be related to the relative lack of specific essential amino acids in plant- as opposed to animal-based proteins. Furthermore, most plant proteins have a relatively low leucine content, which may further reduce their anabolic properties when compared with animal proteins. However, few studies have actually assessed the postprandial muscle protein synthetic response to the ingestion of plant proteins, with soy and wheat protein being the primary sources studied. Despite the proposed lower anabolic properties of plant vs. animal proteins, various strategies may be applied to augment the anabolic properties of plant proteins. These may include the following: 1) fortification of plant-based protein sources with the amino acids methionine, lysine, and/or leucine; 2) selective breeding of plant sources to improve amino acid profiles; 3) consumption of greater amounts of plant-based protein sources; or 4) ingesting multiple protein sources to provide a more balanced amino acid profile. However, the efficacy of such dietary strategies on postprandial muscle protein synthesis remains to be studied. Future research comparing the anabolic properties of a variety of plant-based proteins should define the preferred protein sources to be used in nutritional interventions to support skeletal muscle mass gain or maintenance in both healthy and clinical populations. PMID:26224750

  10. Pressure-jump-induced kinetics reveals a hydration dependent folding/unfolding mechanism of ribonuclease A.

    PubMed

    Font, J; Torrent, J; Ribó, M; Laurents, D V; Balny, C; Vilanova, M; Lange, R

    2006-09-15

    Pressure-jump (p-jump)-induced relaxation kinetics was used to explore the energy landscape of protein folding/unfolding of Y115W, a fluorescent variant of ribonuclease A. Pressure-jumps of 40 MPa amplitude (5 ms dead-time) were conducted both to higher (unfolding) and to lower (folding) pressure, in the range from 100 to 500 MPa, between 30 and 50 degrees C. Significant deviations from the expected symmetrical protein relaxation kinetics were observed. Whereas downward p-jumps resulted always in single exponential kinetics, the kinetics induced by upward p-jumps were biphasic in the low pressure range and monophasic at higher pressures. The relative amplitude of the slow phase decreased as a function of both pressure and temperature. At 50 degrees C, only the fast phase remained. These results can be interpreted within the framework of a two-dimensional energy surface containing a pressure- and temperature-dependent barrier between two unfolded states differing in the isomeric state of the Asn-113-Pro-114 bond. Analysis of the activation volume of the fast kinetic phase revealed a temperature-dependent shift of the unfolding transition state to a larger volume. The observed compensation of this effect by glycerol offers an explanation for its protein stabilizing effect. PMID:16798802

  11. The Unfolding MD Simulations of Cyclophilin: Analyzed by Surface Contact Networks and Their Associated Metrics

    PubMed Central

    Roy, Sourav; Basu, Sankar; Dasgupta, Dipak; Bhattacharyya, Dhananjay; Banerjee, Rahul

    2015-01-01

    Currently, considerable interest exists with regard to the dissociation of close packed aminoacids within proteins, in the course of unfolding, which could result in either wet or dry moltenglobules. The progressive disjuncture of residues constituting the hydrophobic core ofcyclophilin from L. donovani (LdCyp) has been studied during the thermal unfolding of the molecule, by molecular dynamics simulations. LdCyp has been represented as a surface contactnetwork (SCN) based on the surface complementarity (Sm) of interacting residues within themolecular interior. The application of Sm to side chain packing within proteins make it a very sensitive indicator of subtle perturbations in packing, in the thermal unfolding of the protein. Network based metrics have been defined to track the sequential changes in the disintegration ofthe SCN spanning the hydrophobic core of LdCyp and these metrics prove to be highly sensitive compared to traditional metrics in indicating the increased conformational (and dynamical) flexibility in the network. These metrics have been applied to suggest criteria distinguishing DMG, WMG and transition state ensembles and to identify key residues involved in crucial conformational/topological events during the unfolding process. PMID:26545107

  12. Heat, Acid and Chemically Induced Unfolding Pathways, Conformational Stability and Structure-Function Relationship in Wheat ?-Amylase

    PubMed Central

    Singh, Kritika; Shandilya, Manish; Kundu, Suman; Kayastha, Arvind M.

    2015-01-01

    Wheat ?-amylase, a multi-domain protein with immense industrial applications, belongs to ?+? class of proteins with native molecular mass of 32 kDa. In the present study, the pathways leading to denaturation and the relevant unfolded states of this multi-domain, robust enzyme from wheat were discerned under the influence of temperature, pH and chemical denaturants. The structural and functional aspects along with thermodynamic parameters for ?-amylase unfolding were probed and analyzed using fluorescence, circular dichroism and enzyme assay methods. The enzyme exhibited remarkable stability up to 70°C with tendency to aggregate at higher temperature. Acid induced unfolding was also incomplete with respect to the structural content of the enzyme. Strong ANS binding at pH 2.0 suggested the existence of a partially unfolded intermediate state. The enzyme was structurally and functionally stable in the pH range 4.0–9.0 with 88% recovery of hydrolytic activity. Careful examination of biophysical properties of intermediate states populated in urea and GdHCl induced denaturation suggests that ?-amylase unfolding undergoes irreversible and non-coincidental cooperative transitions, as opposed to previous reports of two-state unfolding. Our investigation highlights several structural features of the enzyme in relation to its catalytic activity. Since, ?-amylase has been comprehensively exploited for use in a range of starch-based industries, in addition to its physiological significance in plants and animals, knowledge regarding its stability and folding aspects will promote its biotechnological applications. PMID:26053142

  13. Protein-Folding Homeostasis in the Endoplasmic Reticulum and Nutritional Regulation

    PubMed Central

    Ron, David; Harding, Heather P.

    2012-01-01

    The flux of newly synthesized proteins entering the endoplasmic reticulum (ER) is under negative regulation by the ER-localized PKR-like ER kinase (PERK). PERK is activated by unfolded protein stress in the ER lumen and inhibits new protein synthesis by the phosphorylation of translation initiation factor eIF2?. This homeostatic mechanism, shared by all animal cells, has proven to be especially important to the well-being of professional secretory cells, notably the endocrine pancreas. PERK, its downstream effectors, and the allied branches of the unfolded protein response intersect broadly with signaling pathways that regulate nutrient assimilation, and ER stress and the response to it have been implicated in the development of the metabolic syndrome accompanying obesity in mammals. Here we review our current understanding of the cell biology underlying these relationships. PMID:23209157

  14. Intracellular proteins produced by mammalian cells in response to environmental stress

    NASA Technical Reports Server (NTRS)

    Goochee, Charles F.; Passini, Cheryl A.

    1988-01-01

    The nature of the response of mammalian cells to environmental stress is examined by reviewing results of studies where cultured mouse L cells and baby hamster kidney cells were exposed to heat shock and the synthesis of heat-shock proteins and stress-response proteins (including HSP70, HSC70, HSP90, ubiquitin, and GRP70) in stressed and unstressed cells was evaluated using 2D-PAGE. The intracellular roles of the individual stress response proteins are discussed together with the regulation of the stress response system.

  15. Microcomputer-Analyzed Initial Rate Kinetics of the Benzene-Enhanced Unfolding of Myoglobin: A Biophysical Chemistry Experiment.

    ERIC Educational Resources Information Center

    Schuh, Merlyn D.

    1988-01-01

    Describes a biophysical chemistry experiment that introduces students to globular protein conformation and microcomputer analysis of initial rate data for the unfolding of proteins. Presents background, materials needed and methodology. Uses a visible spectrometer for analysis. Lists educational benefits derived from the experiment. (ML)

  16. Declining global warming effects on the phenology of spring leaf unfolding.

    PubMed

    Fu, Yongshuo H; Zhao, Hongfang; Piao, Shilong; Peaucelle, Marc; Peng, Shushi; Zhou, Guiyun; Ciais, Philippe; Huang, Mengtian; Menzel, Annette; Peñuelas, Josep; Song, Yang; Vitasse, Yann; Zeng, Zhenzhong; Janssens, Ivan A

    2015-10-01

    Earlier spring leaf unfolding is a frequently observed response of plants to climate warming. Many deciduous tree species require chilling for dormancy release, and warming-related reductions in chilling may counteract the advance of leaf unfolding in response to warming. Empirical evidence for this, however, is limited to saplings or twigs in climate-controlled chambers. Using long-term in situ observations of leaf unfolding for seven dominant European tree species at 1,245 sites, here we show that the apparent response of leaf unfolding to climate warming (ST, expressed in days advance of leaf unfolding per °C warming) has significantly decreased from 1980 to 2013 in all monitored tree species. Averaged across all species and sites, ST decreased by 40% from 4.0 ± 1.8 days °C(-1) during 1980-1994 to 2.3 ± 1.6 days °C(-1) during 1999-2013. The declining ST was also simulated by chilling-based phenology models, albeit with a weaker decline (24-30%) than observed in situ. The reduction in ST is likely to be partly attributable to reduced chilling. Nonetheless, other mechanisms may also have a role, such as 'photoperiod limitation' mechanisms that may become ultimately limiting when leaf unfolding dates occur too early in the season. Our results provide empirical evidence for a declining ST, but also suggest that the predicted strong winter warming in the future may further reduce ST and therefore result in a slowdown in the advance of tree spring phenology. PMID:26416746

  17. Role of cavities and hydration in the pressure unfolding of T4 lysozyme

    PubMed Central

    Nucci, Nathaniel V.; Fuglestad, Brian; Athanasoula, Evangelia A.; Wand, A. Joshua

    2014-01-01

    It is well known that high hydrostatic pressures can induce the unfolding of proteins. The physical underpinnings of this phenomenon have been investigated extensively but remain controversial. Changes in solvation energetics have been commonly proposed as a driving force for pressure-induced unfolding. Recently, the elimination of void volumes in the native folded state has been argued to be the principal determinant. Here we use the cavity-containing L99A mutant of T4 lysozyme to examine the pressure-induced destabilization of this multidomain protein by using solution NMR spectroscopy. The cavity-containing C-terminal domain completely unfolds at moderate pressures, whereas the N-terminal domain remains largely structured to pressures as high as 2.5 kbar. The sensitivity to pressure is suppressed by the binding of benzene to the hydrophobic cavity. These results contrast to the pseudo-WT protein, which has a residual cavity volume very similar to that of the L99A–benzene complex but shows extensive subglobal reorganizations with pressure. Encapsulation of the L99A mutant in the aqueous nanoscale core of a reverse micelle is used to examine the hydration of the hydrophobic cavity. The confined space effect of encapsulation suppresses the pressure-induced unfolding transition and allows observation of the filling of the cavity with water at elevated pressures. This indicates that hydration of the hydrophobic cavity is more energetically unfavorable than global unfolding. Overall, these observations point to a range of cooperativity and energetics within the T4 lysozyme molecule and illuminate the fact that small changes in physical parameters can significantly alter the pressure sensitivity of proteins. PMID:25201963

  18. Force-Induced Unfolding of Leucine-Rich Repeats of Glycoprotein Ib? Strengthens Ligand Interaction.

    PubMed

    Ju, Lining; Lou, Jizhong; Chen, Yunfeng; Li, Zhenhai; Zhu, Cheng

    2015-11-01

    Leucine-rich repeat (LRR) is a versatile motif widely present in adhesive proteins and signal-transducing receptors. The concave structure formed by a group of LRRs is thought to facilitate binding to globular protein domains with increased affinities. However, little is known about the conformational dynamics of LRRs in such a structure, e.g., whether and how force induces conformational changes in LRRs to regulate protein binding and signal transduction. Here we investigated the platelet glycoprotein Ib? (GPIb?), a demonstrated mechanoreceptor with known crystal structures for the N-terminal domain (GPIb?N), as a model for LRR-containing proteins using a combined method of steered molecular dynamics simulations and single-molecule force spectroscopy with a biomembrane force probe. We found that force-induced unfolding of GPIb?N starts with LRR2-4 and propagates to other LRRs. Importantly, force-dependent lifetimes of individual VWF-A1 bonds with GPIb? are prolonged after LRR unfolding. Enhancement of protein-protein interactions by force-induced LRR unfolding may be a phenomenon of interest in biology. PMID:26536255

  19. Folding and unfolding of a photoswitchable peptide from picoseconds to microseconds

    PubMed Central

    Ihalainen, Janne A.; Bredenbeck, Jens; Pfister, Rolf; Helbing, Jan; Chi, Lei; van Stokkum, Ivo H. M.; Woolley, G. Andrew; Hamm, Peter

    2007-01-01

    Using time-resolved IR spectroscopy, we monitored the kinetics of folding and unfolding processes of a photoswitchable 16-residue alanine-based ?-helical peptide on a timescale from few picoseconds to almost 40 ?s and over a large temperature range (279–318 K). The folding and unfolding processes were triggered by an ultrafast laser pulse that isomerized the cross linker within a few picoseconds. The main folding and unfolding times (700 ns and 150 ns, respectively, at room temperature) are in line with previous T-jump experiments obtained from similar peptides. However, both processes show complex, strongly temperature-dependent spectral kinetics that deviate clearly from a single-exponential behavior. Whereas in the unfolding experiment the ensemble starts from a well defined folded state, the starting ensemble in the folding experiment is more heterogeneous, which leads to distinctly different kinetics of the experiments, because they are sensitive to different regions of the energy surface. A qualitative agreement with the experimental data-set can be obtained by a model where the unfolded states act as a hub connected to several separated “misfolded” states with a distribution of rates. We conclude that a rather large spread of rates (k1 : kn ? 9) is needed to explain the experimentally observed stretched exponential response with stretching factor ? = 0.8 at 279 K. PMID:17372213

  20. Unfolding of differential energy spectra in the MAGIC experiment

    NASA Astrophysics Data System (ADS)

    Albert, J.; Aliu, E.; Anderhub, H.; Antoranz, P.; Armada, A.; Asensio, M.; Baixeras, C.; Barrio, J. A.; Bartko, H.; Bastieri, D.; Becker, J.; Bednarek, W.; Berger, K.; Bigongiari, C.; Biland, A.; Bock, R. K.; Bordas, P.; Bosch-Ramon, V.; Bretz, T.; Britvitch, I.; Camara, M.; Carmona, E.; Chilingarian, A.; Ciprini, S.; Coarasa, J. A.; Commichau, S.; Contreras, J. L.; Cortina, J.; Costado, M. T.; Curtef, V.; Danielyan, V.; Dazzi, F.; de Angelis, A.; Delgado, C.; de Los Reyes, R.; de Lotto, B.; Domingo-Santamaría, E.; Dorner, D.; Doro, M.; Errando, M.; Fagiolini, M.; Ferenc, D.; Fernández, E.; Firpo, R.; Flix, J.; Fonseca, M. V.; Font, L.; Fuchs, M.; Galante, N.; García-López, R. J.; Garczarczyk, M.; Gaug, M.; Giller, M.; Goebel, F.; Hakobyan, D.; Hayashida, M.; Hengstebeck, T.; Herrero, A.; Höhne, D.; Hose, J.; Hsu, C. C.; Jacon, P.; Jogler, T.; Kosyra, R.; Kranich, D.; Kritzer, R.; Laille, A.; Lindfors, E.; Lombardi, S.; Longo, F.; López, J.; López, M.; Lorenz, E.; Majumdar, P.; Maneva, G.; Mannheim, K.; Mansutti, O.; Mariotti, M.; Martínez, M.; Mazin, D.; Merck, C.; Meucci, M.; Meyer, M.; Miranda, J. M.; Mirzoyan, R.; Mizobuchi, S.; Moralejo, A.; Nieto, D.; Nilsson, K.; Ninkovic, J.; Oña-Wilhelmi, E.; Otte, N.; Oya, I.; Panniello, M.; Paoletti, R.; Paredes, J. M.; Pasanen, M.; Pascoli, D.; Pauss, F.; Pegna, R.; Persic, M.; Peruzzo, L.; Piccioli, A.; Puchades, N.; Prandini, E.; Raymers, A.; Rhode, W.; Ribó, M.; Rico, J.; Rissi, M.; Robert, A.; Rügamer, S.; Saggion, A.; Saito, T.; Sánchez, A.; Sartori, P.; Scalzotto, V.; Scapin, V.; Schmitt, R.; Schweizer, T.; Shayduk, M.; Shinozaki, K.; Shore, S. N.; Sidro, N.; Sillanpää, A.; Sobczynska, D.; Stamerra, A.; Stark, L. S.; Takalo, L.; Temnikov, P.; Tescaro, D.; Teshima, M.; Torres, D. F.; Turini, N.; Vankov, H.; Vitale, V.; Wagner, R. M.; Wibig, T.; Wittek, W.; Zandanel, F.; Zanin, R.; Zapatero, J.

    2007-12-01

    The paper describes the different methods, used in the MAGIC experiment, to unfold experimental energy distributions of cosmic ray particles (?-rays). Questions and problems related to the unfolding are discussed. Various procedures are proposed which can help to make the unfolding robust and reliable. The different methods and procedures are implemented in the MAGIC software and are used in most of the analyses.

  1. Markov Chain Monte Carlo Estimation of Item Parameters for the Generalized Graded Unfolding Model

    ERIC Educational Resources Information Center

    de la Torre, Jimmy; Stark, Stephen; Chernyshenko, Oleksandr S.

    2006-01-01

    The authors present a Markov Chain Monte Carlo (MCMC) parameter estimation procedure for the generalized graded unfolding model (GGUM) and compare it to the marginal maximum likelihood (MML) approach implemented in the GGUM2000 computer program, using simulated and real personality data. In the simulation study, test length, number of response

  2. Folding and Unfolding Linkages, Paper, and Polyhedra

    E-print Network

    Demaine, Erik

    Folding and Unfolding Linkages, Paper, and Polyhedra Erik D. Demaine Department of Computer Science in these problems in discrete and computational geometry. This paper gives a brief survey of some of the recent work in this area, subdivided into three sections based on the type of object being folded: linkages, paper

  3. Unfolding Orthogonal Polyhedra with Quadratic Refinement: The Delta-Unfolding Algorithm

    E-print Network

    Damian, Mirela

    We show that every orthogonal polyhedron homeomorphic to a sphere can be unfolded without overlap while using only polynomially many (orthogonal) cuts. By contrast, the best previous such result used exponentially many ...

  4. Unfolding spinor wave functions and expectation values of general operators: Introducing the unfolding-density operator

    NASA Astrophysics Data System (ADS)

    Medeiros, Paulo V. C.; Tsirkin, Stepan S.; Stafström, Sven; Björk, Jonas

    2015-01-01

    We show that the spectral weights Wm K ?(k ?) used for the unfolding of two-component spinor eigenstates | ?mK ? SC>=|? >| ?mK ? SC,? >+|? >| ?mK ? SC,? > can be decomposed as the sum of the partial spectral weights WmK ? ?(k ?) calculated for each component ? =? ,? independently, effortlessly turning a possibly complicated problem involving two coupled quantities into two independent problems of easy solution. Furthermore, we define the unfolding-density operator ??K ?(k ?;? ) , which unfolds the primitive cell expectation values ?pc(k ?;? ) of any arbitrary operator ? ? according to ?pc(k ?;? ) =Tr( ??K ?(k ?;? ) ? ?) . As a proof of concept, we apply the method to obtain the unfolded band structures, as well as the expectation values of the Pauli spin matrices, for prototypical physical systems described by two-component spinor eigenfunctions.

  5. 2-D DIGE analysis of UV-C radiation-responsive proteins in globe artichoke leaves.

    PubMed

    Falvo, Sara; Di Carli, Mariasole; Desiderio, Angiola; Benvenuto, Eugenio; Moglia, Andrea; America, Twan; Lanteri, Sergio; Acquadro, Alberto

    2012-02-01

    Plants respond to ultraviolet stress inducing a self-defence through the regulation of specific gene family members. The UV acclimation is the result of biochemical and physiological processes, such as enhancement of the antioxidant enzymatic system and accumulation of UV-absorbing phenolic compounds (e.g. flavonoids). Globe artichoke is an attractive species for studying the protein network involved in UV stress response, being characterized by remarkable levels of inducible antioxidants. Proteomic tools can assist the evaluation of the expression patterns of UV-responsive proteins and we applied the difference in-gel electrophoresis (DIGE) technology for monitoring the globe artichoke proteome variation at four time points following an acute UV-C exposure. A total of 145 UV-C-modulated proteins were observed and 119 were identified by LC-MS/MS using a ?144,000 customized Compositae protein database, which included about 19,000 globe artichoke unigenes. Proteins were Gene Ontology (GO) categorized, visualized on their pathways and their behaviour was discussed. A predicted protein interaction network was produced and highly connected hub-like proteins were highlighted. Most of the proteins differentially modulated were chloroplast located, involved in photosynthesis, sugar metabolisms, protein folding and abiotic stress. The identification of UV-C-responsive proteins may contribute to shed light on the molecular mechanisms underlying plant responses to UV stress. PMID:22162389

  6. Evaluating Common Humoral Responses against Fungal Infections with Yeast Protein Microarrays.

    PubMed

    Coelho, Paulo S R; Im, Hogune; Clemons, Karl V; Snyder, Michael P; Stevens, David A

    2015-09-01

    We profiled the global immunoglobulin response against fungal infection by using yeast protein microarrays. Groups of CD-1 mice were infected systemically with human fungal pathogens (Coccidioides posadasii, Candida albicans, or Paracoccidioides brasiliensis) or inoculated with PBS as a control. Another group was inoculated with heat-killed yeast (HKY) of Saccharomyces cerevisiae. After 30 days, serum from mice in the groups were collected and used to probe S. cerevisiae protein microarrays containing 4800 full-length glutathione S-transferase (GST)-fusion proteins. Antimouse IgG conjugated with Alexafluor 555 and anti-GST antibody conjugated with Alexafluor 647 were used to detect antibody-antigen interactions and the presence of GST-fusion proteins, respectively. Serum after infection with C. albicans reacted with 121 proteins: C. posadasii, 81; P. brasiliensis, 67; and after HKY, 63 proteins on the yeast protein microarray, respectively. We identified a set of 16 antigenic proteins that were shared across the three fungal pathogens. These include retrotransposon capsid proteins, heat shock proteins, and mitochondrial proteins. Five of these proteins were identified in our previous study of fungal cell wall by mass spectrometry (Ann. N. Y. Acad. Sci. 2012, 1273, 44-51). The results obtained give a comprehensive view of the immunological responses to fungal infections at the proteomic level. They also offer insight into immunoreactive protein commonality among several fungal pathogens and provide a basis for a panfungal vaccine. PMID:26258609

  7. Dengue-2 Structural Proteins Associate with Human Proteins to Produce a Coagulation and Innate Immune Response Biased Interactome

    PubMed Central

    2011-01-01

    Background Dengue virus infection is a public health threat to hundreds of millions of individuals in the tropical regions of the globe. Although Dengue infection usually manifests itself in its mildest, though often debilitating clinical form, dengue fever, life-threatening complications commonly arise in the form of hemorrhagic shock and encephalitis. The etiological basis for the virus-induced pathology in general, and the different clinical manifestations in particular, are not well understood. We reasoned that a detailed knowledge of the global biological processes affected by virus entry into a cell might help shed new light on this long-standing problem. Methods A bacterial two-hybrid screen using DENV2 structural proteins as bait was performed, and the results were used to feed a manually curated, global dengue-human protein interaction network. Gene ontology and pathway enrichment, along with network topology and microarray meta-analysis, were used to generate hypothesis regarding dengue disease biology. Results Combining bioinformatic tools with two-hybrid technology, we screened human cDNA libraries to catalogue proteins physically interacting with the DENV2 virus structural proteins, Env, cap and PrM. We identified 31 interacting human proteins representing distinct biological processes that are closely related to the major clinical diagnostic feature of dengue infection: haemostatic imbalance. In addition, we found dengue-binding human proteins involved with additional key aspects, previously described as fundamental for virus entry into cells and the innate immune response to infection. Construction of a DENV2-human global protein interaction network revealed interesting biological properties suggested by simple network topology analysis. Conclusions Our experimental strategy revealed that dengue structural proteins interact with human protein targets involved in the maintenance of blood coagulation and innate anti-viral response processes, and predicts that the interaction of dengue proteins with a proposed human protein interaction network produces a modified biological outcome that may be behind the hallmark pathologies of dengue infection. PMID:21281507

  8. Insulin-like growth factor (IGF) binding protein enhances the biologic response to IGF-I

    SciTech Connect

    Elgin, R.G.; Busby, H.W. Jr.; Clemmons, D.R.

    1987-05-01

    The insulin-like growth factors IGF-I and IGF-II circulate in blood bound to carrier proteins. The higher molecular mass IGF-binding protein complex (150 kDa) is composed of subunits, and one subunits that forms this complex is growth hormone dependent. In addition, many cell types and tissues secrete another form of IGF binding protein that is not growth hormone dependent. Both forms of the IGF binding protein are believed to inactivate the IGFs and to function as delivery systems to tissues. This conclusion was based on studies that determined the effects of impure preparations of these binding proteins or that examined the effect of these proteins only on the insulin-like actions of the IGFs. The authors report here that a pure preparation of the extracellular form of the IGF binding protein (purified from human amniotic fluid) markedly potentiated replication of several cell types in response to human IGF-I. Secondary cultures of human, mouse, and chicken embryo fibroblasts as well as porcine aortic smooth muscle cells showed marked enhancement of their DNA synthesis response to IGF-I in the presence of this protein. The binding protein not only potentiated the DNA synthesis response but also enhanced the increase in cell number in response to IGF-I. This stimulation is specific for growth factors that bind to the binding protein since incubation with insulin, which binds to the type I IGF receptor but not to the binding protein, did not result in potentiation of this response. They conclude that a form of IGF binding protein that is present in extracellular fluids and is secreted by many types of cells can markedly potentiate the cellular response to IGF-I.

  9. A Single Destabilizing Mutation (F9S) Promotes Concerted Unfolding of an Entire Globular Domain in ?S-Crystallin

    PubMed Central

    Lee, Soojin; Mahler, Bryon; Toward, Jodie; Jones, Blake; Wyatt, Keith; Dong, Lijin; Wistow, Graeme; Wu, Zhengrong

    2010-01-01

    Conformational change and aggregation of native proteins are associated with many serious age-related and neurological diseases. ?S-Crystallin is a highly stable, abundant structural component of vertebrate eye lens. A single F9S mutation in the N-terminal domain of mouse ?S-crystallin causes the severe Opj cataract, with disruption of cellular organization and appearance of fibrillar structures in the lens. Although the mutant protein has a near-native fold at room temperature, significant increases in hydrogen/deuterium exchange rates were observed by NMR for all the well-protected ?-sheet core residues throughout the entire N-terminal domain of the mutant protein, resulting in up to a 3.5-kcal/mol reduction in the free energy of the folding/unfolding equilibrium. No difference was detected for the C-terminal domain. At a higher temperature, this effect further increases to allow for a much more uniform exchange rate among the N-terminal core residues and those of the least well-structured surface loops. This suggests a concerted unfolding intermediate of the N-terminal domain, while the C-terminal domain stays intact. Increasing concentrations of guanidinium chloride produced two transitions for the Opj mutant, with an unfolding intermediate at ~1 M guanidinium chloride. The consequence of this partial unfolding, whether by elevated temperature or by denaturant, is the formation of thioflavin T staining aggregates, which demonstrated fibril-like morphology by atomic force microscopy. Seeding with the already unfolded protein enhanced the formation of fibrils. The Opj mutant protein provides a model for stress-related unfolding of an essentially normally folded protein and production of aggregates with some of the characteristics of amyloid fibrils. PMID:20382156

  10. Protein Folding and Mechanisms of Proteostasis

    PubMed Central

    Díaz-Villanueva, José Fernando; Díaz-Molina, Raúl; García-González, Victor

    2015-01-01

    Highly sophisticated mechanisms that modulate protein structure and function, which involve synthesis and degradation, have evolved to maintain cellular homeostasis. Perturbations in these mechanisms can lead to protein dysfunction as well as deleterious cell processes. Therefore in recent years the etiology of a great number of diseases has been attributed to failures in mechanisms that modulate protein structure. Interconnections among metabolic and cell signaling pathways are critical for homeostasis to converge on mechanisms associated with protein folding as well as for the preservation of the native structure of proteins. For instance, imbalances in secretory protein synthesis pathways lead to a condition known as endoplasmic reticulum (ER) stress which elicits the adaptive unfolded protein response (UPR). Therefore, taking this into consideration, a key part of this paper is developed around the protein folding phenomenon, and cellular mechanisms which support this pivotal condition. We provide an overview of chaperone protein function, UPR via, spatial compartmentalization of protein folding, proteasome role, autophagy, as well as the intertwining between these processes. Several diseases are known to have a molecular etiology in the malfunction of mechanisms responsible for protein folding and in the shielding of native structure, phenomena which ultimately lead to misfolded protein accumulation. This review centers on our current knowledge about pathways that modulate protein folding, and cell responses involved in protein homeostasis. PMID:26225966

  11. Protein Folding and Mechanisms of Proteostasis.

    PubMed

    Díaz-Villanueva, José Fernando; Díaz-Molina, Raúl; García-González, Victor

    2015-01-01

    Highly sophisticated mechanisms that modulate protein structure and function, which involve synthesis and degradation, have evolved to maintain cellular homeostasis. Perturbations in these mechanisms can lead to protein dysfunction as well as deleterious cell processes. Therefore in recent years the etiology of a great number of diseases has been attributed to failures in mechanisms that modulate protein structure. Interconnections among metabolic and cell signaling pathways are critical for homeostasis to converge on mechanisms associated with protein folding as well as for the preservation of the native structure of proteins. For instance, imbalances in secretory protein synthesis pathways lead to a condition known as endoplasmic reticulum (ER) stress which elicits the adaptive unfolded protein response (UPR). Therefore, taking this into consideration, a key part of this paper is developed around the protein folding phenomenon, and cellular mechanisms which support this pivotal condition. We provide an overview of chaperone protein function, UPR via, spatial compartmentalization of protein folding, proteasome role, autophagy, as well as the intertwining between these processes. Several diseases are known to have a molecular etiology in the malfunction of mechanisms responsible for protein folding and in the shielding of native structure, phenomena which ultimately lead to misfolded protein accumulation. This review centers on our current knowledge about pathways that modulate protein folding, and cell responses involved in protein homeostasis. PMID:26225966

  12. Proof-of-principle to unfold an angle-energy dependent source from forward and adjoint calculations

    SciTech Connect

    Pace, J.V. III

    2001-01-10

    For many years there has existed a discrepancy between the measured and calculated responses from the Little Boy weapon in Hiroshima. A myriad of solutions have been proposed, but to no avail. If one can rationalize to himself that it does not really matter exactly what happened with the weapon when it exploded, and if sufficient information exist about the measurements, one should be able to unfold the source. Moreover, if a source can be unfolded in a controlled environment, then it should be possible to unfold a more complicated source, for example, the Little Boy source. This report records the findings of a proof-of-principle test to unfold a source in the controlled environment.

  13. Nuclear Cytoplasmic Trafficking of Proteins is a Major Response of Human Fibroblasts to Oxidative Stress

    PubMed Central

    Baqader, Noor O.; Radulovic, Marko; Crawford, Mark; Stoeber, Kai; Godovac-Zimmermann, Jasminka

    2014-01-01

    We have used a subcellular spatial razor approach based on LC–MS/MS-based proteomics with SILAC isotope labeling to determine changes in protein abundances in the nuclear and cytoplasmic compartments of human IMR90 fibroblasts subjected to mild oxidative stress. We show that response to mild tert-butyl hydrogen peroxide treatment includes redistribution between the nucleus and cytoplasm of numerous proteins not previously associated with oxidative stress. The 121 proteins with the most significant changes encompass proteins with known functions in a wide variety of subcellular locations and of cellular functional processes (transcription, signal transduction, autophagy, iron metabolism, TCA cycle, ATP synthesis) and are consistent with functional networks that are spatially dispersed across the cell. Both nuclear respiratory factor 2 and the proline regulatory axis appear to contribute to the cellular metabolic response. Proteins involved in iron metabolism or with iron/heme as a cofactor as well as mitochondrial proteins are prominent in the response. Evidence suggesting that nuclear import/export and vesicle-mediated protein transport contribute to the cellular response was obtained. We suggest that measurements of global changes in total cellular protein abundances need to be complemented with measurements of the dynamic subcellular spatial redistribution of proteins to obtain comprehensive pictures of cellular function. PMID:25133973

  14. The PB1 Domain in Auxin Response Factor and Aux/IAA Proteins: A Versatile Protein Interaction Module in the Auxin Response[OPEN

    PubMed Central

    2015-01-01

    An integral part of auxin-regulated gene expression involves the interplay of two types of transcription factors, the DNA binding auxin response factor (ARF) activators and the interacting auxin/indole acetic acid (Aux/IAA) repressors. Insight into the mechanism of how these transcription factors interact with one another has recently been revealed from crystallographic information on ARF5 and ARF7 C-terminal domains (i.e., a protein-protein interaction domain referred to as domain III/IV that is related to domain III/IV in Aux/IAA proteins). Three-dimensional structures showed that this domain in ARF5 and ARF7 conforms to a well-known PB1 (Phox and Bem1) domain that confers protein-protein interactions with other PB1 domain proteins through electrostatic contacts. Experiments verifying the importance of charged amino acids in conferring ARF and Aux/IAA interactions have confirmed the PB1 domain structure. Some in planta experiments designed to test the validity of PB1 interactions in the auxin response have led to updated models for auxin-regulated gene expression and raised many questions that will require further investigation. In addition to the PB1 domain, a second protein interaction module that functions in ARF-ARF dimerization and facilitates DNA binding has recently been revealed from crystallography studies on the ARF1 and ARF5 DNA binding domains. PMID:25604444

  15. Photoinduced Localized Unfolding of Tubulin Dimers Bound to a Water Soluble Porphyrin and the Search for Binding Location Using Docking Simulations Guided by a Combination of Resonance Raman Spectroscopy and Density Functional Theory

    NASA Astrophysics Data System (ADS)

    McMicken, Brady; Brancaleon, Lorenzo; Thomas, Robert

    2014-03-01

    The ability to modify the conformation of a protein by controlling its partial unfolding may have practical applications such as inhibiting its function. One method of locally unfolding a protein involves the use of a photosensitizer non-covalently bound to a protein which triggers photochemical reactions upon irradiation leading to protein conformational changes. We investigate the photoinduced conformational changes of tubulin mediated by a bound water soluble porphyrin which acts as a photosensitizer. Also of interest is how conformational changes of tubulin affect its function such as forming microtubules and the mechanism responsible for the structural change. To better understand the conformational change we must find the binding location between the porphyrin and protein. Density functional theory calculations will be combined with Resonance Raman spectroscopy to correlate the changes in vibrational modes of the porphyrin with changes in its physical structure upon binding to tubulin. This will allow us to determine the distorted conformation of the porphyrin when bound to tubulin which will subsequently be used in docking simulations to find the most likely binding configuration. Consortium Research Fellows Program.

  16. Increased sialylation and defucosylation of plasma proteins are early events in the acute phase response.

    PubMed

    Chavan, Manasi M; Kawle, Poonam D; Mehta, Narendra G

    2005-09-01

    Within hours of turpentine injection to stimulate the acute phase (AP) response in rats, the N-acetylneuraminic acid content of plasma proteins increases and that of fucose decreases, each by about 60%. The two changes are inversely related (r = -0.97). The NeuAc/Gal ratio increases from the normal 0.75 to 1.0 on day 2 of the AP. Whereas 50% of the isolated oligosaccharides of normal plasma proteins are retarded on immobilized Ricinus communis agglutinin, those from day 2 AP plasma fail to do so. This indicates that NeuAc caps the normally Gal-terminated chains. alpha1-Acid glycoprotein (a positive AP protein), alpha1-macroglobulin (a non-AP protein), and alpha1-inhibitor3 (a negative AP protein) also show similar alterations in NeuAc/Gal ratio and decreases in Fuc. alpha2-Macroglobulin, which arises only during the AP, does not contain significant amounts of Fuc. Sambucus nigra agglutinin (alpha2,6-linked NeuAc-specific) binds a majority of plasma proteins, and binding is increased during the AP response. Maackia amurensis lectin (alpha2,3-linked NeuAc-specific) binds only three proteins in normal plasma and three additional proteins in AP plasma. The Fuc-specific Aleuria aurantia agglutinin and Lens culinaris agglutinin each detect five proteins in normal plasma. Their binding decreases during the AP response. These results show that: (1) sialylation and defucosylation of preexisting plasma proteins occur rapidly in the AP response; (2) sialylation caps the preexisting Gal-terminating oligosaccharides; and (3) the oligosaccharides of even the non-AP and negative AP proteins are modified. These changes are distinct from the elevation in the levels of protein-bound monosaccharides and the altered concanavalin A-binding profile the oligosaccharides of AP proteins acquire in diseases. PMID:15858075

  17. Inhibition of endoplasmic reticulum-associated degradation rescues native folding in loss of function protein misfolding diseases.

    PubMed

    Wang, Fan; Song, Wensi; Brancati, Giovanna; Segatori, Laura

    2011-12-16

    Lysosomal storage disorders are often caused by mutations that destabilize native folding and impair trafficking of secretory proteins. We demonstrate that endoplasmic reticulum (ER)-associated degradation (ERAD) prevents native folding of mutated lysosomal enzymes in patient-derived fibroblasts from two clinically distinct lysosomal storage disorders, namely Gaucher and Tay-Sachs disease. Prolonging ER retention via ERAD inhibition enhanced folding, trafficking, and activity of these unstable enzyme variants. Furthermore, combining ERAD inhibition with enhancement of the cellular folding capacity via proteostasis modulation resulted in synergistic rescue of mutated enzymes. ERAD inhibition was achieved by cell treatment with small molecules that interfere with recognition (kifunensine) or retrotranslocation (eeyarestatin I) of misfolded substrates. These different mechanisms of ERAD inhibition were shown to enhance ER retention of mutated proteins but were associated with dramatically different levels of ER stress, unfolded protein response activation, and unfolded protein response-induced apoptosis. PMID:22006919

  18. Processing of protein ADP-ribosylation by Nudix hydrolases.

    PubMed

    Palazzo, Luca; Thomas, Benjamin; Jemth, Ann-Sofie; Colby, Thomas; Leidecker, Orsolya; Feijs, Karla L H; Zaja, Roko; Loseva, Olga; Puigvert, Jordi Carreras; Matic, Ivan; Helleday, Thomas; Ahel, Ivan

    2015-06-01

    ADP-ribosylation is a post-translational modification (PTM) of proteins found in organisms from all kingdoms of life which regulates many important biological functions including DNA repair, chromatin structure, unfolded protein response and apoptosis. Several cellular enzymes, such as macrodomain containing proteins PARG [poly(ADP-ribose) glycohydrolase] and TARG1 [terminal ADP-ribose (ADPr) protein glycohydrolase], reverse protein ADP-ribosylation. In the present study, we show that human Nudix (nucleoside diphosphate-linked moiety X)-type motif 16 (hNUDT16) represents a new enzyme class that can process protein ADP-ribosylation in vitro, converting it into ribose-5'-phosphate (R5P) tags covalently attached to the modified proteins. Furthermore, our data show that hNUDT16 enzymatic activity can be used to trim ADP-ribosylation on proteins in order to facilitate analysis of ADP-ribosylation sites on proteins by MS. PMID:25789582

  19. The modulus of unfoldings of cusps in conformal geometry

    NASA Astrophysics Data System (ADS)

    Rousseau, C.

    In this paper we give the complete classification of generic 1-parameter unfoldings of germs of real analytic curves with a cuspidal point under conformal equivalence. A cusp is obtained by squaring an analytic curve having contact of order 1 with a line through the origin. We show that this point of view can be extended to the unfolding. This allows to reduce the classification of unfoldings of cusps to the classification of unfoldings of a pair of curves having a contact of order 1 at the origin, one being obtained from the other through a reflection with respect to the origin. This unfolding can be studied in the same way as an unfolding of a curvilinear angle with zero angle, called a horn. We then classify the unfoldings of the special horns corresponding to cusps by means of the associated diffeomorphisms. We interpret the results geometrically.

  20. The nucleocapsid protein of measles virus blocks host interferon response

    SciTech Connect

    Takayama, Ikuyo; Sato, Hiroki; Watanabe, Akira; Omi-Furutani, Mio; Sugai, Akihiro; Kanki, Keita; Yoneda, Misako; Kai, Chieko

    2012-03-01

    Measles virus (MV) belongs to the genus Morbillivirus of the family Paramyxoviridae. A number of paramyxoviruses inhibit host interferon (IFN) signaling pathways in host immune systems by various mechanisms. Inhibition mechanisms have been described for many paramyxoviruses. Although there are inconsistencies among previous reports concerning MV, it appears that P/V/C proteins interfere with the pathways. In this study, we confirmed the effects of MV P gene products of a wild MV strain on IFN pathways and examined that of other viral proteins on it. Interestingly, we found that N protein acts as an IFN-{alpha}/{beta} and {gamma}-antagonist as strong as P gene products. We further investigated the mechanisms of MV-N inhibition, and revealed that MV-N blocks the nuclear import of activated STAT without preventing STAT and Jak activation or STAT degradation, and that the nuclear translocation of MV-N is important for the inhibition. The inhibitory effect of the N protein was observed as a common feature of other morbilliviruses. The results presented in this report suggest that N protein of MV as well as P/V/C proteins is involved in the inhibition of host IFN signaling pathways.

  1. Autophagy proteins regulate innate immune response by inhibiting NALP3 inflammasome-mediated mitochondrial DNA release

    PubMed Central

    Nakahira, Kiichi; Haspel, Jeffrey Adam; Rathinam, Vijay AK; Lee, Seon-Jin; Dolinay, Tamas; Lam, Hilaire C; Englert, Joshua A; Rabinovitch, Marlene; Cernadas, Manuela; Kim, Hong Pyo; Fitzgerald, Katherine A; Ryter, Stefan W; Choi, Augustine MK

    2010-01-01

    Autophagy, a cellular process for organelle and protein turnover, regulates innate immune responses. We demonstrate that depletion of autophagic proteins microtubule associated protein-1 light chain 3B (LC3B) and Beclin 1 enhances caspase-1 activation and secretion of interleukin-1? and interleukin-18. Autophagic protein depletion promoted accumulation of dysfunctional mitochondria and cytosolic translocation of mitochondrial DNA (mtDNA) in response to lipopolysaccharide (LPS) and ATP in macrophages. Release of mtDNA into the cytosol depended on the NALP3 inflammasome and mitochondrial ROS. Cytosolic mtDNA contributed to IL-1? and IL-18 secretion in response to LPS and ATP. LC3B-deficient mice produced more caspase-1-dependent cytokines in two sepsis models and were susceptible to LPS-induced mortality. Our study suggests that autophagic proteins regulate NALP3-dependent inflammation by preserving mitochondrial integrity. PMID:21151103

  2. Mapping the energy landscape for second-stage folding of a single membrane protein.

    PubMed

    Min, Duyoung; Jefferson, Robert E; Bowie, James U; Yoon, Tae-Young

    2015-12-01

    Membrane proteins are designed to fold and function in a lipid membrane, yet folding experiments within a native membrane environment are challenging to design. Here we show that single-molecule forced unfolding experiments can be adapted to study helical membrane protein folding under native-like bicelle conditions. Applying force using magnetic tweezers, we find that a transmembrane helix protein, Escherichia coli rhomboid protease GlpG, unfolds in a highly cooperative manner, largely unraveling as one physical unit in response to mechanical tension above 25 pN. Considerable hysteresis is observed, with refolding occurring only at forces below 5 pN. Characterizing the energy landscape reveals only modest thermodynamic stability (?G = 6.5 kBT) but a large unfolding barrier (21.3 kBT) that can maintain the protein in a folded state for long periods of time (t1/2 ?3.5 h). The observed energy landscape may have evolved to limit the existence of troublesome partially unfolded states and impart rigidity to the structure. PMID:26479439

  3. Using the Heat-Shock Response To Discover Anticancer Compounds that Target Protein Homeostasis

    E-print Network

    Santagata, Sandro

    Unlike normal tissues, cancers experience profound alterations in protein homeostasis. Powerful innate adaptive mechanisms, especially the transcriptional response regulated by Heat Shock Factor 1 (HSF1), are activated in ...

  4. Native and Unfolded Cytochrome c – Comparison of Dynamics using 2D-IR Vibrational Echo Spectroscopy

    PubMed Central

    Kim, Seongheun; Chung, Jean K.; Kwak, Kyungwon; Bowman, Sarah E. J.; Bren, Kara L.; Bagchi, Biman; Fayer, M. D.

    2009-01-01

    Unfolded vs. native carbon monoxide coordinated horse heart cytochrome c (h-cyt c) and a heme axial methionine mutant cyt c552 from Hydrogenobacter thermophilus (Ht-M61A) are studied by IR absorption spectroscopy and ultrafast 2D-IR vibrational echo spectroscopy of the CO stretching mode. The unfolding is induced by guanidinium hydrochloride (GuHCl). The CO IR absorption spectra for both h-cyt c and Ht-M61A shift to the red as the GuHCl concentration is increased through the concentration region over which unfolding occurs. The spectra for the unfolded state are substantially broader than the spectra for the native proteins. A plot of the CO peak position vs. GuHCl concentration produces a sigmoidal curve that overlays the concentration dependent circular dichroism (CD) data of the CO-coordinated forms of both Ht-M61A and h-cyt c within experimental error. The coincidence of the CO peak shift curve with the CD curves demonstrates that the CO vibrational frequency is sensitive to the structural changes induced by the denaturant. 2D-IR vibrational echo experiments are performed on native Ht-M61A and on the protein in low and high concentration GuHCl solutions. The 2D-IR vibrational echo is sensitive to the global protein structural dynamics on time scales from sub-picosecond to greater than one hundred picoseconds through the change in the shape of the 2D spectrum with time (spectral diffusion). At the high GuHCl concentration (5.1 M), at which Ht-M61A is essentially fully denatured as judged by CD, a very large reduction in dynamics is observed compared to the native protein within the ~100 ps time window of the experiment. The results suggest the denatured protein may be in a glassy like state involving hydrophobic collapse around the heme. PMID:18646797

  5. Thermal stabilization of dihydrofolate reductase using monte carlo unfolding simulations and its functional consequences.

    PubMed

    Tian, Jian; Woodard, Jaie C; Whitney, Anna; Shakhnovich, Eugene I

    2015-04-01

    Design of proteins with desired thermal properties is important for scientific and biotechnological applications. Here we developed a theoretical approach to predict the effect of mutations on protein stability from non-equilibrium unfolding simulations. We establish a relative measure based on apparent simulated melting temperatures that is independent of simulation length and, under certain assumptions, proportional to equilibrium stability, and we justify this theoretical development with extensive simulations and experimental data. Using our new method based on all-atom Monte-Carlo unfolding simulations, we carried out a saturating mutagenesis of Dihydrofolate Reductase (DHFR), a key target of antibiotics and chemotherapeutic drugs. The method predicted more than 500 stabilizing mutations, several of which were selected for detailed computational and experimental analysis. We find a highly significant correlation of r=0.65-0.68 between predicted and experimentally determined melting temperatures and unfolding denaturant concentrations for WT DHFR and 42 mutants. The correlation between energy of the native state and experimental denaturation temperature was much weaker, indicating the important role of entropy in protein stability. The most stabilizing point mutation was D27F, which is located in the active site of the protein, rendering it inactive. However for the rest of mutations outside of the active site we observed a weak yet statistically significant positive correlation between thermal stability and catalytic activity indicating the lack of a stability-activity tradeoff for DHFR. By combining stabilizing mutations predicted by our method, we created a highly stable catalytically active E. coli DHFR mutant with measured denaturation temperature 7.2°C higher than WT. Prediction results for DHFR and several other proteins indicate that computational approaches based on unfolding simulations are useful as a general technique to discover stabilizing mutations. PMID:25905910

  6. Thermal Stabilization of Dihydrofolate Reductase Using Monte Carlo Unfolding Simulations and Its Functional Consequences

    PubMed Central

    Whitney, Anna; Shakhnovich, Eugene I.

    2015-01-01

    Design of proteins with desired thermal properties is important for scientific and biotechnological applications. Here we developed a theoretical approach to predict the effect of mutations on protein stability from non-equilibrium unfolding simulations. We establish a relative measure based on apparent simulated melting temperatures that is independent of simulation length and, under certain assumptions, proportional to equilibrium stability, and we justify this theoretical development with extensive simulations and experimental data. Using our new method based on all-atom Monte-Carlo unfolding simulations, we carried out a saturating mutagenesis of Dihydrofolate Reductase (DHFR), a key target of antibiotics and chemotherapeutic drugs. The method predicted more than 500 stabilizing mutations, several of which were selected for detailed computational and experimental analysis. We find a highly significant correlation of r = 0.65–0.68 between predicted and experimentally determined melting temperatures and unfolding denaturant concentrations for WT DHFR and 42 mutants. The correlation between energy of the native state and experimental denaturation temperature was much weaker, indicating the important role of entropy in protein stability. The most stabilizing point mutation was D27F, which is located in the active site of the protein, rendering it inactive. However for the rest of mutations outside of the active site we observed a weak yet statistically significant positive correlation between thermal stability and catalytic activity indicating the lack of a stability-activity tradeoff for DHFR. By combining stabilizing mutations predicted by our method, we created a highly stable catalytically active E. coli DHFR mutant with measured denaturation temperature 7.2°C higher than WT. Prediction results for DHFR and several other proteins indicate that computational approaches based on unfolding simulations are useful as a general technique to discover stabilizing mutations. PMID:25905910

  7. Multi-layered molecular mechanisms of polypeptide holding, unfolding and disaggregation by HSP70/HSP110 chaperones

    PubMed Central

    Finka, Andrija; Sharma, Sandeep K.; Goloubinoff, Pierre

    2015-01-01

    Members of the HSP70/HSP110 family (HSP70s) form a central hub of the chaperone network controlling all aspects of proteostasis in bacteria and the ATP-containing compartments of eukaryotic cells. The heat-inducible form HSP70 (HSPA1A) and its major cognates, cytosolic HSC70 (HSPA8), endoplasmic reticulum BIP (HSPA5), mitochondrial mHSP70 (HSPA9) and related HSP110s (HSPHs), contribute about 3% of the total protein mass of human cells. The HSP70s carry out a plethora of housekeeping cellular functions, such as assisting proper de novo folding, assembly and disassembly of protein complexes, pulling polypeptides out of the ribosome and across membrane pores, activating and inactivating signaling proteins and controlling their degradation. The HSP70s can induce structural changes in alternatively folded protein conformers, such as clathrin cages, hormone receptors and transcription factors, thereby regulating vesicular trafficking, hormone signaling and cell differentiation in development and cancer. To carry so diverse cellular housekeeping and stress-related functions, the HSP70s act as ATP-fuelled unfolding nanomachines capable of switching polypeptides between different folded states. During stress, the HSP70s can bind (hold) and prevent the aggregation of misfolding proteins and thereafter act alone or in collaboration with other unfolding chaperones to solubilize protein aggregates. Here, we discuss the common ATP-dependent mechanisms of holding, unfolding-by-clamping and unfolding-by-entropic pulling, by which the HSP70s can apparently convert various alternatively folded and misfolded polypeptides into differently active conformers. Understanding how HSP70s can prevent the formation of cytotoxic protein aggregates, pull, unfold, and solubilize them into harmless species is central to the design of therapies against protein conformational diseases. PMID:26097841

  8. Death-specific protein in a marine diatom regulates photosynthetic responses to iron and light availability

    E-print Network

    Death-specific protein in a marine diatom regulates photosynthetic responses to iron and light, using reverse genetics, that a death-specific protein (DSP; previously named for its apparent association with cell death) in the coastal diatom Thalassiosira pseudonana (TpDSP1) localizes to the plastid

  9. Protein carbon content evolves in response to carbon availability and may influence

    E-print Network

    Wagner, Andreas

    Protein carbon content evolves in response to carbon availability and may influence the fate that ancestral yeast strains preferentially express proteins with low carbon content during carbon limitation, relative to strains selected in the laboratory under carbon limitation. The likely reason

  10. Specific Nongluten Proteins of Wheat Are Novel Target Antigens in Celiac Disease Humoral Response

    PubMed Central

    2014-01-01

    While the antigenic specificity and pathogenic relevance of immunologic reactivity to gluten in celiac disease have been extensively researched, the immune response to nongluten proteins of wheat has not been characterized. We aimed to investigate the level and molecular specificity of antibody response to wheat nongluten proteins in celiac disease. Serum samples from patients and controls were screened for IgG and IgA antibody reactivity to a nongluten protein extract from the wheat cultivar Triticum aestivum Butte 86. Antibodies were further analyzed for reactivity to specific nongluten proteins by two-dimensional gel electrophoresis and immunoblotting. Immunoreactive molecules were identified by tandem mass spectrometry. Compared with healthy controls, patients exhibited significantly higher levels of antibody reactivity to nongluten proteins. The main immunoreactive nongluten antibody target proteins were identified as serpins, purinins, ?-amylase/protease inhibitors, globulins, and farinins. Assessment of reactivity toward purified recombinant proteins further confirmed the presence of antibody response to specific antigens. The results demonstrate that, in addition to the well-recognized immune reaction to gluten, celiac disease is associated with a robust humoral response directed at a specific subset of the nongluten proteins of wheat. PMID:25329597

  11. Viral Proteins That Bind Double-Stranded RNA: Countermeasures Against Host Antiviral Responses

    PubMed Central

    2014-01-01

    Several animal viruses encode proteins that bind double-stranded RNA (dsRNA) to counteract host dsRNA-dependent antiviral responses. This article discusses the structure and function of the dsRNA-binding proteins of influenza A virus and Ebola viruses (EBOVs). PMID:24905203

  12. Dynamics and unfolding pathway of chimeric azurin variants: insights from molecular dynamics simulation.

    PubMed

    Evoli, Stefania; Guzzi, Rita; Rizzuti, Bruno

    2013-10-01

    The spectroscopic, thermal, and functional properties of blue copper proteins can be modulated by mutations in the metal binding loop. Molecular dynamics simulation was used to compare the conformational properties of azurin and two chimeric variants, which were obtained by inserting into the azurin scaffold the copper binding loop of amicyanin and plastocyanin, respectively. Simulations at room temperature show that the proteins retain their overall structure and exhibit concerted motions among specific inner regions, as revealed by principal component analysis. Molecular dynamics at high temperature indicates that the first events in the unfolding pathway are structurally similar in the three proteins and unfolding starts from the region of the ?-helix that is far from the metal binding loop. The results provide details of the denaturation process that are consistent with experimental data and in close agreement with other computational approaches, suggesting a distinct mechanism of unfolding of azurin and its chimeric variants. Moreover, differences observed in the dynamics of specific regions in the three proteins correlate with their thermal behavior, contributing to the determination of the basic factors that influence the stability. PMID:23838900

  13. Tauroursodeoxycholic acid prevents stress induced aggregation of proteins in vitro and promotes PERK activation in HepG2 cells.

    PubMed

    Gani, Amina R; Uppala, Jagadeesh Kumar; Ramaiah, Kolluru V A

    2015-02-15

    Tauroursodeoxycholic acid (TUDCA) a bile salt and chemical chaperone reduces stress-induced aggregation of proteins; activates PERK [PKR (RNA-dependent protein kinase)-like ER (endoplasmic reticulum) kinase] or EIF2AK3, one of the hall marks of ER stress induced unfolded protein response (UPR) in human hepatoblastoma HepG2 cells; prevents heat and dithiothreitol (DTT) induced aggregation of BSA (bovine serum albumin), and reduces ANS (1-anilino-naphthalene-8-sulfonate) bound BSA fluorescence in vitro. TUDCA inactivates heat treated, but not the native EcoR1 enzyme, and reduces heat-induced aggregation and activity of COX-1 (cyclooxygenase enzyme-1) in vitro. These findings suggest that TUDCA binds to the hydrophobic regions of proteins and prevents their subsequent aggregation. This may stabilize unfolded proteins that can mount UPR or facilitate their degradation through cellular degradation pathways. PMID:25579883

  14. Dissecting FMR1, the protein responsible for fragile X syndrome, in its structural and functional domains.

    PubMed Central

    Adinolfi, S; Bagni, C; Musco, G; Gibson, T; Mazzarella, L; Pastore, A

    1999-01-01

    FMR1 is an RNA-binding protein that is either absent or mutated in patients affected by the fragile X syndrome, the most common inherited cause of mental retardation in humans. Sequence analysis of the FMR1 protein has suggested that RNA binding is related to the presence of two K-homologous (KH) modules and an RGG box. However, no attempt has been so far made to map the RNA-binding sites along the protein sequence and to identify possible differential RNA-sequence specificity. In the present article, we describe work done to dissect FMR1 into regions with structurally and functionally distinct properties. A semirational approach was followed to identify four regions: an N-terminal stretch of 200 amino acids, the two KH regions, and a C-terminal stretch. Each region was produced as a recombinant protein, purified, and probed for its state of folding by spectroscopical techniques. Circular dichroism and NMR spectra of the N-terminus show formation of secondary structure with a strong tendency to aggregate. Of the two homologous KH motifs, only the first one is folded whereas the second remains unfolded even when it is extended both N- and C-terminally. The C-terminus is, as expected from its amino acid composition, nonglobular. Binding assays were then performed using the 4-nt homopolymers. Our results show that only the first KH domain but not the second binds to RNA, and provide the first direct evidence for RNA binding of both the N-terminal and the C-terminal regions. RNA binding for the N-terminus could not be predicted from sequence analysis because no known RNA-binding motif is identifiable in this region. Different sequence specificity was observed for the fragments: both the N-terminus of the protein and KH1 bind preferentially to poly-(rG). The C-terminal region, which contains the RGG box, is nonspecific, as it recognizes the bases with comparable affinity. We therefore conclude that FMR1 is a protein with multiple sites of interaction with RNA: sequence specificity is most likely achieved by the whole block that comprises the first approximately 400 residues, whereas the C-terminus provides a nonspecific binding surface. PMID:10496225

  15. Mechanism of Fibrin(ogen) Forced Unfolding

    PubMed Central

    Zhmurov, Artem; Brown, Andre E.X.; Litvinov, Rustem I.; Dima, Ruxandra I.; Weisel, John W.; Barsegov, Valeri

    2012-01-01

    SUMMARY Fibrinogen, upon enzymatic conversion to monomeric fibrin, provides the building blocks for fibrin polymer, the scaffold of blood clots and thrombi. Little has been known about the force-induced unfolding of fibrin(ogen), even though it is the foundation for the mechanical and rheological properties of fibrin, which are essential for hemostasis. We determined mechanisms and mapped the free energy landscape of the elongation of fibrin(ogen) monomers and oligomers through combined experimental and theoretical studies of the nanomechanical properties of fibrin(ogen), using atomic force microscopy-based single-molecule unfolding and simulations in the experimentally relevant timescale. We have found that mechanical unraveling of fibrin(ogen) is determined by the combined molecular transitions that couple stepwise unfolding of the ? chain nodules and reversible extension-contraction of the ?-helical coiled-coil connectors. These findings provide important characteristics of the fibrin(ogen) nanomechanics necessary to understand the molecular origins of fibrin viscoelasticity at the fiber and whole clot levels. PMID:22078561

  16. Cardiac fiber unfolding by semidefinite programming.

    PubMed

    Li, Hongying; Robini, Marc C; Yang, Feng; Magnin, Isabelle; Zhu, Yuemin

    2015-02-01

    Diffusion-tensor imaging allows noninvasive assessment of the myocardial fiber architecture, which is fundamental in understanding the mechanics of the heart. In this context, tractography techniques are often used for representing and visualizing cardiac fibers, but their output is only qualitative. We introduce here a new framework toward a more quantitative description of the cardiac fiber architecture from tractography results. The proposed approach consists in taking three-dimensional (3-D) fiber tracts as inputs, and then unfolding these fibers in the Euclidean plane under local isometry constraints using semidefinite programming. The solution of the unfolding problem takes the form of a Gram matrix which defines the two-dimensional (2-D) embedding of the fibers and whose spectrum provides quantitative information on their organization. Experiments on synthetic and real data show that unfolding makes it easier to observe and to study the cardiac fiber architecture. Our conclusion is that 2-D embedding of cardiac fibers is a promising approach to supplement 3-D rendering for understanding the functioning of the heart. PMID:25291787

  17. Peanut proteins in periodate specific anion sensing: An ensuing reduction in allergic response.

    PubMed

    Ghatak, Sumanta Kumar; Sen, Souvik; Majumdar, Dipanwita; Singha, Achintya; Sen, Kamalika

    2016-04-15

    Peanut proteins conarachin II, conarachin I and arachin were found to behave as highly selective fluorescence sensors for periodate amongst a set of different anions. The interactions of the proteins with periodate were also confirmed by other spectral methods and enzyme linked immunosorbent assay (ELISA). The results indicate a selective interaction of peanut proteins with periodate amongst chloride, sulphate, iodide, phosphate, nitrate, nitrite, bromide, fluoride, persulphate, acetate, thiosulphate, arsenite, arsenate, sulphite, and iodide. Periodate sensing using different synthesized organic molecules are already reported in the literature. In this article we report the efficiency of peanut proteins as anion sensor which are bioactive and inexpensive too. The protein periodate interactions have also resulted in a simultaneous reduction in allergenicity of the peanut proteins. A change in the secondary structure of the protein was found responsible for this change which was further established with the help of circular dichroism and Raman spectroscopy. PMID:26675870

  18. Innate immune responses of immune proteins in juvenile channel catfish

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Channel catfish (Ictalurus punctatus) are raised for aquaculture in the Southeast U.S. and are susceptible to many bacterial and viral infections acquired from their pond environment. The innate immune proteins mannose-binding lectin (MBL) and lysozyme were studied during two consecutive years in ch...

  19. A Mutation in Caenorhabditis elegans NDUF-7 Activates the Mitochondrial Stress Response and Prolongs Lifespan via ROS and CED-4

    PubMed Central

    Rauthan, Manish; Ranji, Parmida; Abukar, Ragda; Pilon, Marc

    2015-01-01

    The mevalonate pathway is responsible for the synthesis of cholesterol, coenzyme Q, and prenyl groups essential for small GTPase modification and function, and for the production of dolichols important for protein glycosylation. Statins, i.e., cholesterol-lowering drugs that inhibit the rate-limiting enzyme in the mevalonate pathway, HMG-CoA reductase, are lethal to Caenorhabditis elegans even though this animal lacks the branch of the mevalonate pathway that leads to cholesterol synthesis. To better understand the effects of statins that are not related to cholesterol, we have adopted the strategy of isolating statin-resistant C. elegans mutants. Previously, we showed that such mutants often have gain-of-function mutations in ATFS-1, a protein that activates the mitochondrial unfolded protein response. Here, we describe the isolation of a statin-resistant mutant allele of the NDUF-7 protein, which is a component of complex I in the mitochondrial electron transport chain. The novel nduf-7(et19) mutant also exhibits constitutive and ATFS-1-dependent activation of the mitochondrial unfolded protein response (UPRmt) and prolonged life span, both of which are mediated through production of ROS. Additionally, lifespan extension, but not activation, of the mitochondrial unfolded protein response was dependent on the pro-apoptotic gene ced-4. We conclude that the nduf-7(et19) mutant allele causes an increase in reactive oxygen species that activate ATFS-1, hence UPRmt-mediated statin resistance, and extends life span via CED-4. PMID:26038366

  20. Highly precise quantification of protein molecules per cell during stress and starvation responses in Bacillus subtilis.

    PubMed

    Maa?, Sandra; Wachlin, Gerhild; Bernhardt, Jörg; Eymann, Christine; Fromion, Vincent; Riedel, Katharina; Becher, Dörte; Hecker, Michael

    2014-09-01

    Systems biology based on high quality absolute quantification data, which are mandatory for the simulation of biological processes, successively becomes important for life sciences. We provide protein concentrations on the level of molecules per cell for more than 700 cytosolic proteins of the Gram-positive model bacterium Bacillus subtilis during adaptation to changing growth conditions. As glucose starvation and heat stress are typical challenges in B. subtilis' natural environment and induce both, specific and general stress and starvation proteins, these conditions were selected as models for starvation and stress responses. Analyzing samples from numerous time points along the bacterial growth curve yielded reliable and physiologically relevant data suitable for modeling of cellular regulation under altered growth conditions. The analysis of the adaptational processes based on protein molecules per cell revealed stress-specific modulation of general adaptive responses in terms of protein amount and proteome composition. Furthermore, analysis of protein repartition during glucose starvation showed that biomass seems to be redistributed from proteins involved in amino acid biosynthesis to enzymes of the central carbon metabolism. In contrast, during heat stress most resources of the cell, namely those from amino acid synthetic pathways, are used to increase the amount of chaperones and proteases. Analysis of dynamical aspects of protein synthesis during heat stress adaptation revealed, that these proteins make up almost 30% of the protein mass accumulated during early phases of this stress. PMID:24878497

  1. Highly Precise Quantification of Protein Molecules per Cell During Stress and Starvation Responses in Bacillus subtilis *

    PubMed Central

    Maa?, Sandra; Wachlin, Gerhild; Bernhardt, Jörg; Eymann, Christine; Fromion, Vincent; Riedel, Katharina; Becher, Dörte; Hecker, Michael

    2014-01-01

    Systems biology based on high quality absolute quantification data, which are mandatory for the simulation of biological processes, successively becomes important for life sciences. We provide protein concentrations on the level of molecules per cell for more than 700 cytosolic proteins of the Gram-positive model bacterium Bacillus subtilis during adaptation to changing growth conditions. As glucose starvation and heat stress are typical challenges in B. subtilis' natural environment and induce both, specific and general stress and starvation proteins, these conditions were selected as models for starvation and stress responses. Analyzing samples from numerous time points along the bacterial growth curve yielded reliable and physiologically relevant data suitable for modeling of cellular regulation under altered growth conditions. The analysis of the adaptational processes based on protein molecules per cell revealed stress-specific modulation of general adaptive responses in terms of protein amount and proteome composition. Furthermore, analysis of protein repartition during glucose starvation showed that biomass seems to be redistributed from proteins involved in amino acid biosynthesis to enzymes of the central carbon metabolism. In contrast, during heat stress most resources of the cell, namely those from amino acid synthetic pathways, are used to increase the amount of chaperones and proteases. Analysis of dynamical aspects of protein synthesis during heat stress adaptation revealed, that these proteins make up almost 30% of the protein mass accumulated during early phases of this stress. PMID:24878497

  2. PROTEOMICS ANALYSIS OF OVEREXPRESSED PLASMA PROTEINS IN RESPONSE TO COLD ACCLIMATION IN Ostrinia furnacalis.

    PubMed

    Shang, Qingli; Pan, Yiou; Peng, Tianfei; Yang, Shuang; Lu, Xin; Wang, Zhenying; Xi, Jinghui

    2015-12-01

    Many insects in temperate regions overwinter in diapause. In these insects, one of the metabolic adaptations to cold stress is the synthesis of responsive proteins. Using proteomic analysis, an investigation aimed to a better understanding of the molecular adaptation mechanisms to cold stress was carried out in Ostrinia furnacalis larva. Proteins were extracted from the larval hemolymph collected from both control and overwintering larva. By polyethylene glycol precipitation, approximately 560 protein spots were separated and visualized on two-dimensional (2D) gels after silver staining. Eighteen protein spots were found to be upregulated in overwinter larval plasma in different patterns. As an initial work, 13 of these proteins were identified using MALDI TOF/TOF MS. The differentially overexpressed proteins include heat shock 70 kDa cognate protein, small heat shock protein (sHSP), putative aliphatic nitrilase, arginine kinase, phosphoglyceromutase, triosephosphateisomerase, and glutathione transferase. Alterations in the levels of these proteins were further confirmed by qPCR. This study is the first analysis of differentially expressed plasma proteins in O. furnacalis diapause larvae under extremely low temperature conditions and gives new insights into the acclimation mechanisms responsive to cold stress. Our results also support the idea that energy metabolism, alanine and proline metabolism, and antioxidative reaction act in the cold acclimation of O. furnacalis diapause larvae. PMID:26440752

  3. Thermally responsive silicon nanowire arrays for native/denatured-protein separation

    NASA Astrophysics Data System (ADS)

    Wang, Hongwei; Wang, Yanwei; Yuan, Lin; Wang, Lei; Yang, Weikang; Wu, Zhaoqiang; Li, Dan; Chen, Hong

    2013-03-01

    We present our findings of the selective adsorption of native and denatured proteins onto thermally responsive, native-protein resistant poly(N-isopropylacrylamide) (PNIPAAm) decorated silicon nanowire arrays (SiNWAs). The PNIPAAm-SiNWAs surface, which shows very low levels of native-protein adsorption, favors the adsorption of denatured proteins. The amount of denatured-protein adsorption is higher at temperatures above the lower critical solution temperature (LCST) of PNIPAAm. Temperature cycling surrounding the LCST, which ensures against thermal denaturation of native proteins, further increases the amount of denatured-protein adsorption. Moreover, the PNIPAAm-SiNWAs surface is able to selectively adsorb denatured protein even from mixtures of different protein species; meanwhile, the amount of native proteins in solution is kept nearly at its original level. It is believed that these results will not only enrich current understanding of protein interactions with PNIPAAm-modified SiNWAs surfaces, but may also stimulate applications of PNIPAAm-SiNWAs surfaces for native/denatured protein separation.

  4. Dynamic heterogeneity in the folding/unfolding transitions of FiP35

    NASA Astrophysics Data System (ADS)

    Mori, Toshifumi; Saito, Shinji

    2015-04-01

    Molecular dynamics simulations have become an important tool in studying protein dynamics over the last few decades. Atomistic simulations on the order of micro- to milliseconds are becoming feasible and are used to study the state-of-the-art experiments in atomistic detail. Yet, analyzing the high-dimensional-long-temporal trajectory data is still a challenging task and sometimes leads to contradictory results depending on the analyses. To reveal the dynamic aspect of the trajectory, here we propose a simple approach which uses a time correlation function matrix and apply to the folding/unfolding trajectory of FiP35 WW domain [Shaw et al., Science 330, 341 (2010)]. The approach successfully characterizes the slowest mode corresponding to the folding/unfolding transitions and determines the free energy barrier indicating that FiP35 is not an incipient downhill folder. The transition dynamics analysis further reveals that the folding/unfolding transition is highly heterogeneous, e.g., the transition path time varies by ˜100 fold. We identify two misfolded states and show that the dynamic heterogeneity in the folding/unfolding transitions originates from the trajectory being trapped in the misfolded and half-folded intermediate states rather than the diffusion driven by a thermal noise. The current results help reconcile the conflicting interpretations of the folding mechanism and highlight the complexity in the folding dynamics. This further motivates the need to understand the transition dynamics beyond a simple free energy picture using simulations and single-molecule experiments.

  5. Dynamic heterogeneity in the folding/unfolding transitions of FiP35

    SciTech Connect

    Mori, Toshifumi Saito, Shinji

    2015-04-07

    Molecular dynamics simulations have become an important tool in studying protein dynamics over the last few decades. Atomistic simulations on the order of micro- to milliseconds are becoming feasible and are used to study the state-of-the-art experiments in atomistic detail. Yet, analyzing the high-dimensional-long-temporal trajectory data is still a challenging task and sometimes leads to contradictory results depending on the analyses. To reveal the dynamic aspect of the trajectory, here we propose a simple approach which uses a time correlation function matrix and apply to the folding/unfolding trajectory of FiP35 WW domain [Shaw et al., Science 330, 341 (2010)]. The approach successfully characterizes the slowest mode corresponding to the folding/unfolding transitions and determines the free energy barrier indicating that FiP35 is not an incipient downhill folder. The transition dynamics analysis further reveals that the folding/unfolding transition is highly heterogeneous, e.g., the transition path time varies by ?100 fold. We identify two misfolded states and show that the dynamic heterogeneity in the folding/unfolding transitions originates from the trajectory being trapped in the misfolded and half-folded intermediate states rather than the diffusion driven by a thermal noise. The current results help reconcile the conflicting interpretations of the folding mechanism and highlight the complexity in the folding dynamics. This further motivates the need to understand the transition dynamics beyond a simple free energy picture using simulations and single-molecule experiments.

  6. Temperature induced structural transitions from native to unfolded aggregated states of tobacco etch virus protease

    NASA Astrophysics Data System (ADS)

    Zhu, Guo-Fei; Ren, Si-Yan; Xi, Lei; Du, Lin-Fang; Zhu, Xiao-Feng

    2015-02-01

    Tobacco etch virus protease (TEVp) is widely used to remove fusion tags from recombinant proteins because of its high and unique specificity. This work describes the conformational and the thermodynamic properties in the unfolding/refolding process of TEVp3M (three-point mutant: L56V/S135G/S219V) induced by temperature. With temperature increasing from 20 to 100 °C, the CD spectra showed a transition trend from ?-helix to ?-sheet, and the fluorescence emission, synchronous fluorescence, ANS and RLS spectroscopy consistently revealed that the temperature-induced unfolding process behaved in a three-state manner, for there was a relatively stable intermediate state observed around 50 °C. The reversibility of thermal unfolding of TEVp3M further showed that the transition from the native to the intermediate state was reversible (below 50 °C), however the transition from the intermediate to the unfolded state was irreversible (above 60 °C). Moreover, aggregates were observed above 60 °C as revealed by SDS-PAGE, Thioflavin-T fluorescence and Congo red absorbance.

  7. ATP-dependent G-quadruplex unfolding by Bloom helicase exhibits low processivity

    PubMed Central

    Budhathoki, Jagat B.; Stafford, Edward J.; Yodh, Jaya G.; Balci, Hamza

    2015-01-01

    Various helicases and single stranded DNA (ssDNA) binding proteins unfold G-quadruplex (GQ) structures. However, the underlying mechanisms of this activity have only recently come to focus. We report kinetic studies on Bloom (BLM) helicase and human telomeric GQ interactions using single-molecule Förster resonance energy transfer (smFRET). Using partial duplex DNA (pdDNA) constructs with different 5? ssDNA overhangs, we show that BLM localizes in the vicinity of ssDNA/double-stranded DNA (dsDNA) junction and reels in the ssDNA overhang in an ATP-dependent manner. A comparison of DNA constructs with or without GQ in the overhang shows that GQ unfolding is achieved in 50–70% of reeling attempts under physiological salt and pH conditions. The unsuccessful attempts often result in dissociation of BLM from DNA which slows down the overall BLM activity. BLM-mediated GQ unfolding is typically followed by refolding of the GQ, a pattern that is repeated several times before BLM dissociates from DNA. BLM is significantly less processive compared to the highly efficient GQ destabilizer Pif1 that can repeat GQ unfolding activity hundreds of times before dissociating from DNA. Despite the variations in processivity, our studies point to possible common patterns used by different helicases in minimizing the duration of stable GQ formation. PMID:25990739

  8. Regulating the ethylene response of a plant by modulation of F-box proteins

    SciTech Connect

    Guo, Hongwei; Ecker, Joseph R.

    2011-03-08

    The invention relates to transgenic plants having reduced sensitivity to ethylene as a result of having a recombinant nucleic acid encoding an F-box protein that interacts with a EIN3 involved in an ethylene response of plants, and a method of producing a transgenic plant with reduced ethylene sensitivity by transforming the plant with a nucleic acid sequence encoding an F-box protein. The inventions also relates to methods of altering the ethylene response in a plant by modulating the activity or expression of an F-box protein.

  9. The non-uniform early structural response of globular proteins to cold denaturing conditions: A case study with Yfh1

    SciTech Connect

    Chatterjee, Prathit; Bagchi, Sayan E-mail: s.bagchi@ncl.res.in; Sengupta, Neelanjana E-mail: s.bagchi@ncl.res.in

    2014-11-28

    The mechanism of cold denaturation in proteins is often incompletely understood due to limitations in accessing the denatured states at extremely low temperatures. Using atomistic molecular dynamics simulations, we have compared early (nanosecond timescale) structural and solvation properties of yeast frataxin (Yfh1) at its temperature of maximum stability, 292 K (T{sub s}), and the experimentally observed temperature of complete unfolding, 268 K (T{sub c}). Within the simulated timescales, discernible “global” level structural loss at T{sub c} is correlated with a distinct increase in surface hydration. However, the hydration and the unfolding events do not occur uniformly over the entire protein surface, but are sensitive to local structural propensity and hydrophobicity. Calculated infrared absorption spectra in the amide-I region of the whole protein show a distinct red shift at T{sub c} in comparison to T{sub s}. Domain specific calculations of IR spectra indicate that the red shift primarily arises from the beta strands. This is commensurate with a marked increase in solvent accessible surface area per residue for the beta-sheets at T{sub c}. Detailed analyses of structure and dynamics of hydration water around the hydrophobic residues of the beta-sheets show a more bulk water like behavior at T{sub c} due to preferential disruption of the hydrophobic effects around these domains. Our results indicate that in this protein, the surface exposed beta-sheet domains are more susceptible to cold denaturing conditions, in qualitative agreement with solution NMR experimental results.

  10. ECM Protein Nanofibers and Nanostructures Engineered Using Surface-initiated Assembly

    PubMed Central

    Szymanski, John M.; Jallerat, Quentin; Feinberg, Adam W.

    2014-01-01

    The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo. PMID:24799041

  11. ECM protein nanofibers and nanostructures engineered using surface-initiated assembly.

    PubMed

    Szymanski, John M; Jallerat, Quentin; Feinberg, Adam W

    2014-01-01

    The extracellular matrix (ECM) in tissues is synthesized and assembled by cells to form a 3D fibrillar, protein network with tightly regulated fiber diameter, composition and organization. In addition to providing structural support, the physical and chemical properties of the ECM play an important role in multiple cellular processes including adhesion, differentiation, and apoptosis. In vivo, the ECM is assembled by exposing cryptic self-assembly (fibrillogenesis) sites within proteins. This process varies for different proteins, but fibronectin (FN) fibrillogenesis is well-characterized and serves as a model system for cell-mediated ECM assembly. Specifically, cells use integrin receptors on the cell membrane to bind FN dimers and actomyosin-generated contractile forces to unfold and expose binding sites for assembly into insoluble fibers. This receptor-mediated process enables cells to assemble and organize the ECM from the cellular to tissue scales. Here, we present a method termed surface-initiated assembly (SIA), which recapitulates cell-mediated matrix assembly using protein-surface interactions to unfold ECM proteins and assemble them into insoluble fibers. First, ECM proteins are adsorbed onto a hydrophobic polydimethylsiloxane (PDMS) surface where they partially denature (unfold) and expose cryptic binding domains. The unfolded proteins are then transferred in well-defined micro- and nanopatterns through microcontact printing onto a thermally responsive poly(N-isopropylacrylamide) (PIPAAm) surface. Thermally-triggered dissolution of the PIPAAm leads to final assembly and release of insoluble ECM protein nanofibers and nanostructures with well-defined geometries. Complex architectures are possible by engineering defined patterns on the PDMS stamps used for microcontact printing. In addition to FN, the SIA process can be used with laminin, fibrinogen and collagens type I and IV to create multi-component ECM nanostructures. Thus, SIA can be used to engineer ECM protein-based materials with precise control over the protein composition, fiber geometry and scaffold architecture in order to recapitulate the structure and composition of the ECM in vivo. PMID:24799041

  12. Priming of protein expression in the defence response of Zantedeschia aethiopica to Pectobacterium carotovorum.

    PubMed

    Luzzatto-Knaan, Tal; Kerem, Zohar; Doron-Faigenboim, Adi; Yedidia, Iris

    2014-05-01

    The defence response of Zantedeschia aethiopica, a natural rhizomatous host of the soft rot bacterium Pectobacterium carotovorum, was studied following the activation of common induced resistance pathways—systemic acquired resistance and induced systemic resistance. Proteomic tools were used, together with in?vitro quantification and in?situ localization of selected oxidizing enzymes. In total, 527 proteins were analysed by label-free mass spectrometry (MS) and annotated against the National Center for Biotechnology Information (NCBI) nonredundant (nr) protein database of rice (Oryza sativa). Of these, the fore most differentially expressed group comprised 215 proteins that were primed following application of methyl jasmonate (MJ) and subsequent infection with the pathogen. Sixty-five proteins were down-regulated following MJ treatments. The application of benzothiadiazole (BTH) increased the expression of 23 proteins; however, subsequent infection with the pathogen repressed their expression and did not induce priming. The sorting of primed proteins by Gene Ontology protein function category revealed that the primed proteins included nucleic acid-binding proteins, cofactor-binding proteins, ion-binding proteins, transferases, hydrolases and oxidoreductases. In line with the highlighted involvement of oxidoreductases in the defence response, we determined their activities, priming pattern and localization in?planta. Increased activities were confined to the area surrounding the pathogen penetration site, associating these enzymes with the induced systemic resistance afforded by the jasmonic acid signalling pathway. The results presented here demonstrate the concerted priming of protein expression following MJ treatment, making it a prominent part of the defence response of Z.?aethiopica to P.?carotovorum. PMID:24822269

  13. A Comparison of the LR and DFIT Frameworks of Differential Functioning Applied to the Generalized Graded Unfolding Model

    ERIC Educational Resources Information Center

    Carter, Nathan T.; Zickar, Michael J.

    2011-01-01

    Recently, applied psychological measurement researchers have become interested in the application of the generalized graded unfolding model (GGUM), a parametric item response theory model that posits an ideal point conception of the relationship between latent attributes and observed item responses. Little attention has been given to…

  14. Surface Plasmon Resonance Imaging of Transcription Factor Proteins: Interactions of Bacterial Response

    E-print Network

    sequences of known DNA binding regions for two response regulators: OmpR, which controls gene expression that underlies their role in the control of gene expression. TraditionalmethodsthathavebeenusedtoanalyzeDNA the sequence specific binding of these two response regulator proteins to the DNA arrays. The promoter regions

  15. Microbial response to single-cell protein production and brewery wastewater treatment

    E-print Network

    Microbial response to single-cell protein production and brewery wastewater treatment Jackson Z in wastewater bioreactors. In this study, we investigate microbial response across a full-scale brewery wastewater treatment plant and a parallel pilot bioreactor modified to produce an SCP product

  16. Interaction network of proteins associated with abiotic stress response and development in wheat

    E-print Network

    Sarhan, Fathey

    Interaction network of proteins associated with abiotic stress response and development in wheat online: 9 January 2007 Ó Springer Science+Business Media B.V. 2007 Abstract Wheat is the most widely associated with abi- otic stress response and development in wheat was generated using the yeast two

  17. Inversion of aerotactic response in Escherichia coli deficient in cheB protein methylesterase.

    PubMed Central

    Dang, C V; Niwano, M; Ryu, J; Taylor, B L

    1986-01-01

    Mutants of Escherichia coli and Salmonella typhimurium that were deficient in protein methylesterase activity encoded by cheB had an inverted response to oxygen; they were repelled by concentrations of oxygen that attract wild-type bacteria. Normal responses to oxygen and phosphotransferase substrates were observed in mutants that were deficient in protein methyltransferase (CheR) and the methyl-accepting transducing proteins (Tsr, Tar, Trg). However, the methylation-independent response to oxygen was modified by the loss of esterase activity. The inversion was apparently effected by the amidated Tsr protein present in cheB tsr+ mutants because aerotaxis was normal in cheB tsr strains. Chemotaxis to phosphotransferase sugars was normal in cheB mutants provided the extreme clockwise bias of the flagellar motors was modified to increase the probability of counterclockwise rotation. PMID:3007436

  18. Understanding the Structural Ensembles of a Highly Extended Disordered Protein

    PubMed Central

    Daughdrill, Gary W.; Kashtanov, Stepan; Stancik, Amber; Hill, Shannon E.; Helms, Gregory; Muschol, Martin

    2013-01-01

    Developing a comprehensive description of the equilibrium structural ensembles for intrinsically disordered proteins (IDPs) is essential to understanding their function. The p53 transactivation domain (p53TAD) is an IDP that interacts with multiple protein partners and contains numerous phosphorylation sites. Multiple techniques were used to investigate the equilibrium structural ensemble of p53TAD in its native and chemically unfolded states. The results from these experiments show that the native state of p53TAD has dimensions similar to a classical random coil while the chemically unfolded state is more extended. To investigate the molecular properties responsible for this behavior, a novel algorithm that generates diverse and unbiased structural ensembles of IDPs was developed. This algorithm was used to generate a large pool of plausible p53TAD structures that were reweighted to identify a subset of structures with the best fit to small angle X-ray scattering data. High weight structures in the native state ense