Sample records for unfolded protein response

  1. The unfolded-protein-response

    E-print Network

    Walter, Peter

    The unfolded- protein-response pathway in yeast The accumulation of unfolded proteins in the endoplasmic reticulum (ER) triggers the increased production of several ER- resident proteins. This signalling in structure to growth-factor receptorkinases has been identified as a key component of the unfolded-protein

  2. Unfolded Protein Responses With or Without Unfolded Proteins?

    PubMed Central

    Snapp, Erik L.

    2012-01-01

    The endoplasmic reticulum (ER) is the site of secretory protein biogenesis. The ER quality control (QC) machinery, including chaperones, ensures the correct folding of secretory proteins. Mutant proteins and environmental stresses can overwhelm the available QC machinery. To prevent and resolve accumulation of misfolded secretory proteins in the ER, cells have evolved integral membrane sensors that orchestrate the Unfolded Protein Response (UPR). The sensors, Ire1p in yeast and IRE1, ATF6, and PERK in metazoans, bind the luminal ER chaperone BiP during homeostasis. As unfolded secretory proteins accumulate in the ER lumen, BiP releases, and the sensors activate. The mechanisms of activation and attenuation of the UPR sensors have exhibited unexpected complexity. A growing body of data supports a model in which Ire1p, and potentially IRE1, directly bind unfolded proteins as part of the activation process. However, evidence for an unfolded protein-independent mechanism has recently emerged, suggesting that UPR can be activated by multiple modes. Importantly, dysregulation of the UPR has been linked to human diseases including Type II diabetes, heart disease, and cancer. The existence of alternative regulatory pathways for UPR sensors raises the exciting possibility for the development of new classes of therapeutics for these medically important proteins. PMID:24710536

  3. Proteostasis control by the unfolded protein response.

    PubMed

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

    2015-06-30

    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

  4. Unfolding the Unfolded Protein Response: Unique Insights into Brain Ischemia

    PubMed Central

    Sanderson, Thomas H.; Gallaway, Molly; Kumar, Rita

    2015-01-01

    The endoplasmic reticulum (ER) is responsible for processing of proteins that are destined to be secreted, enclosed in a vesicle, or incorporated in the plasma membrane. Nascent peptides that enter the ER undergo a series of highly regulated processing steps to reach maturation as they transit the ER. Alterations in the intracellular environment that induce ER stress are thought to interrupt these processing steps, and result in unfolding of proteins in the ER. Accumulation of unfolded proteins concurrently activates three transmembrane stress sensors, IRE1, ATF6 and PERK, and is referred to as the Unfolded Protein Response (UPR). Our understanding of the mechanisms of UPR induction has been assembled primarily from experiments inducing ER stress with chemical and genetic manipulations. However, physiological stress often induces activation of ER stress sensors in a distinct manner from the canonical UPR. The unique activation profiles in vivo have prompted us to examine the mechanism of UPR activation in neurons following cerebral ischemia. PMID:25830481

  5. Cell Metabolism The Unfolded Protein Response

    E-print Network

    Campbell, Kevin P.

    Cell Metabolism Article The Unfolded Protein Response Mediates Adaptation to Exercise in Skeletal that maintains ER homeostasis upon luminal stress, is activated in skeletal muscle during exercise and adapts skeletal muscle to exercise training. The transcriptional coac- tivator PGC-1a, which regulates several

  6. The Unfolded Protein Response and Diabetic Retinopathy

    PubMed Central

    Wang, Josh J.; Zhang, Sarah X.

    2014-01-01

    Diabetic retinopathy, a common complication of diabetes, is the leading cause of blindness in adults. Diabetes chronically damages retinal blood vessels and neurons likely through multiple pathogenic pathways such as oxidative stress, inflammation, and endoplasmic reticulum (ER) stress. To relieve ER stress, the cell activates an adaptive mechanism known as the unfolded protein response (UPR). The UPR coordinates the processes of protein synthesis, protein folding, and degradation to ensure proteostasis, which is vital for cell survival and activity. Emerging evidence suggests that diabetes can activate all three UPR branches in retinal cells, among which the PERK/ATF4 pathway is the most extensively studied in the development of diabetic retinopathy. X-box binding protein 1 (XBP1) is a major transcription factor in the core UPR pathway and also regulates a variety of genes involved in cellular metabolism, redox state, autophagy, inflammation, cell survival, and vascular function. The exact function and implication of XBP1 in the pathogenesis of diabetic retinopathy remain elusive. Focusing on this less studied pathway, we summarize recent progress in studies of the UPR pertaining to diabetic changes in retinal vasculature and neurons, highlighting the perspective of XBP1 as a potential therapeutic target in diabetic retinopathy. PMID:25530974

  7. The unfolded protein response and diabetic retinopathy.

    PubMed

    Ma, Jacey Hongjie; Wang, Josh J; Zhang, Sarah X

    2014-01-01

    Diabetic retinopathy, a common complication of diabetes, is the leading cause of blindness in adults. Diabetes chronically damages retinal blood vessels and neurons likely through multiple pathogenic pathways such as oxidative stress, inflammation, and endoplasmic reticulum (ER) stress. To relieve ER stress, the cell activates an adaptive mechanism known as the unfolded protein response (UPR). The UPR coordinates the processes of protein synthesis, protein folding, and degradation to ensure proteostasis, which is vital for cell survival and activity. Emerging evidence suggests that diabetes can activate all three UPR branches in retinal cells, among which the PERK/ATF4 pathway is the most extensively studied in the development of diabetic retinopathy. X-box binding protein 1 (XBP1) is a major transcription factor in the core UPR pathway and also regulates a variety of genes involved in cellular metabolism, redox state, autophagy, inflammation, cell survival, and vascular function. The exact function and implication of XBP1 in the pathogenesis of diabetic retinopathy remain elusive. Focusing on this less studied pathway, we summarize recent progress in studies of the UPR pertaining to diabetic changes in retinal vasculature and neurons, highlighting the perspective of XBP1 as a potential therapeutic target in diabetic retinopathy. PMID:25530974

  8. Signal integration in the endoplasmic reticulum unfolded protein response

    Microsoft Academic Search

    David Ron; Peter Walter

    2007-01-01

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

  9. The Unfolded Protein Response and Gastrointestinal Disease

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  11. Endoplasmic Reticulum Stress: Signaling the Unfolded Protein Response

    NSDL National Science Digital Library

    2007-06-01

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

  12. Virus-induced ER stress and the unfolded protein response

    PubMed Central

    Zhang, Lingrui; Wang, Aiming

    2012-01-01

    The accumulation of unfolded or misfolded proteins in the lumen of the endoplasmic reticulum (ER) results in ER stress that triggers cytoprotective signaling pathways, termed the unfolded protein response (UPR), to restore and maintain homeostasis in the ER or to induce apoptosis if ER stress remains unmitigated. The UPR signaling network encompasses three core elements, i.e., PKR-like ER kinase (PERK), activating transcription factor 6 (ATF6), and inositol-requiring protein-1 (IRE1). Activation of these three branch pathways of the UPR leads to the translation arrest and degradation of misfolded proteins, the expression of ER molecular chaperones, and the expansion of the ER membrane to decrease the load of proteins and increase the protein-folding capacity in the ER. Recently, the essential roles of the UPR have been implicated in a number of mammalian diseases, particularly viral diseases. In virus-infected cells, the cellular translation machinery is hijacked by the infecting virus to produce large amounts of viral proteins, which inevitably perturbs ER homeostasis and causes ER stress. This review summarizes current knowledge about the UPR signaling pathways, highlights two identified UPR pathways in plants, and discuss progress in elucidating the UPR in virus-infected cells and its functional roles in viral infection. PMID:23293645

  13. Targeting unfolded protein response in cancer and diabetes.

    PubMed

    Mkrtchian, Souren

    2015-06-01

    The maturation of secretory and membrane proteins in the endoplasmic reticulum (ER) is tightly regulated by the unfolded protein response (UPR), a signal transduction pathway maintaining ER protein folding homeostasis. However, certain ER states are incompatible with cell survival and therefore the UPR may choose to eliminate severely disrupted cells by apoptosis. This is accomplished primarily through the activation of the transcription factor CCAAT-enhancer-binding protein homologous protein (CHOP). In the April 2015 issue of Endocrine-Related Cancer, researchers from the universities of South Carolina and Athens (Greece) suggested a novel mechanism of CHOP-mediated apoptosis connected with the suppression of a prominent cell cycle regulator with anti-apoptotic activity, p21. These findings and suggested clinical applications, such as potentiation of cancer chemotherapy and a novel therapeutic approach for type 2 diabetes, are discussed in the context of UPR. PMID:25792543

  14. Multiple Mechanisms of Unfolded Protein Response-Induced Cell Death.

    PubMed

    Hiramatsu, Nobuhiko; Chiang, Wei-Chieh; Kurt, Timothy D; Sigurdson, Christina J; Lin, Jonathan H

    2015-07-01

    Eukaryotic cells fold and assemble membrane and secreted proteins in the endoplasmic reticulum (ER), before delivery to other cellular compartments or the extracellular environment. Correctly folded proteins are released from the ER, and poorly folded proteins are retained until they achieve stable conformations; irreparably misfolded proteins are targeted for degradation. Diverse pathological insults, such as amino acid mutations, hypoxia, or infection, can overwhelm ER protein quality control, leading to misfolded protein buildup, causing ER stress. To cope with ER stress, eukaryotic cells activate the unfolded protein response (UPR) by increasing levels of ER protein-folding enzymes and chaperones, enhancing the degradation of misfolded proteins, and reducing protein translation. In mammalian cells, three ER transmembrane proteins, inositol-requiring enzyme-1 (IRE1; official name ERN1), PKR-like ER kinase (PERK; official name EIF2AK3), and activating transcription factor-6, control the UPR. The UPR signaling triggers a set of prodeath programs when the cells fail to successfully adapt to ER stress or restore homeostasis. ER stress and UPR signaling are implicated in the pathogenesis of diverse diseases, including neurodegeneration, cancer, diabetes, and inflammation. This review discusses the current understanding in both adaptive and apoptotic responses as well as the molecular mechanisms instigating apoptosis via IRE1 and PERK signaling. We also examine how IRE1 and PERK signaling may be differentially used during neurodegeneration arising in retinitis pigmentosa and prion infection. PMID:25956028

  15. REGULATION OF THE UNFOLDED PROTEIN RESPONSE BY MICRORNAS

    PubMed Central

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

    2013-01-01

    The unfolded protein response (UPR) is an adaptive response to stress that is caused by an accumulation of misfolded proteins in the lumen of endoplasmic reticulum (ER) and is therefore an important component of cellular homeostasis. During ER stress, the UPR increases the protein folding capacity of the endoplasmic reticulum to relieve the stress, and failure to recover leads to apoptosis. Specific cellular mechanisms, therefore, are required for the cellular recovery phase after UPR activation. In this review, we discuss the potential role of microRNAs as key regulators of this pathway. Using bioinformatics, we identified a number of microRNAs that are predicted to decrease the mRNA expression levels for a number of critical components of the UPR and discuss how microRNAs may play an essential role in turning off the UPR after the stress has subsided. PMID:24092331

  16. Unfolded protein response in hepatitis C virus infection

    PubMed Central

    Chan, Shiu-Wan

    2014-01-01

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

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

    Microsoft Academic Search

    Shiu-Wan Chan; Philip Anthony Egan

    2005-01-01

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

  18. Unfolded protein response in filamentous fungi-implications in biotechnology.

    PubMed

    Heimel, Kai

    2015-01-01

    The unfolded protein response (UPR) represents a mechanism to preserve endoplasmic reticulum (ER) homeostasis that is conserved in eukaryotes. ER stress caused by the accumulation of potentially toxic un- or misfolded proteins in the ER triggers UPR activation and the induction of genes important for protein folding in the ER, ER expansion, and transport from and to the ER. Along with this adaptation, the overall capacity for protein secretion is markedly increased by the UPR. In filamentous fungi, various approaches to employ the UPR for improved production of homologous and heterologous proteins have been investigated. As the effects on protein production were strongly dependent on the expressed protein, generally applicable strategies have to be developed. A combination of transcriptomic approaches monitoring secretion stress and basic research on the UPR mechanism provided novel and important insight into the complex regulatory cross-connections between UPR signalling, cellular physiology, and developmental processes. It will be discussed how this increasing knowledge on the UPR might stimulate the development of novel strategies for using the UPR as a tool in biotechnology. PMID:25384707

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

    Microsoft Academic Search

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

    1999-01-01

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

  20. The mitochondrial unfolded protein response in mammalian physiology.

    PubMed

    Mottis, Adrienne; Jovaisaite, Virginija; Auwerx, Johan

    2014-10-01

    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, which encodes 13 subunits of the oxidative phosphorylation 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, a 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 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 resolve the local stress but also can have long-lasting effects on whole body metabolism, fitness, and longevity. A delicate tuning of its activation levels might constitute a treatment of various diseases, such as metabolic diseases, cancer, and neurodegenerative disorders. PMID:24898297

  1. The unfolded protein response is required for dendrite morphogenesis

    PubMed Central

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

    2015-01-01

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

  2. 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

  3. 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

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

    Microsoft Academic Search

    Claudio Hetz

    2012-01-01

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

  5. The Unfolded Protein Response Triggers Site-Specific Regulatory Ubiquitylation of 40S Ribosomal Proteins.

    PubMed

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

    2015-07-01

    Insults to ER homeostasis activate the unfolded protein response (UPR), which elevates protein folding and degradation capacity and attenuates protein synthesis. While a role for ubiquitin in regulating the degradation of misfolded ER-resident proteins is well described, ubiquitin-dependent regulation of translational reprogramming during the UPR remains uncharacterized. Using global quantitative ubiquitin proteomics, we identify evolutionarily conserved, site-specific regulatory ubiquitylation of 40S ribosomal proteins. We demonstrate that these events occur on assembled cytoplasmic ribosomes and are stimulated by both UPR activation and translation inhibition. We further show that ER stress-stimulated regulatory 40S ribosomal ubiquitylation occurs on a timescale similar to eIF2? phosphorylation, is dependent upon PERK signaling, and is required for optimal cell survival during chronic UPR activation. In total, these results reveal regulatory 40S ribosomal ubiquitylation as an important facet of eukaryotic translational control. PMID:26051182

  6. Translational Control Is Required for the Unfolded Protein Response and In Vivo Glucose Homeostasis

    Microsoft Academic Search

    Donalyn Scheuner; Benbo Song; Edward McEwen; Chuan Liu; Ross Laybutt; Patrick Gillespie; Thom Saunders; Susan Bonner-Weir; Randal J Kaufman

    2001-01-01

    The accumulation of unfolded protein in the endoplasmic reticulum (ER) attenuates protein synthesis initiation through phosphorylation of the ? subunit of eukaryotic translation initiation factor 2 (eIF2?) at Ser51. Subsequently, transcription of genes encoding adaptive functions including the glucose-regulated proteins is induced. We show that eIF2? phosphorylation is required for translation attenuation, transcriptional induction, and survival in response to ER

  7. West Nile Virus Infection Activates the Unfolded Protein Response, Leading to CHOP Induction and Apoptosis

    Microsoft Academic Search

    Guruprasad R. Medigeshi; Alissa M. Lancaster; Alec J. Hirsch; Thomas Briese; W. Ian Lipkin; Victor DeFilippis; Klaus Fruh; Peter W. Mason; Janko Nikolich-Zugich; Jay A. Nelson

    2007-01-01

    West Nile virus (WNV)-mediated neuronal death is a hallmark of WNV meningitis and encephalitis. However, the mechanisms of WNV-induced neuronal damage are not well understood. We investigated WNV neuropathogenesis by using human neuroblastoma cells and primary rat hippocampal neurons. We observed that WNV activates multiple unfolded protein response (UPR) pathways, leading to transcrip- tional and translational induction of UPR target

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

    PubMed Central

    Mao, Xianrong R.; Crowder, C. Michael

    2010-01-01

    Prolonged cellular hypoxia results in energy failure and ultimately cell death. However, less-severe hypoxia can induce a cytoprotective response termed hypoxic preconditioning (HP). The unfolded protein response pathway (UPR) has been known for some time to respond to hypoxia and regulate hypoxic sensitivity; however, the role of the UPR, if any, in HP essentially has been unexplored. We have shown previously that a sublethal hypoxic exposure of the nematode Caenorhabditis elegans induces a protein chaperone component of the UPR (L. L. Anderson, X. Mao, B. A. Scott, and C. M. Crowder, Science 323:630-633, 2009). Here, we show that HP induces the UPR and that the pharmacological induction of misfolded proteins is itself sufficient to stimulate a delayed protective response to hypoxic injury that requires the UPR pathway proteins IRE-1, XBP-1, and ATF-6. HP also required IRE-1 but not XBP-1 or ATF-6; instead, GCN-2, which is known to suppress translation and induce an adaptive transcriptional response under conditions of UPR activation or amino acid deprivation, was required for HP. The phosphorylation of the translation factor eIF2?, an established mechanism of GCN-2-mediated translational suppression, was not necessary for HP. These data suggest a model where hypoxia-induced misfolded proteins trigger the activation of IRE-1, which along with GCN-2 controls an adaptive response that is essential to HP. PMID:20733002

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

    Microsoft Academic Search

    Saeed U. Khan; Martin Schröder

    2008-01-01

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

  10. The unfolded protein response is activated in connexin 50 mutant mouse lenses.

    PubMed

    Alapure, Bhagwat V; Stull, Jaime K; Firtina, Zeynep; Duncan, Melinda K

    2012-09-01

    The unfolded protein response is a set of cell signaling pathways recently recognized to be activated in the lens during both normal development and endoplasmic reticulum stress induced by either unfolded proteins or oxidative damage. While mutations in the gene for connexin 50 are known to cause autosomal dominant cataracts, it has not been previously reported whether mutant connexins can activate the unfolded protein response in the lens. Mice homozygous for the S50P or G22R mutation of connexin 50 have reduced amounts of connexin 50 protein at the cell membrane, with some intracellular staining consistent with retention in the endoplasmic reticulum. Connexin 50 mutants have elevated levels of BiP expression in both lens epithelial and fiber cells from E15.5 with the most robust elevation detected in newborns. While this elevation decreases in magnitude postnatally, BiP expression is still abnormally high in adults, particularly in the perinuclear endoplasmic reticulum of cell nuclei that are inappropriately retained in adult homozygous mutant lenses. Xbp1 splicing was elevated in lenses from both connexin mutants studied, while Atf4 and Atf6 levels were not majorly affected. Overall, these data suggest that UPR may be a contributing factor to the phenotype of connexin 50 mutant lenses even though the relatively modest extent of the response suggests that it is unlikely to be a major driver of the pathology. PMID:22713599

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed

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

    2011-04-01

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

  13. Intracellular Signaling by the Unfolded Protein

    E-print Network

    Mullins, Dyche

    Intracellular Signaling by the Unfolded Protein Response Sebasti´an Bernales,1 Feroz R. Papa,2 reticulum stress, signal transduction, organelle homeostasis, protein folding, regulated mRNA splicing, translational control Abstract The unfolded protein response (UPR) is an intracellular signaling pathway

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

    PubMed Central

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

    2012-01-01

    Failure to maintain protein homeostasis (proteostasis) leads to accumulation of unfolded proteins and contributes to the pathogenesis of many human diseases. Accumulation of unfolded proteins in the endoplasmic reticulum (ER) elicits unfolded protein response (UPR) that serves to attenuate protein translation, and increase protein refolding or degradation. In contrast to UPR in the ER, the regulatory molecules operative in cytosolic responses and their potential relation to ER stress are not well elucidated. Aggresome-like induced structures (ALIS) have been described as transient aggregation of ubiquitinated proteins in the cytosol. In this study, we show that cells respond to inflammation, infection or ER stress by cytosolic formation of ALIS, indicating that ALIS formation represents an early event in cellular adjustment to altered proteostasis that occurs under these conditions. This response was aided by rapid transcriptional up-regulation of polyubiqutin-binding protein p62. NF-?B and mTOR activation were also required for ALIS formation. Importantly, we show a cross talk between UPR in the ER and cytosolic ALIS. Down-regulation of ER UPR in XBP1 deficient cells increases cyotosolic ALIS formation. Furthermore, lysosomal activity but not macroautophagy is responsible for ALIS clearance. This study reveals the underlying regulatory mechanisms of ALIS formation and clearance, and provides a previously unrecognized common adaptive mechanism for cellular responses against inflammation and ER stress. PMID:22518844

  15. 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

  16. 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

  17. Unfolded protein response and activated degradative pathways regulation in GNE myopathy.

    PubMed

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

    2013-01-01

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

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

    SciTech Connect

    Honma, Yuichi; Harada, Masaru, E-mail: msrharada@med.uoeh-u.ac.jp

    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.

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

    PubMed

    Krivoruchko, Anastasia; Storey, Kenneth B

    2013-02-01

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

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

    PubMed

    Halliday, Mark; Mallucci, Giovanna R

    2015-06-01

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

  2. Modulation of the unfolded protein response by the human hepatitis B virus

    PubMed Central

    Lazar, Catalin; Uta, Mihaela; Branza-Nichita, Norica

    2014-01-01

    During productive viral infection the host cell is confronted with synthesis of a vast amount of viral proteins which must be folded, quality controlled, assembled and secreted, perturbing the normal function of the endoplasmic reticulum (ER). To counteract the ER stress, cells activate specific signaling pathways, designated as the unfolded proteins response (UPR), which essentially increase their folding capacity, arrest protein translation, and degrade the excess of misfolded proteins. This cellular defense mechanism may, in turn, affect significantly the virus life-cycle. This review highlights the current understanding of the mechanisms of the ER stress activation by Human Hepatitis B virus (HBV), a deadly pathogen affecting more than 350 million people worldwide. Further discussion addresses the latest discoveries regarding the adaptive strategies developed by HBV to manipulate the UPR for its own benefits, the controversies in the field and future perspectives. PMID:25191311

  3. Vertebrate unfolded protein response: mammalian signaling pathways are conserved in Medaka fish.

    PubMed

    Ishikawa, Tokiro; Taniguchi, Yoshihito; Okada, Tetsuya; Takeda, Shunichi; Mori, Kazutoshi

    2011-01-01

    The accumulation of unfolded proteins in the endoplasmic reticulum (ER) activates the unfolded protein response (UPR). The ER stress signal is sensed and transmitted by a transmembrane protein(s) in the ER. The number of these transducers has increased with evolution, one in yeast, three in worm and fly, and five in mammals. Here, we examined medaka fish, Oryzias latipes, as a vertebrate model organism, and found that the medaka genome encodes five UPR transducers. Analysis of a medaka embryonic cell line revealed that the mammalian UPR signaling mechanisms are very well conserved. Thus, XBP1 mRNA, which encodes the transcription factor XBP1 downstream of the IRE1 pathway, was spliced in response to ER stress, resulting in production of the active form of XBP1. Translation was generally attenuated in response to ER stress, which paradoxically induced the translation of ATF4, the transcription factor downstream of the PERK pathway. ATF6 was constitutively synthesized as a transmembrane protein and activated by ER stress-induced proteolysis. Results obtained with the overexpression of active ATF6?, ATF6?, and XBP1 strongly suggested that ATF6? plays a major role in upregulating the major ER chaperone BiP, contrary to the case in non-vertebrates, in which the IRE1 pathway is essential to the induction of BiP. Physiological ER stress occurring during embryonic development was visualized using transgenic medaka carrying the enhanced green fluorescent protein gene under the control of the BiP promoter. Thus, analysis of the vertebrate UPR using medaka will help provide a more comprehensive understanding of the biology and physiology of the UPR. PMID:22067999

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

    PubMed Central

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

    2014-01-01

    Endoplasmic reticulum stress from unfolded proteins is associated with the proliferation of pancreatic tumor cells, making the many regulatory molecules of this pathway appealing targets for therapy. The objective of our study was to assess potential therapeutic efficacy of inhibitors of unfolded protein response (UPR) in pancreatic cancers focusing on IRE1? inhibitors. IRE1?-mediated XBP-1 mRNA splicing encodes a transcription factor that enhances transcription of chaperone proteins in order to reverse UPR. Proliferation assays using a panel of 14 pancreatic cancer cell lines showed a dose- and time-dependent growth inhibition by IRE1?-specific inhibitors (STF-083010, 2-Hydroxy-1-naphthaldehyde, 3-Ethoxy-5,6-dibromosalicylaldehyde, toyocamycin). Growth inhibition was also noted using a clonogenic growth assay in soft agar, as well as a xenograft in vivo model of pancreatic cancer. Cell cycle analysis showed that these IRE1? inhibitors caused growth arrest at either the G1 or G2/M phases (SU8686, MiaPaCa2) and induced apoptosis (Panc0327, Panc0403). Western blot analysis showed cleavage of caspase 3 and PARP, and prominent induction of the apoptotic molecule BIM. In addition, synergistic effects were found between either STF-083010, 2-Hydroxy-1-naphthaldehyde, 3-Ethoxy-5,6-dibromosalicylaldehyde, or toyocamycin and either gemcitabine or bortezomib. Our data suggest that use of an IRE1? inhibitor is a novel therapeutic approach for treatment of pancreatic cancers. PMID:24952679

  5. 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

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

    PubMed

    Zhu, Genyuan; Lee, Amy S

    2015-07-01

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

  7. Lifespan Extension Conferred by Endoplasmic Reticulum Secretory Pathway Deficiency Requires Induction of the Unfolded Protein Response

    PubMed Central

    Labunskyy, Vyacheslav M.; Gerashchenko, Maxim V.; Delaney, Joe R.; Kaya, Alaattin; Kennedy, Brian K.; Kaeberlein, Matt; Gladyshev, Vadim N.

    2014-01-01

    Cells respond to accumulation of misfolded proteins in the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR) signaling pathway. The UPR restores ER homeostasis by degrading misfolded proteins, inhibiting translation, and increasing expression of chaperones that enhance ER protein folding capacity. Although ER stress and protein aggregation have been implicated in aging, the role of UPR signaling in regulating lifespan remains unknown. Here we show that deletion of several UPR target genes significantly increases replicative lifespan in yeast. This extended lifespan depends on a functional ER stress sensor protein, Ire1p, and is associated with constitutive activation of upstream UPR signaling. We applied ribosome profiling coupled with next generation sequencing to quantitatively examine translational changes associated with increased UPR activity and identified a set of stress response factors up-regulated in the long-lived mutants. Besides known UPR targets, we uncovered up-regulation of components of the cell wall and genes involved in cell wall biogenesis that confer resistance to multiple stresses. These findings demonstrate that the UPR is an important determinant of lifespan that governs ER stress and identify a signaling network that couples stress resistance to longevity. PMID:24391512

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

    PubMed Central

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

    2011-01-01

    The endoplasmic reticulum (ER) is the cellular organelle responsible for protein folding and assembly, lipid and sterol biosynthesis, and calcium storage. The unfolded protein response (UPR) is an adaptive intracellular stress response to accumulation of unfolded or misfolded proteins in the ER. In this study, we show that the most conserved UPR sensor inositol-requiring enzyme 1 ? (IRE1?), an ER transmembrane protein kinase/endoribonuclease, is required to maintain hepatic lipid homeostasis under ER stress conditions through repressing hepatic lipid accumulation and maintaining lipoprotein secretion. To elucidate physiological roles of IRE1?-mediated signalling in the liver, we generated hepatocyte-specific Ire1?-null mice by utilizing an albumin promoter-controlled Cre recombinase-mediated deletion. Deletion of Ire1? caused defective induction of genes encoding functions in ER-to-Golgi protein transport, oxidative protein folding, and ER-associated degradation (ERAD) of misfolded proteins, and led to selective induction of pro-apoptotic UPR trans-activators. We show that IRE1? is required to maintain the secretion efficiency of selective proteins. In the absence of ER stress, mice with hepatocyte-specific Ire1? deletion displayed modest hepatosteatosis that became profound after induction of ER stress. Further investigation revealed that IRE1? represses expression of key metabolic transcriptional regulators, including CCAAT/enhancer-binding protein (C/EBP) ?, C/EBP?, peroxisome proliferator-activated receptor ? (PPAR?), and enzymes involved in triglyceride biosynthesis. IRE1? was also found to be required for efficient secretion of apolipoproteins upon disruption of ER homeostasis. Consistent with a role for IRE1? in preventing intracellular lipid accumulation, mice with hepatocyte-specific deletion of Ire1? developed severe hepatic steatosis after treatment with an ER stress-inducing anti-cancer drug Bortezomib, upon expression of a misfolding-prone human blood clotting factor VIII, or after partial hepatectomy. The identification of IRE1? as a key regulator to prevent hepatic steatosis provides novel insights into ER stress mechanisms in fatty liver diseases associated with toxic liver injuries. PMID:21407177

  9. LUMINAL Ca2+ DEPLETION DURING THE UNFOLDED PROTEIN RESPONSE IN Xenopus OOCYTES: CAUSE AND CONSEQUENCE

    PubMed Central

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

    2013-01-01

    The Endoplasmic Reticulum (ER) is a Ca2+ storing organelle that plays a critical role in the synthesis, folding and post-translational modifications of many proteins. The ER enters into a condition of stress when the load of newly synthesized proteins exceeds its folding and processing capacity. This activates a signal transduction pathway called the Unfolded Protein Response (UPR) that attempts to restore homeostasis. The precise role of ER Ca2+ in the initiation of the UPR has not been defined. Specifically, it has not been established whether ER Ca2+ dysregulation is a cause or consequence of ER stress. Here, we report that partial depletion of ER Ca2+ stores induces a significant induction of the UPR, and leads to the retention of a normally secreted protein Carboxypeptidase Y. Moreover, inhibition of protein glycosylation by tunicamycin rapidly induced an ER Ca2+ leak into the cytosol. However, blockade of the translocon with emetine inhibited the tunicamycin-induced Ca2+ release. Furthermore, emetine treatment blocked elF2? phosphorylation and reduced expression of the chaperone BiP. These findings suggest that Ca2+ may be both a cause and a consequence of ER protein misfolding. Thus, it appears that ER Ca2+ leak is a significant co-factor for the initiation of the UPR. PMID:23415071

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

    PubMed

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

    2011-06-01

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

  11. Polychlorinated Biphenyl Quinone Induces Endoplasmic Reticulum Stress, Unfolded Protein Response, and Calcium Release.

    PubMed

    Xu, Demei; Su, Chuanyang; Song, Xiufang; Shi, Qiong; Fu, Juanli; Hu, Lihua; Xia, Xiaomin; Song, Erqun; Song, Yang

    2015-06-15

    Organisms are able to respond to environmental insult to maintain cellular homeostasis, which include the activation of a wide range of cellular adaptive responses with tightly controlled mechanisms. The endoplasmic reticulum (ER) is an organelle responsible for protein folding and calcium storage. ER stress leads to the accumulation of unfolded proteins in the ER lumen. To be against or respond to this effect, cells have a comprehensive signaling system, called unfolded protein response (UPR), to restore homeostasis and normal ER function or activate the cell death program. Therefore, it is critical to understand how environmental insult regulates the ingredients of ER stress and UPR signalings. Previously, we have demonstrated that polychlorinated biphenyl (PCB) quinone caused oxidative stress, cytotoxicity, genotoxicity, and apoptosis in HepG2 cells. Here, we investigated the role of a PCB quinone, PCB29-pQ on ER stress, UPR, and calcium release. PCB29-pQ markedly increased the hallmark genes of ER stress, namely, glucose-regulated protein 78 (GRP78), GRP94, and C/EBP homologous protein (CHOP) on both protein and mRNA levels in HepG2 cells. We also confirmed PCB29-pQ induced ER morphological defects by using transmission electron microscopy. Moreover, PCB29-pQ induced intracellular calcium accumulation and calpain activity, which were significantly inhibited by the pretreatment of BAPTA-AM (Ca(2+) chelator). These results were correlated with the outcome that PCB29-pQ induces ER stress-related apoptosis through caspase family gene 12, while salubrinal and Z-ATAD-FMK (a specific inhibitor of caspase 12) partially ameliorated this effect, respectively. N-Acetyl-l-cysteine (NAC) scavenged ROS formation and consequently alleviated PCB29-pQ-induced expression of ER stress-related genes. In conclusion, our result demonstrated for the first time that PCB quinone leads to ROS-dependent induction of ER stress, and UPR and calcium release in HepG2 cells, and the evaluation of the perturbations of ER stress, UPR, and calcium signaling provide further information on the mechanisms of PCB-induced toxicity. PMID:25950987

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

    PubMed Central

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

    2013-01-01

    In Alzheimer’s disease (AD), the mechanisms of neuronal loss remain largely unknown. While tau pathology is closely correlated with neuronal loss, how its accumulation may lead to activation of neurotoxic pathways is unclear. Here we show that tau increased the levels of ubiquitinated proteins in the brain and that this triggered activation of the Unfolded Protein Response (UPR). This suggested that tau was interfering with protein quality control in the endoplasmic reticulum (ER). Consistent with this, ubiquitin was found to associate with the ER in human AD brains and rTg4510 tau transgenic mouse brains, but this was not always co-localized with tau. The increased levels of ubiquitinated protein were accompanied by increased levels of phosphorylated PERK, a marker that indicates UPR activation. Importantly, depleting soluble tau levels in cells and brain could reverse UPR activation. Tau accumulation facilitated its deleterious interaction with ER membrane and associated proteins that are essential for ER-associated degradation (ERAD), including VCP and Hrd1. Based on this, the effects of tau accumulation on ERAD efficiency were evaluated using the CD3? reporter, an ERAD substrate. Indeed, CD3? accumulated in both in vitro and in vivo models of tau over-expression and AD brains. These data suggest that soluble tau impairs ERAD, and the result is activation of the UPR. The reversibility of this process, however, suggests that tau-based therapeutics could significantly delay this type of cell death and consequently disease progression. PMID:23719816

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

    PubMed Central

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

    2014-01-01

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

  14. Restoration of the Unfolded Protein Response in Pancreatic ? Cells Protects Mice Against Type 1 Diabetes

    PubMed Central

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

    2014-01-01

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

  15. Proteasome inhibitors induce a terminal unfolded protein response in multiple myeloma cells

    PubMed Central

    Obeng, Esther A.; Carlson, Louise M.; Gutman, Delia M.; Harrington, William J.; Lee, Kelvin P.; Boise, Lawrence H.

    2006-01-01

    Multiple myeloma (MM) is an incurable plasma cell malignancy. The 26S proteasome inhibitor, bortezomib, selectively induces apoptosis in MM cells; however, the nature of its selectivity remains unknown. Here we demonstrate that 5 different MM cell lines display similar patterns of sensitivity to 3 proteasome inhibitors (PIs) but respond differently to specific NF-?B inhibition. We further show that PIs initiate the unfolded protein response (UPR), a signaling pathway activated by the accumulation of misfolded proteins within the endoplasmic reticulum (ER). Consistent with reports that prosurvival/physiologic UPR components are required for B-cell differentiation into antibody-secreting cells, we found that MM cells inherently expressed the ER chaperones GRP78/Bip and GRP94/gp96. However, bortezomib rapidly induced components of the proapoptotic/terminal UPR, including PERK, the ER stress–specific eIF-2? kinase; ATF4, an ER stress–induced transcription factor; and its proapoptotic target, CHOP/GADD153. Consistent with our hypothesis that PIs induce the accumulation of misfolded ER-processed proteins, we found that the amount of immunoglobulin subunits retained within MM cells correlated with their sensitivity to PIs. These findings suggest that MM cells have a lower threshold for PI-induced UPR induction and ER stress–induced apoptosis because they constitutively express ER stress survival factors to function as secretory cells. PMID:16507771

  16. USP19 deubiquitinating enzyme inhibits muscle cell differentiation by suppressing unfolded-protein response signaling

    PubMed Central

    Wiles, Benjamin; Miao, Miao; Coyne, Erin; Larose, Louise; Cybulsky, Andrey V.; Wing, Simon S.

    2015-01-01

    The USP19 deubiquitinating enzyme modulates the expression of myogenin and myofibrillar proteins in L6 muscle cells. This raised the possibility that USP19 might regulate muscle cell differentiation. We therefore tested the effects of adenoviral-mediated overexpression or small interfering RNA (siRNA)-mediated silencing of either the cytoplasmic or endoplasmic reticulum (ER)-localized isoforms of USP19. Only the ER-localized isoform of USP19 (USP19-ER) modulated myoblast fusion as well as the expression of myogenin and myofibrillar proteins, and these effects were also dependent on USP19 catalytic activity. USP19-ER inhibited muscle cell differentiation and the induction of CHOP, a transcription factor in the unfolded-protein response (UPR) that is activated during differentiation. Inducing the UPR by creating mild ER stress with thapsigargin was able to reverse the defect in myoblast fusion caused by the overexpression of USP19-ER, suggesting strongly that USP19 exerts its effects on fusion through its effects on UPR signaling. USP19 also functions similarly in vivo, as USP19?/? mice display improved muscle regeneration concomitant with enhanced expression of CHOP. Collectively these results implicate a deubiquitinating enzyme as a regulator of the UPR. They also suggest that inhibition of USP19 may be a therapeutic approach for the enhancement of muscle growth following injury. PMID:25568336

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

    PubMed Central

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

    2013-01-01

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

  18. Inhibition of the Unfolded Protein Response by metformin in renal proximal tubular epithelial cells.

    PubMed

    Thériault, Jimmy R; Palmer, Helen J; Pittman, Debra D

    2011-06-10

    Metformin (Met), an AMP-activated protein kinase (AMPK) inducer, is primarily transported by organic cation transporters expressed at the surface of renal proximal tubular epithelial cells. However, the implication of Met in renal function remains poorly understood. Interestingly, AICAR, another AMPK inducer, has been shown to inhibit the Unfolded Protein Response (UPR) generated by tunicamycin in cardiomyocytes in an AMPK-kinase dependent fashion suggesting metformin may also block the UPR. In this work, we have examined the effect of metformin on the expression of UPR-related markers (GRP94 and CHOP) induced by glucosamine (GlcN), 2-deoxyglucose (2-DOG) and tunicamycin (TUNI) in renal proximal tubular epithelial cells and in murine mesangial cells. Met attenuated GRP94 and CHOP expression induced by GlcN and 2-DOG, but not TUNI only in renal epithelial cells, even though the AMPK activation was observed in both renal epithelial and mesangial cells. Met did not require the contribution of its AMPK kinase inducing activity to block UPR markers expression. This report has identified a novel inhibitory function of metformin on UPR, which may have a beneficial impact on kidney homeostatic function. PMID:21600878

  19. Disruption of the unfolded protein response (UPR) by lead compound selectively suppresses cancer cell growth.

    PubMed

    Huang, Hejing; Liu, Huanan; Liu, Changmei; Fan, Lixia; Zhang, Xinwen; Gao, Anhui; Hu, Xiaobei; Zhang, Kunzhi; Cao, Xianchao; Jiang, Kailong; Zhou, Yubo; Hou, Jian; Nan, Fajun; Li, Jia

    2015-05-01

    Identifying chemotherapy candidates with high selectivity against cancer cells is a major challenge in cancer treatment. Tumor microenvironments cause chronic endoplasmic reticulum (ER) stress and activate the unfolded protein response (UPR) as an adaptive response. Here, one novel small-molecule compound, 17#, was discovered as a potent pan-UPR inhibitor. It exhibited good selection for growth inhibition when cancer cells were cultured in 2-deoxy-D-glucose (2DG), mimicking an in vitro glucose-deprived status. Additionally, 17# alone could mildly suppress the growth of HeLa tumor xenografts, and a synergistic anti-cancer effect was observed when 17# was combined with 2DG. A mechanistic study showed that 17#-induced selective anti-cancer effects were highly dependent on UPR inhibition, and overexpressing GRP78 or XBP1s reversed the 17#-induced growth inhibition and cell cycle arrest, partially by delaying the downregulation of the cell cycle regulator cyclin B1. Furthermore, 17# improved the sensitivity of anti-cancer drugs such as doxorubicin or etoposide. Our study presents evidence that disrupting the UPR has selective therapeutic potential and may enhance drug sensitivity. PMID:25721085

  20. 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

  1. 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

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

    PubMed Central

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

    2012-01-01

    Ubiquitin (Ub) is a vital regulatory component in various cellular processes, including cellular responses to viral infection. As obligate intracellular pathogens, viruses have the capacity to manipulate the ubiquitin (Ub) cycle to their advantage by encoding Ub-modifying proteins including deubiquitinases (DUBs). However, how cellular DUBs modulate specific viral infections, such as norovirus, is poorly understood. To examine the role of DUBs during norovirus infection, we used WP1130, a small molecule inhibitor of a subset of cellular DUBs. Replication of murine norovirus in murine macrophages and the human norovirus Norwalk virus in a replicon system were significantly inhibited by WP1130. Chemical proteomics identified the cellular DUB USP14 as a target of WP1130 in murine macrophages, and pharmacologic inhibition or siRNA-mediated knockdown of USP14 inhibited murine norovirus infection. USP14 is a proteasome-associated DUB that also binds to inositol-requiring enzyme 1 (IRE1), a critical mediator of the unfolded protein response (UPR). WP1130 treatment of murine macrophages did not alter proteasome activity but activated the X-box binding protein-1 (XBP-1) through an IRE1-dependent mechanism. In addition, WP1130 treatment or induction of the UPR also reduced infection of other RNA viruses including encephalomyocarditis virus, Sindbis virus, and La Crosse virus but not vesicular stomatitis virus. Pharmacologic inhibition of the IRE1 endonuclease activity partially rescued the antiviral effect of WP1130. Taken together, our studies support a model whereby induction of the UPR through cellular DUB inhibition blocks specific viral infections, and suggest that cellular DUBs and the UPR represent novel targets for future development of broad spectrum antiviral therapies. PMID:22792064

  3. Antiviral activity of a small molecule deubiquitinase inhibitor occurs via induction of the unfolded protein response.

    PubMed

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

    2012-01-01

    Ubiquitin (Ub) is a vital regulatory component in various cellular processes, including cellular responses to viral infection. As obligate intracellular pathogens, viruses have the capacity to manipulate the ubiquitin (Ub) cycle to their advantage by encoding Ub-modifying proteins including deubiquitinases (DUBs). However, how cellular DUBs modulate specific viral infections, such as norovirus, is poorly understood. To examine the role of DUBs during norovirus infection, we used WP1130, a small molecule inhibitor of a subset of cellular DUBs. Replication of murine norovirus in murine macrophages and the human norovirus Norwalk virus in a replicon system were significantly inhibited by WP1130. Chemical proteomics identified the cellular DUB USP14 as a target of WP1130 in murine macrophages, and pharmacologic inhibition or siRNA-mediated knockdown of USP14 inhibited murine norovirus infection. USP14 is a proteasome-associated DUB that also binds to inositol-requiring enzyme 1 (IRE1), a critical mediator of the unfolded protein response (UPR). WP1130 treatment of murine macrophages did not alter proteasome activity but activated the X-box binding protein-1 (XBP-1) through an IRE1-dependent mechanism. In addition, WP1130 treatment or induction of the UPR also reduced infection of other RNA viruses including encephalomyocarditis virus, Sindbis virus, and La Crosse virus but not vesicular stomatitis virus. Pharmacologic inhibition of the IRE1 endonuclease activity partially rescued the antiviral effect of WP1130. Taken together, our studies support a model whereby induction of the UPR through cellular DUB inhibition blocks specific viral infections, and suggest that cellular DUBs and the UPR represent novel targets for future development of broad spectrum antiviral therapies. PMID:22792064

  4. Atypical, bidirectional regulation of cadmium-induced apoptosis via distinct signaling of unfolded protein response.

    PubMed

    Yokouchi, M; Hiramatsu, N; Hayakawa, K; Kasai, A; Takano, Y; Yao, J; Kitamura, M

    2007-08-01

    Cadmium is a widely distributed nephrotoxic metal that causes renal tubular injury. In this report, we investigated involvement of endoplasmic reticulum (ER) stress and individual unfolded protein responses in cadmium-initiated apoptosis of tubular epithelial cells. Cadmium chloride (CdCl(2)) induced expression of endogenous ER stress markers, GRP78, GRP94 and CHOP in vitro and in vivo, and subsequently caused cytological changes typical of apoptosis. Attenuation of ER stress by transfection with ER chaperone GRP78 or ORP150 suppressed CdCl(2)-triggered apoptosis. In response to CdCl(2), phosphorylation of RNA-dependent protein kinase-like ER kinase (PERK) and eukaryotic translation initiation factor 2alpha (eIF2alpha) was observed. Enhanced phosphorylation of eIF2alpha attenuated, whereas inhibition of eIF2alpha exacerbated CdCl(2)-induced apoptosis. Activating transcription factor 6 (ATF6) was also activated by CdCl(2) and blockade of this process suppressed induction of CHOP and thereby improved cell survival. CdCl(2) also triggered activation of the inositol-requiring ER-to-nucleus signal kinase 1 (IRE1)-X-box-binding protein 1 (XBP1) pathway and inhibition of XBP1 attenuated apoptosis independent of GRP78 and CHOP. c-Jun N-terminal kinase (JNK), another molecule downstream of IRE1, was also phosphorylated by CdCl(2) and its inhibition attenuated apoptosis. These results evidenced bidirectional regulation of apoptosis in cadmium-exposed cells. The ATF6 and IRE1 pathways cooperatively caused apoptosis via induction of CHOP, activation of XBP1 and phosphorylation of JNK, and the PERK-eIF2alpha pathway counteracted the proapoptotic processes. PMID:17464326

  5. Differences in Unfolded Protein Response Pathway Activation in the Lenses of Three Types of Cataracts

    PubMed Central

    Gu, Jianjun; Wang, Yujuan; Guo, Minfei; Liu, Yizhi

    2015-01-01

    Purpose To investigate the activation of three unfolded protein response (UPR) pathways in the lenses of age-related, high myopia-related and congenital cataracts. Methods and Materials Lens specimens were collected from patients during small incision cataract surgery. Lenses from young cadaver eyes were collected as normal controls. Real-time PCR and Western blotting were performed to detect the expression of GRP78, p-eIF2?, spliced XBP1, ATF6, ATF4 and p-IRE1? in the lenses of normal human subjects and patients with age-related, myopia-related or congenital cataracts. Results In the lenses of the age-related and high myopia-related cataract groups, the protein levels of ATF6, p-eIF2? and p-IRE1? and the gene expression levels of spliced XBP1, GRP78, ATF6 and ATF4 were greatly increased. Additionally, in the congenital cataract group, the protein levels of p-eIF2? and p-IRE1? and the gene expression levels of spliced XBP1, GRP78 and ATF4 were greatly increased. However, the protein and gene expression levels of ATF6 were not up-regulated in the congenital cataract group compared with the normal control group. Conclusions The UPR is activated via different pathways in the lenses of age-related, high myopia-related and congenital cataracts. UPR activation via distinct pathways might play important roles in cataractogenesis mechanisms in different types of cataracts. PMID:26091066

  6. Inhibition of mitochondrial genome expression triggers the activation of CHOP-10 by a cell signaling dependent on the integrated stress response but not the mitochondrial unfolded protein response.

    PubMed

    Michel, Sebastien; Canonne, Morgane; Arnould, Thierry; Renard, Patricia

    2015-03-01

    Mitochondria-to-nucleus communication, known as retrograde signaling, is important to adjust the nuclear gene expression in response to organelle dysfunction. Among the transcription factors described to respond to mitochondrial stress, CHOP-10 is activated by respiratory chain inhibition, mitochondrial accumulation of unfolded proteins and mtDNA mutations. In this study, we show that altered/impaired expression of mtDNA induces CHOP-10 expression in a signaling pathway that depends on the eIF2?/ATF4 axis of the integrated stress response rather than on the mitochondrial unfolded protein response. PMID:25643991

  7. Analysis of unfolded protein response during single-chain antibody expression in Saccaromyces cerevisiae reveals different roles for BiP and PDI in folding

    Microsoft Academic Search

    Ping Xu; David Raden; Francis J. Doyle III; Anne Skaja Robinson

    2005-01-01

    The production of recombinant proteins is a critical technology for biotechnology and biomedical research. Heterologous expression of secreted proteins can saturate the cell's capacity to properly fold protein, initiating the unfolded protein response (UPR), and resulting in a loss of protein expression. The overexpression of chaperone binding protein (BiP) and disulfide bond isomerase (PDI) in Saccaromyces cerevisiae can effectively increase

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

    PubMed Central

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

    2013-01-01

    We previously identified SMIP004 (N-(4-butyl-2-methyl-phenyl) acetamide) as a novel inducer of cancer-cell selective apoptosis of human prostate cancer cells. SMIP004 decreased the levels of positive cell cycle regulators, upregulated cyclin-dependent kinase inhibitors, and resulted in G1 arrest, inhibition of colony formation in soft agar, and cell death. However, the mechanism of SMIP004-induced cancer cell selective apoptosis remained unknown. Here, we used chemical genomic and proteomic profiling to unravel a SMIP004-induced pro-apoptotic pathway, which initiates with disruption of mitochondrial respiration leading to oxidative stress. This, in turn, activates two pathways, one eliciting cell cycle arrest by rapidly targeting cyclin D1 for proteasomal degradation and driving the transcriptional downregulation of the androgen receptor, and a second pathway that activates pro-apoptotic signaling through MAPK activation downstream of the unfolded protein response (UPR). SMIP004 potently inhibits the growth of prostate and breast cancer xenografts in mice. Our data suggest that SMIP004, by inducing mitochondrial ROS formation, targets specific sensitivities of prostate cancer cells to redox and bioenergetic imbalances that can be exploited in cancer therapy. PMID:23902736

  9. Divergent androgen regulation of unfolded protein response pathways drives prostate cancer

    PubMed Central

    Sheng, Xia; Arnoldussen, Yke Jildouw; Storm, Margrethe; Tesikova, Martina; Nenseth, Hatice Zeynep; Zhao, Sen; Fazli, Ladan; Rennie, Paul; Risberg, Bjørn; Wæhre, Håkon; Danielsen, Håvard; Mills, Ian G; Jin, Yang; Hotamisligil, Gökhan; Saatcioglu, Fahri

    2015-01-01

    The unfolded protein response (UPR) is a homeostatic mechanism to maintain endoplasmic reticulum (ER) function. The UPR is activated by various physiological conditions as well as in disease states, such as cancer. As androgens regulate secretion and development of the normal prostate and drive prostate cancer (PCa) growth, they may affect UPR pathways. Here, we show that the canonical UPR pathways are directly and divergently regulated by androgens in PCa cells, through the androgen receptor (AR), which is critical for PCa survival. AR bound to gene regulatory sites and activated the IRE1? branch, but simultaneously inhibited PERK signaling. Inhibition of the IRE1? arm profoundly reduced PCa cell growth in vitro as well as tumor formation in preclinical models of PCa in vivo. Consistently, AR and UPR gene expression were correlated in human PCa, and spliced XBP-1 expression was significantly upregulated in cancer compared with normal prostate. These data establish a genetic switch orchestrated by AR that divergently regulates the UPR pathways and suggest that targeting IRE1? signaling may have therapeutic utility in PCa. PMID:25864123

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

    PubMed Central

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

    2014-01-01

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

  11. Activation of the mitochondrial unfolded protein response does not predict longevity in Caenorhabditis elegans

    PubMed Central

    Bennett, Christopher F.; Wende, Helen Vander; Simko, Marissa; Klum, Shannon; Barfield, Sarah; Choi, Haeri; Pineda, Victor V.; Kaeberlein, Matt

    2014-01-01

    Recent studies have propagated the model that the mitochondrial unfolded protein response (UPRmt) is causal for lifespan extension from inhibition of the electron transport chain (ETC) in C. elegans. Here we report a genome-wide RNAi screen for negative regulators of the UPRmt. Lifespan analysis of nineteen RNAi clones that induce the hsp-6p::gfp reporter demonstrate differential effects on longevity. Deletion of atfs-1, which is required for induction of the UPRmt, fails to prevent lifespan extension from knockdown of two genes identified in our screen or following knockdown of the ETC gene cco-1. RNAi knockdown of atfs-1 also has no effect on lifespan extension caused by mutation of the ETC gene isp-1. Constitutive activation of the UPRmt by gain of function mutations in atfs-1 fails to extend lifespan. These observations identify several new factors that promote mitochondrial homeostasis and demonstrate that the UPRmt, as currently defined, is neither necessary nor sufficient for lifespan extension. PMID:24662282

  12. The Unfolded Protein State Revisited

    Microsoft Academic Search

    Patricio A. Carvajal; Tyre C. Lanier

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

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

    PubMed Central

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

    2009-01-01

    Filamentous fungi rely heavily on the secretory pathway, both for the delivery of cell wall components to the hyphal tip and the production and secretion of extracellular hydrolytic enzymes needed to support growth on polymeric substrates. Increased demand on the secretory system exerts stress on the endoplasmic reticulum (ER), which is countered by the activation of a coordinated stress response pathway termed the unfolded protein response (UPR). To determine the contribution of the UPR to the growth and virulence of the filamentous fungal pathogen Aspergillus fumigatus, we disrupted the hacA gene, encoding the major transcriptional regulator of the UPR. The ?hacA mutant was unable to activate the UPR in response to ER stress and was hypersensitive to agents that disrupt ER homeostasis or the cell wall. Failure to induce the UPR did not affect radial growth on rich medium at 37°C, but cell wall integrity was disrupted at 45°C, resulting in a dramatic loss in viability. The ?hacA mutant displayed a reduced capacity for protease secretion and was growth-impaired when challenged to assimilate nutrients from complex substrates. In addition, the ?hacA mutant exhibited increased susceptibility to current antifungal agents that disrupt the membrane or cell wall and had attenuated virulence in multiple mouse models of invasive aspergillosis. These results demonstrate the importance of ER homeostasis to the growth and virulence of A. fumigatus and suggest that targeting the UPR, either alone or in combination with other antifungal drugs, would be an effective antifungal strategy. PMID:19132084

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

    Microsoft Academic Search

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

    2006-01-01

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

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

    PubMed Central

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

    2011-01-01

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

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

    PubMed Central

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

    2009-01-01

    Background Glaucoma is the world's second leading cause of bilateral blindness with progressive loss of vision due to retinal ganglion cell death. Myocilin has been associated with congenital glaucoma and 2–4% of primary open angle glaucoma (POAG) cases, but the pathogenic mechanisms remain largely unknown. Among several hypotheses, activation of the unfolded protein response (UPR) has emerged as a possible disease mechanism. Methodology / Principal Findings We used a transgenic Drosophila model to analyze whole-genome transcriptional profiles in flies that express human wild-type or mutant MYOC in their eyes. The transgenic flies display ocular fluid discharge, reflecting ocular hypertension, and a progressive decline in their behavioral responses to light. Transcriptional analysis shows that genes associated with the UPR, ubiquitination, and proteolysis, as well as metabolism of reactive oxygen species and photoreceptor activity undergo altered transcriptional regulation. Following up on the results from these transcriptional analyses, we used immunoblots to demonstrate the formation of MYOC aggregates and showed that the formation of such aggregates leads to induction of the UPR, as evident from activation of the fluorescent UPR marker, xbp1-EGFP. Conclusions / Significance Our results show that aggregation of MYOC in the endoplasmic reticulum activates the UPR, an evolutionarily conserved stress pathway that culminates in apoptosis. We infer from the Drosophila model that MYOC-associated ocular hypertension in the human eye may result from aggregation of MYOC and induction of the UPR in trabecular meshwork cells. This process could occur at a late age with wild-type MYOC, but might be accelerated by MYOC mutants to account for juvenile onset glaucoma. PMID:19148291

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

    PubMed Central

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

    2014-01-01

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

  18. Evidence for Activation of the Unfolded Protein Response in Collagen IV Nephropathies

    PubMed Central

    Pieri, Myrtani; Stefanou, Charalambos; Zaravinos, Apostolos; Erguler, Kamil; Stylianou, Kostas; Lapathitis, George; Karaiskos, Christos; Savva, Isavella; Paraskeva, Revekka; Dweep, Harsh; Sticht, Carsten; Anastasiadou, Natassa; Zouvani, Ioanna; Goumenos, Demetris; Felekkis, Kyriakos; Saleem, Moin; Voskarides, Konstantinos; Gretz, Norbert

    2014-01-01

    Thin-basement-membrane nephropathy (TBMN) and Alport syndrome (AS) are progressive collagen IV nephropathies caused by mutations in COL4A3/A4/A5 genes. These nephropathies invariably present with microscopic hematuria and frequently progress to proteinuria and CKD or ESRD during long-term follow-up. Nonetheless, the exact molecular mechanisms by which these mutations exert their deleterious effects on the glomerulus remain elusive. We hypothesized that defective trafficking of the COL4A3 chain causes a strong intracellular effect on the cell responsible for COL4A3 expression, the podocyte. To this end, we overexpressed normal and mutant COL4A3 chains (G1334E mutation) in human undifferentiated podocytes and tested their effects in various intracellular pathways using a microarray approach. COL4A3 overexpression in the podocyte caused chain retention in the endoplasmic reticulum (ER) that was associated with activation of unfolded protein response (UPR)–related markers of ER stress. Notably, the overexpression of normal or mutant COL4A3 chains differentially activated the UPR pathway. Similar results were observed in a novel knockin mouse carrying the Col4a3-G1332E mutation, which produced a phenotype consistent with AS, and in biopsy specimens from patients with TBMN carrying a heterozygous COL4A3-G1334E mutation. These results suggest that ER stress arising from defective localization of collagen IV chains in human podocytes contributes to the pathogenesis of TBMN and AS through activation of the UPR, a finding that may pave the way for novel therapeutic interventions for a variety of collagenopathies. PMID:24262798

  19. An activated unfolded protein response promotes retinal degeneration and triggers an inflammatory response in the mouse retina.

    PubMed

    Rana, T; Shinde, V M; Starr, C R; Kruglov, A A; Boitet, E R; Kotla, P; Zolotukhin, S; Gross, A K; Gorbatyuk, M S

    2014-01-01

    Recent studies on the endoplasmic reticulum stress have shown that the unfolded protein response (UPR) is involved in the pathogenesis of inherited retinal degeneration caused by mutant rhodopsin. However, the main question of whether UPR activation actually triggers retinal degeneration remains to be addressed. Thus, in this study, we created a mouse model for retinal degeneration caused by a persistently activated UPR to assess the physiological and morphological parameters associated with this disease state and to highlight a potential mechanism by which the UPR can promote retinal degeneration. We performed an intraocular injection in C57BL6 mice with a known unfolded protein response (UPR) inducer, tunicamycin (Tn) and examined animals by electroretinography (ERG), spectral domain optical coherence tomography (SD-OCT) and histological analyses. We detected a significant loss of photoreceptor function (over 60%) and retinal structure (35%) 30 days post treatment. Analysis of retinal protein extracts demonstrated a significant upregulation of inflammatory markers including interleukin-1? (IL-1?), IL-6, tumor necrosis factor-? (TNF-?), monocyte chemoattractant protein-1 (MCP-1) and IBA1. Similarly, we detected a strong inflammatory response in mice expressing either Ter349Glu or T17M rhodopsin (RHO). These mutant rhodopsin species induce severe retinal degeneration and T17M rhodopsin elicits UPR activation when expressed in mice. RNA and protein analysis revealed a significant upregulation of pro- and anti-inflammatory markers such as IL-1?, IL-6, p65 nuclear factor kappa B (NF-kB) and MCP-1, as well as activation of F4/80 and IBA1 microglial markers in both the retinas expressing mutant rhodopsins. We then assessed if the Tn-induced inflammatory marker IL-1? was capable of inducing retinal degeneration by injecting C57BL6 mice with a recombinant IL-1?. We observed ~19% reduction in ERG a-wave amplitudes and a 29% loss of photoreceptor cells compared with control retinas, suggesting a potential link between pro-inflammatory cytokines and retinal pathophysiological effects. Our work demonstrates that in the context of an established animal model for ocular disease, the persistent activation of the UPR could be responsible for promoting retinal degeneration via the UPR-induced pro-inflammatory cytokine IL-1?. PMID:25522272

  20. An activated unfolded protein response promotes retinal degeneration and triggers an inflammatory response in the mouse retina

    PubMed Central

    Rana, T; Shinde, V M; Starr, C R; Kruglov, A A; Boitet, E R; Kotla, P; Zolotukhin, S; Gross, A K; Gorbatyuk, M S

    2014-01-01

    Recent studies on the endoplasmic reticulum stress have shown that the unfolded protein response (UPR) is involved in the pathogenesis of inherited retinal degeneration caused by mutant rhodopsin. However, the main question of whether UPR activation actually triggers retinal degeneration remains to be addressed. Thus, in this study, we created a mouse model for retinal degeneration caused by a persistently activated UPR to assess the physiological and morphological parameters associated with this disease state and to highlight a potential mechanism by which the UPR can promote retinal degeneration. We performed an intraocular injection in C57BL6 mice with a known unfolded protein response (UPR) inducer, tunicamycin (Tn) and examined animals by electroretinography (ERG), spectral domain optical coherence tomography (SD-OCT) and histological analyses. We detected a significant loss of photoreceptor function (over 60%) and retinal structure (35%) 30 days post treatment. Analysis of retinal protein extracts demonstrated a significant upregulation of inflammatory markers including interleukin-1? (IL-1?), IL-6, tumor necrosis factor-? (TNF-?), monocyte chemoattractant protein-1 (MCP-1) and IBA1. Similarly, we detected a strong inflammatory response in mice expressing either Ter349Glu or T17M rhodopsin (RHO). These mutant rhodopsin species induce severe retinal degeneration and T17M rhodopsin elicits UPR activation when expressed in mice. RNA and protein analysis revealed a significant upregulation of pro- and anti-inflammatory markers such as IL-1?, IL-6, p65 nuclear factor kappa B (NF-kB) and MCP-1, as well as activation of F4/80 and IBA1 microglial markers in both the retinas expressing mutant rhodopsins. We then assessed if the Tn-induced inflammatory marker IL-1? was capable of inducing retinal degeneration by injecting C57BL6 mice with a recombinant IL-1?. We observed ~19% reduction in ERG a-wave amplitudes and a 29% loss of photoreceptor cells compared with control retinas, suggesting a potential link between pro-inflammatory cytokines and retinal pathophysiological effects. Our work demonstrates that in the context of an established animal model for ocular disease, the persistent activation of the UPR could be responsible for promoting retinal degeneration via the UPR-induced pro-inflammatory cytokine IL-1?. PMID:25522272

  1. Novel insights into the unfolded protein response using Pichia pastoris specific DNA microarrays

    PubMed Central

    Graf, Alexandra; Gasser, Brigitte; Dragosits, Martin; Sauer, Michael; Leparc, Germán G; Tüchler, Thomas; Kreil, David P; Mattanovich, Diethard

    2008-01-01

    Background DNA Microarrays are regarded as a valuable tool for basic and applied research in microbiology. However, for many industrially important microorganisms the lack of commercially available microarrays still hampers physiological research. Exemplarily, our understanding of protein folding and secretion in the yeast Pichia pastoris is presently widely dependent on conclusions drawn from analogies to Saccharomyces cerevisiae. To close this gap for a yeast species employed for its high capacity to produce heterologous proteins, we developed full genome DNA microarrays for P. pastoris and analyzed the unfolded protein response (UPR) in this yeast species, as compared to S. cerevisiae. Results By combining the partially annotated gene list of P. pastoris with de novo gene finding a list of putative open reading frames was generated for which an oligonucleotide probe set was designed using the probe design tool TherMODO (a thermodynamic model-based oligoset design optimizer). To evaluate the performance of the novel array design, microarrays carrying the oligo set were hybridized with samples from treatments with dithiothreitol (DTT) or a strain overexpressing the UPR transcription factor HAC1, both compared with a wild type strain in normal medium as untreated control. DTT treatment was compared with literature data for S. cerevisiae, and revealed similarities, but also important differences between the two yeast species. Overexpression of HAC1, the most direct control for UPR genes, resulted in significant new understanding of this important regulatory pathway in P. pastoris, and generally in yeasts. Conclusion The differences observed between P. pastoris and S. cerevisiae underline the importance of DNA microarrays for industrial production strains. P. pastoris reacts to DTT treatment mainly by the regulation of genes related to chemical stimulus, electron transport and respiration, while the overexpression of HAC1 induced many genes involved in translation, ribosome biogenesis, and organelle biosynthesis, indicating that the regulatory events triggered by DTT treatment only partially overlap with the reactions to overexpression of HAC1. The high reproducibility of the results achieved with two different oligo sets is a good indication for their robustness, and underlines the importance of less stringent selection of regulated features, in order to avoid a large number of false negative results. PMID:18713468

  2. Chikungunya virus non-structural protein 2-mediated host shut-off disables the unfolded protein response.

    PubMed

    Fros, Jelke J; Major, Lee D; Scholte, Florine E M; Gardner, Joy; van Hemert, Martijn J; Suhrbier, Andreas; Pijlman, Gorben P

    2015-03-01

    The unfolded protein response (UPR) is a cellular defence mechanism against high concentrations of misfolded protein in the endoplasmic reticulum (ER). In the presence of misfolded proteins, ER-transmembrane proteins PERK and IRE1? become activated. PERK phosphorylates eIF2? leading to a general inhibition of cellular translation, whilst the expression of transcription factor ATF4 is upregulated. Active IRE1? splices out an intron from XBP1 mRNA, to produce a potent transcription factor. Activation of the UPR increases the production of several proteins involved in protein folding, degradation and apoptosis. Here, we demonstrated that transient expression of chikungunya virus (CHIKV) (family Togaviridae, genus Alphavirus) envelope glycoproteins induced the UPR and that CHIKV infection resulted in the phosphorylation of eIF2? and partial splicing of XBP1 mRNA. However, infection with CHIKV did not increase the expression of ATF4 and known UPR target genes (GRP78/BiP, GRP94 and CHOP). Moreover, nuclear XBP1 was not observed during CHIKV infection. Even upon stimulation with tunicamycin, the UPR was efficiently inhibited in CHIKV-infected cells. Individual expression of CHIKV non-structural proteins (nsPs) revealed that nsP2 alone was sufficient to inhibit the UPR. Mutations that rendered nsP2 unable to cause host-cell shut-off prevented nsP2-mediated inhibition of the UPR. This indicates that initial UPR induction takes place in the ER but that expression of functional UPR transcription factors and target genes is efficiently inhibited by CHIKV nsP2. PMID:25395592

  3. Reduced Paneth cell antimicrobial protein levels correlate with activation of the unfolded protein response in the gut of obese individuals.

    PubMed

    Hodin, Caroline M; Verdam, Froukje J; Grootjans, Joep; Rensen, Sander S; Verheyen, Fons K; Dejong, Cornelis H C; Buurman, Wim A; Greve, Jan Willem; Lenaerts, Kaatje

    2011-10-01

    The intestinal microbiota is increasingly acknowledged to play a crucial role in the development of obesity. A shift in intestinal microbiota composition favouring the presence of Firmicutes over Bacteroidetes has been observed in obese subjects. A similar shift has been reported in mice with deficiency of active Paneth cell ?-defensins. We aimed at investigating changes in Paneth cell antimicrobial levels in the gut of obese subjects. Next, we studied activation of the unfolded protein response (UPR) as a possible mechanism involved in altered Paneth cell function. Paneth cell numbers were counted in jejunal sections of 15 severely obese (BMI > 35) and 15 normal weight subjects. Expression of Paneth cell antimicrobials human ?-defensin 5 (HD5) and lysozyme were investigated using immunohistochemistry, qPCR, and western blot. Activation of the UPR was assessed with western blot. Severely obese subjects showed decreased protein levels of both HD5 and lysozyme, while Paneth cell numbers were unchanged. Lysozyme protein levels correlated inversely with BMI. Increased expression of HD5 (DEFA5) and lysozyme (LYZ) transcripts in the intestine of obese subjects prompted us to investigate a possible translational block caused by UPR activation. Binding protein (BiP) and activating transcription factor 4 (ATF4) levels were increased, confirming activation of the UPR in the gut of obese subjects. Furthermore, levels of both proteins correlated with BMI. Involvement of the UPR in the lowered antimicrobial protein levels in obese subjects was strongly suggested by a negative correlation between BiP levels and lysozyme levels. Additionally, indications of ER stress were apparent in Paneth cells of obese subjects. Our findings provide the first evidence for altered Paneth cell function in obesity, which may have important implications for the obesity-associated shift in microbiota composition. In addition, we show activation of the UPR in the intestine of obese subjects, which may underlie the observed Paneth cell compromise. PMID:21630271

  4. Polynomial algebra reveals diverging roles of the unfolded protein response in endothelial cells during ischemia-reperfusion injury.

    PubMed

    Le Pape, Sylvain; Dimitrova, Elena; Hannaert, Patrick; Konovalov, Alexander; Volmer, Romain; Ron, David; Thuillier, Raphaël; Hauet, Thierry

    2014-08-25

    The unfolded protein response (UPR)--the endoplasmic reticulum stress response--is found in various pathologies including ischemia-reperfusion injury (IRI). However, its role during IRI is still unclear. Here, by combining two different bioinformatical methods--a method based on ordinary differential equations (Time Series Network Inference) and an algebraic method (probabilistic polynomial dynamical systems)--we identified the IRE1?-XBP1 and the ATF6 pathways as the main UPR effectors involved in cell's adaptation to IRI. We validated these findings experimentally by assessing the impact of their knock-out and knock-down on cell survival during IRI. PMID:24945730

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

    PubMed Central

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

    2013-01-01

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

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

    Microsoft Academic Search

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

    In heterologous protein production by filamentous fungi, target proteins are expressed as fusions with homologous secretory\\u000a proteins, called carriers, for higher production yields. Although carrier fusion is thought to overcome the bottleneck in\\u000a transcriptional and (post)translational processes during heterologous protein production, there is limited knowledge of its\\u000a physiological effects on the host strain. In this study, we performed DNA microarray

  7. 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

  8. Evaluation of the Unfolded Protein Response: PI 3-Kinase Regulatory Subunits as Regulators of the IRE1? Pathway

    PubMed Central

    Winnay, Jonathon N.; Kahn, C. Ronald

    2015-01-01

    The endoplasmic reticulum (ER) consists of an interconnected, membranous network that is the major site for the synthesis and folding of integral membrane and secretory proteins. Within the ER lumen, protein folding is facilitated by molecular chaperones and a variety of enzymes that ensure that polypeptides obtain their appropriate, tertiary conformation (1,2). Physiological conditions that increase protein synthesis or stimuli that disturb the processes by which proteins obtain their native conformation, create an imbalance between the protein-folding demand and capacity of the ER. This results in the accumulation of unfolded or improperly folded proteins in the ER lumen and a state of ER stress. The cellular response, referred to as the unfolded protein response (UPR), results in activation of three linked signal transduction pathways: PKR-like kinase (PERK), inositol requiring 1 ? (IRE1?) and activating transcription factor 6? (ATF6?) (3,4). Collectively, the combined actions of these signaling cascades serve to reduce ER stress through attenuation of translation to reduce protein synthesis and through activation of transcriptional programs that ultimately serve to increase ER protein folding capacity. Recently, we and Park et. al have characterized a novel function for the p85? and p85? subunits as modulators of the UPR by virtue of their ability to facilitate the nuclear entry of XBP-1s following induction of ER stress (5,6). This chapter describes the recently elucidated role for the regulatory subunits of PI 3-kinase as modulators of the UPR and provides methods to measure UPR pathway activation. PMID:21266249

  9. The Plant Cell, Vol. 15, 561576, February 2003, www.plantcell.org 2003 American Society of Plant Biologists Genomic Analysis of the Unfolded Protein Response

    E-print Network

    Chrispeels, Maarten J.

    are required (XBP-1, ATF6, and ATF4). In mammalian cells, 1% of the genome is induced as part of the UPR, Biologists Genomic Analysis of the Unfolded Protein Response in Arabidopsis Shows Its Connection to Important-associ- ated degradation proteins. Most of the downregulated genes encode extracellular proteins. Therefore

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

    Microsoft Academic Search

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

    2010-01-01

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

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

    PubMed Central

    Szokalska, Angelika; Makowski, Marcin; Nowis, Dominika; Wilczy?ski, Grzegorz M.; Kujawa, Marek; Wójcik, Cezary; M?ynarczuk-Bia?y, Izabela; Salwa, Pawel; Bil, Jacek; Janowska, Sylwia; Agostinis, Patrizia; Verfaillie, Tom; Bugajski, Marek; Gietka, Jan; Issat, Tadeusz; G?odkowska, Eliza; Mrówka, Piotr; Stoklosa, Tomasz; Hamblin, Michael R; Mróz, Pawe?; Jakóbisiak, Marek; Golab, Jakub

    2009-01-01

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

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

    PubMed Central

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

    2015-01-01

    Objective Inflammatory bowel diseases (IBDs) feature multiple cellular stress responses, including endoplasmic reticulum (ER) unfolded protein responses (UPRs). UPRs represent autoregulatory pathways that adjust organelle capacity to cellular demand. A similar mechanism, mitochondrial UPR (mtUPR), has been described for mitochondria. ER UPR in intestinal epithelial cells (IECs) contributes to the development of intestinal inflammation, and since mitochondrial alterations and dysfunction are implicated in the pathogenesis of IBDs, the authors characterised mtUPR in the context of intestinal inflammation. Methods Truncated ornithine transcarbamylase was used to selectively induce mtUPR in a murine IEC line. Dextran sodium sulphate (DSS) was administered to PKR (double-stranded-RNA-activated protein kinase) knockout mice to induce IEC stress in vivo and to test for their susceptibility to DSS-induced colitis. Expression levels of the mitochondrial chaperone chaperonin 60 (CPN60) and PKR were quantified in IECs from patients with IBDs and from murine models of colitis using immunohistochemistry and Western blot analysis. Results Selective mtUPR induction by truncated ornithine transcarbamylase transfection triggered the phosphorylation of eukaryotic translation initiation factor (eIF) 2? and cJun through the recruitment of PKR. Using pharmacological inhibitors and small inhibitory RNA, the authors identified mtUPR-induced eIF2? phosphorylation and transcription factor activation (cJun/AP1) as being dependent on the activities of the mitochondrial protease ClpP and the cytoplasmic kinase PKR. Pkr?/? mice failed to induce CPN60 in IECs upon DSS treatment at early time points and subsequently showed an almost complete resistance to DSS-induced colitis. Under inflammatory conditions, primary IECs from patients with IBDs and two murine models of colitis exhibited a strong induction of the mtUPR marker protein CPN60 associated with enhanced expression of PKR. Conclusion PKR integrates mtUPR into the disease-relevant ER UPR via eIF2? phosphorylation and AP1 activation. Induction of mtUPR and PKR was observed in IECs from murine models and patients with IBDs. The authors’ results indicate that PKR might link mitochondrial stress to intestinal inflammation. PMID:21997551

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

    PubMed

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

    2015-06-01

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

  14. 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

  15. 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

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

    PubMed Central

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

    2011-01-01

    SUMMARY Exercise has been shown to be effective for treating obesity and type 2 diabetes. However, the molecular mechanisms for adaptation to exercise training are not fully understood. Endoplasmic reticulum (ER) stress has been linked to metabolic dysfunction. Here we show that the unfolded protein response (UPR), an adaptive response pathway that maintains ER homeostasis upon luminal stress, is activated in skeletal muscle during exercise and adapts skeletal muscle to exercise training. The transcriptional coactivator PGC-1?, which regulates several exercise-associated aspects of skeletal muscle function, mediates the UPR in myotubes and skeletal muscle through coactivation of ATF6?. Efficient recovery from acute exercise is compromised in ATF6??/? mice. Blocking ER-stress related cell death via deletion of CHOP partially rescues the exercise intolerance phenotype in muscle-specific PGC-1? KO mice. These findings suggest that modulation of the UPR through PGC1? represents an alternative avenue to improve skeletal muscle function and achieve metabolic benefits. PMID:21284983

  17. 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

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

    PubMed

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

    2011-02-01

    Exercise has been shown to be effective for treating obesity and type 2 diabetes. However, the molecular mechanisms for adaptation to exercise training are not fully understood. Endoplasmic reticulum (ER) stress has been linked to metabolic dysfunction. Here we show that the unfolded protein response (UPR), an adaptive response pathway that maintains ER homeostasis upon luminal stress, is activated in skeletal muscle during exercise and adapts skeletal muscle to exercise training. The transcriptional coactivator PGC-1?, which regulates several exercise-associated aspects of skeletal muscle function, mediates the UPR in myotubes and skeletal muscle through coactivation of ATF6?. Efficient recovery from acute exercise is compromised in ATF6?(-/-) mice. Blocking ER-stress-related cell death via deletion of CHOP partially rescues the exercise intolerance phenotype in muscle-specific PGC-1? KO mice. These findings suggest that modulation of the UPR through PGC1? represents an alternative avenue to improve skeletal muscle function and achieve metabolic benefits. PMID:21284983

  19. Plastid-produced interorgannellar stress signal MEcPP potentiates induction of the unfolded protein response in endoplasmic reticulum.

    PubMed

    Walley, Justin; Xiao, Yanmei; Wang, Jin-Zheng; Baidoo, Edward E; Keasling, Jay D; Shen, Zhouxin; Briggs, Steven P; Dehesh, Katayoon

    2015-05-12

    Cellular homeostasis in response to internal and external stimuli requires a tightly coordinated interorgannellar communication network. We recently identified methylerythritol cyclodiphosphate (MEcPP) as a novel stress-specific retrograde signaling metabolite that accumulates in response to environmental perturbations to relay information from plastids to the nucleus. We now demonstrate, using a combination of transcriptome and proteome profiling approaches, that mutant plants (ceh1) with high endogenous levels of MEcPP display increased transcript and protein levels for a subset of the core unfolded protein response (UPR) genes. The UPR is an adaptive cellular response conserved throughout eukaryotes to stress conditions that perturb the endoplasmic reticulum (ER) homeostasis. Our results suggest that MEcPP directly triggers the UPR. Exogenous treatment with MEcPP induces the rapid and transient induction of both the unspliced and spliced forms of the UPR gene bZIP60. Moreover, compared with the parent background (P), ceh1 mutants are less sensitive to the ER-stress-inducing agent tunicamycin (Tm). P and ceh1 plants treated with Tm display similar UPR transcript profiles, suggesting that although MEcPP accumulation causes partial induction of selected UPR genes, full induction is triggered by accumulation of misfolded proteins. This finding refines our perspective of interorgannellar communication by providing a link between a plastidial retrograde signaling molecule and its targeted ensemble of UPR components in ER. PMID:25922532

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

    PubMed

    Valkonen, M; Penttilä, M; Saloheimo, M

    2004-11-01

    A signal transduction pathway called the unfolded protein response is activated when increased levels of misfolded proteins or incorrectly assembled subunits accumulate in the endoplasmic reticulum (ER). The expression of several genes for ER-resident foldases and chaperones, as well as genes encoding proteins that are involved in functions associated with the secretory process, are induced by this pathway. This paper describes the cloning and characterisation of genes for two components of the pathway, ire1 and ptc2, from the filamentous fungus Trichoderma reesei (Hypocrea jecorina). The data presented demonstrates that the T. reesei genes can complement Saccharomyces cerevisiae mutants that are deficient in the corresponding homologues. The T. reesei IREI protein has intrinsic kinase activity, as revealed by an in vitro autophosphorylation assay. Overexpression of ire1 in a T. reesei strain that expresses a foreign protein (laccase 1 from Phlebia radiata), results in up-regulation of the UPR pathway, as indicated by the increased expression levels of the known UPR target genes bip1 and pdi1. Splicing of the mRNA encoding the transcription factor HAC1 is also observed. Other genes encoding proteins from different parts of the secretory pathway also respond to ire1 overexpression. PMID:15480788

  1. An alternatively spliced heat shock transcription factor, OsHSFA2dI, functions in the heat stress-induced unfolded protein response in rice.

    PubMed

    Cheng, Q; Zhou, Y; Liu, Z; Zhang, L; Song, G; Guo, Z; Wang, W; Qu, X; Zhu, Y; Yang, D

    2015-03-01

    As sessile organisms, plants have evolved a wide range of defence pathways to cope with environmental stress such as heat shock. However, the molecular mechanism of these defence pathways remains unclear in rice. In this study, we found that OsHSFA2d, a heat shock transcriptional factor, encodes two main splice variant proteins, OsHSFA2dI and OsHSFA2dII in rice. Under normal conditions, OsHSFA2dII is the dominant but transcriptionally inactive spliced form. However, when the plant suffers heat stress, OsHSFA2d is alternatively spliced into a transcriptionally active form, OsHSFA2dI, which participates in the heat stress response (HSR). Further study found that this alternative splicing was induced by heat shock rather than photoperiod. We found that OsHSFA2dI is localised to the nucleus, whereas OsHSFA2dII is localised to the nucleus and cytoplasm. Moreover, expression of the unfolded protein response (UNFOLDED PROTEIN RESPONSE) sensors, OsIRE1, OsbZIP39/OsbZIP60 and the UNFOLDED PROTEIN RESPONSE marker OsBiP1, was up-regulated. Interestingly, OsbZIP50 was also alternatively spliced under heat stress, indicating that UNFOLDED PROTEIN RESPONSE signalling pathways were activated by heat stress to re-establish cellular protein homeostasis. We further demonstrated that OsHSFA2dI participated in the unfolded protein response by regulating expression of OsBiP1. PMID:25255693

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

    PubMed

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

    2015-08-01

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

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

    PubMed Central

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

    2012-01-01

    Asparagine-linked protein glycosylation is a hallmark for glycoprotein structure and function. Its impairment by tunicamycin [a competitive inhibitor of N-acetylglucosaminyl 1-phosphate transferase (GPT)] has been known to inhibit neo-vascularization (i.e., angiogenesis) in humanized breast tumor due to an induction of ER stress-mediated unfolded protein response (UPR). The studies presented here demonstrate that (i) tunicamycin (i) inhibits capillary endothelial cell proliferation in a dose dependent manner; (ii) treated cells are incapable of forming colonies upon its withdrawal; and (iii) tunicamycin treatment causes nuclear fragmentation. Tunicamycin-induced ER stress-mediated UPR event in these cells was studied with the aid of Raman spectroscopy, in particular, the interpretation of bands at 1672, 1684 and 1694 cm?1, which are characteristics of proteins and originate from C=O stretching vibrations of mono-substituted amides. In tunicamycin-treated cells these bands decreased in area as follows: at 1672 cm?1 by 41.85% at 3 h and 55.39% at 12 h; at 1684 cm?1 by 20.63% at 3 h and 40.08% at 12 h; and also at 1994 cm?1 by 33.33% at 3 h and 32.92% at 12 h, respectively. Thus, in the presence of tunicamycin, newly synthesized protein chains fail to arrange properly into their final secondary and/or tertiary structures, and the random coils they form had undergone further degradation. PMID:22936838

  4. 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

  5. Protection of injured retinal ganglion cell dendrites and unfolded protein response resolution after long-term dietary resveratrol.

    PubMed

    Lindsey, James D; Duong-Polk, Karen X; Hammond, Dustin; Leung, Christopher Kai-Shun; Weinreb, Robert N

    2015-05-01

    Long-term dietary supplementation with resveratrol protects against cardiovascular disease, osteoporesis, and metabolic decline. This study determined how long-term dietary resveratrol treatment protects against retinal ganglion cell (RGC) dendrite loss after optic nerve injury and alters the resolution of the unfolded protein response. Associated changes in markers of endoplasmic reticulum stress in RGCs also were investigated. Young-adult Thy1-yellow fluorescent protein (YFP) and C57BL/6 mice received either control diet or diet containing resveratrol for approximately 1 year. Both groups then received optic nerve crush (ONC). Fluorescent RGC dendrites in the Thy1-YFP mice were imaged weekly for 4 weeks after ONC. There was progressive loss of dendrite length in all RGC types within the mice that received control diet. Resveratrol delayed loss of dendrite complexity and complete dendrite loss for most RGC types. However, there were variations in the rate of retraction among different RGC types. Three weeks after ONC, cytoplasmic binding immunoglobulin protein (BiP) suppression observed in control diet ganglion cell layer neurons was reversed in mice that received resveratrol, nuclear C/EBP homologous protein (CHOP) was near baseline in control diet eyes but was moderately increased by resveratrol; and increased nuclear X-box-binding protein-1 (XBP-1) observed in control diet eyes was reduced in eyes that received resveratrol to the same level as in control diet uncrushed eyes. These results indicate that protection of dendrites by resveratrol after ONC differs among RGC types and suggest that alterations in long-term expression of binding immunoglobulin protein, CHOP, and XBP-1 may contribute to the resveratrol-mediated protection of RGC dendrites after ONC. PMID:25772060

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

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-04-01

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

  8. Unfolding protein response signaling is involved in development, maintenance, and regression of the corpus luteum during the bovine estrous cycle.

    PubMed

    Park, Hyo-Jin; Park, Sun-Ji; Koo, Deog-Bon; Kong, Il-Keun; Kim, Min Kyu; Kim, Jin-Man; Choi, Myung-Sook; Park, Young-Ho; Kim, Sun-Uk; Chang, Kyu-Tae; Park, Choon-Keun; Chae, Jung-Il; Lee, Dong-Seok

    2013-11-15

    The corpus luteum (CL) is a transient endocrine organ. Development, maintenance, and regression of CL are effectively controlled by dynamic changes in gene expression. However, it is unknown what types of gene are affected during the CL life span of the estrous cycle in bovine. Here, we determined whether unfolded protein response (UPR) signaling via eIF2?/ATF4/GADD34, p90ATF6/p50ATF6, and IRE1/XBP1, which is a cellular stress response associated with the endoplasmic reticulum (ER), is involved in the bovine CL life span. Our results indicated that expression of Grp78/Bip, the master UPR regulator, was increased during the maintenance stage and rapidly decreased at the regression stage. Additionally, UPR signaling pathways genes were found to be involved in luteal phase progression during the estrous cycle. Our findings suggested that Grp78/Bip, ATF6, and XBP1 act as ER chaperones for initiating CL development and maintaining the CL. In addition, we investigated whether ER stress-mediated apoptosis is occurred through three UPR signaling pathways in CL regression stage. Interestingly, pIRE1 and CHOP were found to be involved in both the adaptive response and ER stress-mediated apoptosis. During the CL regression stage, increased expression of pJNK and CHOP, two components of ER stress-mediated apoptotic cascades, occurred before increased level of cleaved caspase 3 were observed. The present investigation was performed to identify a functional link between UPR signaling and CL life span during the bovine estrous cycle. Taken together, results from this study demonstrated that UPR protein/gene expression levels were different at various stages of the bovine CL life span. Variations in the expression of these protein/genes may play important roles in luteal stage progression during the estrous cycle. PMID:24161737

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

    PubMed

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

    2014-11-26

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2012-01-01

    The molecular chaperone GRP78/BiP is a key regulator of protein folding in the endoplasmic reticulum, and it plays a pivotal role in cancer cell survival and chemoresistance. Inhibition of its function has therefore been an important strategy for inhibiting tumor cell growth in cancer therapy. Previous efforts to achieve this goal have used peptides that bind to GRP78/BiP conjugated to pro-drugs or cell-death-inducing sequences. Here, we describe a peptide that induces prostate tumor cell death without the need of any conjugating sequences. This peptide is a sequence derived from the cochaperone Bag-1. We have shown that this sequence interacts with and inhibits the refolding activity of GRP78/BiP. Furthermore, we have demonstrated that it modulates the unfolded protein response in ER stress resulting in PARP and caspase-4 cleavage. Prostate cancer cells stably expressing this peptide showed reduced growth and increased apoptosis in in vivo xenograft tumor models. Amino acid substitutions that destroyed binding of the Bag-1 peptide to GRP78/BiP or downregulation of the expression of GRP78 compromised the inhibitory effect of this peptide. This sequence therefore represents a candidate lead peptide for anti-tumor therapy. PMID:23049684

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

    PubMed Central

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

    2013-01-01

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

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

    PubMed

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

    2013-11-01

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

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

    PubMed Central

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

    2013-01-01

    SUMMARY Secretory pathway dysfunction and lipid accumulation (steatosis) are the two most common responses of hepatocytes to ethanol exposure and are major factors in the pathophysiology of alcoholic liver disease (ALD). However, the mechanisms by which ethanol elicits these cellular responses are not fully understood. Recent data indicates that activation of the unfolded protein response (UPR) in response to secretory pathway dysfunction can cause steatosis. Here, we examined the relationship between alcohol metabolism, oxidative stress, secretory pathway stress and steatosis using zebrafish larvae. We found that ethanol was immediately internalized and metabolized by larvae, such that the internal ethanol concentration in 4-day-old larvae equilibrated to 160 mM after 1 hour of exposure to 350 mM ethanol, with an average ethanol metabolism rate of 56 ?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

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

    PubMed

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

    2015-01-01

    Two ER membrane-resident transmembrane kinases, IRE1 and PERK, function as stress sensors in the unfolded protein response. IRE1 also has an endoribonuclease activity, which initiates a non-conventional mRNA splicing reaction, while PERK phosphorylates eIF2?. We engineered a potent small molecule, IPA, that binds to IRE1's ATP-binding pocket and predisposes the kinase domain to oligomerization, activating its RNase. IPA also inhibits PERK but, paradoxically, activates it at low concentrations, resulting in a bell-shaped activation profile. We reconstituted IPA-activation of PERK-mediated eIF2? phosphorylation from purified components. We estimate that under conditions of maximal activation less than 15% of PERK molecules in the reaction are occupied by IPA. We propose that IPA binding biases the PERK kinase towards its active conformation, which trans-activates apo-PERK molecules. The mechanism by which partial occupancy with an inhibitor can activate kinases may be wide-spread and carries major implications for design and therapeutic application of kinase inhibitors. PMID:25986605

  16. EBNA3C interacts with Gadd34 and counteracts the unfolded protein response

    Microsoft Academic Search

    Jose L Garrido; Seijii Maruo; Kenzo Takada; Adam Rosendorff

    2009-01-01

    EBNA3C is an EBV-encoded nuclear protein, essential for proliferation of EBV infected B-lymphocytes. Using EBNA3C amino acids 365-545 in a yeast two hybrid screen, we found an interaction with the Growth Arrest and DNA-damage protein, Gadd34. When both proteins are overexpressed, Gadd34 can interact with EBNA3C in both nuclear and cytoplasmic compartments. Amino acids 483-610 of Gadd34, including the two

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

    PubMed Central

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

    2011-01-01

    Up-regulation of the dolichol pathway, a “hallmark” of asparagine-linked protein glycosylation, enhances angiogenesis in vitro. The dynamic relationship between these two processes is now evaluated with tunicamycin. Capillary endothelial cells treated with tunicamycin were growth inhibited and could not be reversed with exogenous VEGF165. Inhibition of angiogenesis is supported by down-regulation of (i) phosphorylated VEGFR1 and VEGFR2 receptors; (ii) VEGF165-specific phosphotyrosine kinase activity; and (iii) MatrigelTM invasion and chemotaxis. In vivo, tunicamycin prevented the vessel development in MatrigelTM implants in athymic Balb/c (nu/nu) mice. Immunohistochemical analysis of CD34 (p < 0.001) and CD144 (p < 0.001) exhibited reduced vascularization. A 3.8-fold increased expression of TSP-1, an endogenous angiogenesis inhibitor in MatrigelTM implants correlated with that in tunicamycin (32 h)-treated capillary endothelial cells. Intravenous injection of tunicamycin (0.5 mg/kg to 1.0 mg/kg) per week slowed down a double negative (MDA-MB-435) grade III breast adenocarcinoma growth by ?50–60% in 3 weeks. Histopathological analysis of the paraffin sections indicated significant reduction in vessel size, the microvascular density and tumor mitotic index. Ki-67 and VEGF expression in tumor tissue were also reduced. A significant reduction of N-glycan expression in tumor microvessel was also observed. High expression of GRP-78 in CD144-positive cells supported unfolded protein response-mediated ER stress in tumor microvasculature. ?65% reduction of a triple negative (MDA-MB-231) breast tumor xenograft in 1 week with tunicamycin (0.25 mg/kg) given orally and the absence of systemic and/or organ failure strongly supported tunicamycin's potential for a powerful glycotherapeutic treatment of breast cancer in the clinic. PMID:21676868

  18. 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 ...

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

    PubMed Central

    Jwa, Miri; Chang, Paul

    2012-01-01

    Summary Poly(ADP-ribose) polymerases (PARPs) (also known as ADP-ribosyl transferase D proteins) modify acceptor proteins with ADP-ribose modifications of varying length (reviewed in refs 1–3). PARPs regulate key stress response pathways, including DNA damage repair and the cytoplasmic stress response2,3,4,5,6. Here, we show that PARPs also regulate the unfolded protein response (UPR) of the endoplasmic reticulum (ER). Human PARP16/ARTD15 is a tail-anchored ER transmembrane protein required for activation of the functionally related ER stress sensors PERK and IRE1? during the UPR. The third identified ER stress sensor, ATF6, is not regulated by PARP16. Similar to other PARPs that function during stress, PARP16 enzymatic activity is up-regulated during ER stress when it (ADP-ribosyl)ates itself, PERK and IRE1?. (ADP-ribosyl)ation by PARP16 is sufficient for activating PERK and IRE1? in the absence of ER stress, and is required for PERK and IRE1 ? activation during the UPR. Modification of PERK and IRE1? by PARP16 increases their kinase activities and the endonuclease activity of IRE1?. Interestingly, the C-terminal luminal tail of PARP16 is required for PARP16 function during ER stress, suggesting that it transduces stress signals to the cytoplasmic PARP catalytic domain. PMID:23103912

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

    PubMed

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

    2013-05-01

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

  1. The unfolded protein response : integrating stress signals from the endoplasmic reticulum to the nucleolus

    E-print Network

    DuRose, Jenny Bratlien

    2008-01-01

    of rRNA transcription and protein translation with growthprotein. Figure 15. Model of PERK Pathway Controlling rRNA Transcription and Translationprotein synthesis and rRNA transcription occurs simultaneously. The major pathway known to simultaneously regulate rRNA transcription and translation

  2. 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.

  3. The unfolded protein response regulator GRP78/BiP is required for endoplasmic

    E-print Network

    Bedwell, David M.

    responsables du stage : Charis Quay E-mail : charis.quay@u-psud.fr Téléphone : 01 69 15 53 62 Page web : http://users.lps.u-psud.fr/NS2/ Possibilité de poursuivre en thèse : oui non Titre : Vie et mort de spins et charges dans un

  4. Involvement of selective reactive oxygen species upstream of proapoptotic branches of unfolded protein response.

    PubMed

    Yokouchi, Makiko; Hiramatsu, Nobuhiko; Hayakawa, Kunihiro; Okamura, Maro; Du, Shuqi; Kasai, Ayumi; Takano, Yosuke; Shitamura, Akihiro; Shimada, Tsuyoshi; Yao, Jian; Kitamura, Masanori

    2008-02-15

    Cadmium triggers apoptosis of LLC-PK1 cells through induction of endoplasmic reticulum (ER) stress. We found that cadmium caused generation of reactive oxygen species (ROS) and that cadmium-induced ER stress was inhibited by antioxidants. In contrast, suppression of ER stress did not attenuate cadmium-triggered oxidative stress, suggesting that ER stress occurs downstream of oxidative stress. Exposure of the cells to either O(2)(*), H(2)O(2), or ONOO(-) caused apoptosis, whereas ER stress was induced only by O(2)(*) or ONOO(-). Transfection with manganese superoxide dismutase significantly attenuated cadmium-induced ER stress and apoptosis, whereas pharmacological inhibition of ONOO(-) was ineffective. Interestingly, transfection with catalase attenuated cadmium-induced apoptosis without affecting the level of ER stress. O(2)(*) caused activation of the activating transcription factor 6-CCAAT/enhancer-binding protein-homologous protein (CHOP) and the inositol-requiring ER-to-nucleus signal kinase 1-X-box-binding protein 1 (XBP1) proapoptotic cascades, and overexpression of manganese superoxide dismutase attenuated cadmium-triggered induction of both pathways. Furthermore, phosphorylation of proapoptotic c-Jun N-terminal kinase by O(2)(*) or cadmium was suppressed by dominant-negative inhibition of XBP1. These data elucidated 1) cadmium caused ER stress via generation of ROS, 2) O(2)(*) was selectively involved in cadmium-triggered, ER stress-mediated apoptosis through activation of the activating transcription factor 6-CHOP and inositol-requiring ER-to-nucleus signal kinase 1-XBP1 pathways, and 3) phosphorylation of JNK was caused by O(2)(*)-triggered activation of XBP1. PMID:18086661

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

    PubMed Central

    2011-01-01

    Background To investigate the molecular and cellular pathogenesis underlying myocarditis, we used an experimental autoimmune myocarditis (EAM)-induced heart failure rat model that represents T cell mediated postinflammatory heart disorders. Results By performing unbiased 2-dimensional electrophoresis of protein extracts from control rat heart tissues and EAM rat heart tissues, followed by nano-HPLC-ESI-QIT-MS, 67 proteins were identified from 71 spots that exhibited significantly altered expression levels. The majority of up-regulated proteins were confidently associated with unfolded protein responses (UPR), while the majority of down-regulated proteins were involved with the generation of precursor metabolites and energy metabolism in mitochondria. Although there was no difference in AKT signaling between EAM rat heart tissues and control rat heart tissues, the amounts and activities of extracellular signal-regulated kinase (ERK)-1/2 and ribosomal protein S6 (rpS6) were significantly increased. By comparing our data with the previously reported myocardial proteome of the Coxsackie viruses of group B (CVB)-mediated myocarditis model, we found that UPR-related proteins were commonly up-regulated in two murine myocarditis models. Even though only two out of 29 down-regulated proteins in EAM rat heart tissues were also dysregulated in CVB-infected rat heart tissues, other proteins known to be involved with the generation of precursor metabolites and energy metabolism in mitochondria were also dysregulated in CVB-mediated myocarditis rat heart tissues, suggesting that impairment of mitochondrial functions may be a common underlying mechanism of the two murine myocarditis models. Conclusions UPR, ERK-1/2 and S6RP signaling were activated in both EAM- and CVB-induced myocarditis murine models. Thus, the conserved components of signaling pathways in two murine models of acute myocarditis could be targets for developing new therapeutic drugs or methods aimed at treating enigmatic myocarditis. PMID:22014063

  6. The Unfolded Protein Response Transducer ATF6 Represents a Novel Transmembrane-type Endoplasmic Reticulum-associated Degradation Substrate Requiring Both Mannose Trimming and SEL1L Protein*

    PubMed Central

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

    2013-01-01

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

  7. Extracellular deposition of mouse senile AApoAII amyloid fibrils induced different unfolded protein responses in the liver, kidney, and heart.

    PubMed

    Luo, Hongmin; Sawashita, Jinko; Tian, Geng; Liu, Yingye; Li, Lin; Ding, Xin; Xu, Zhe; Yang, Mu; Miyahara, Hiroki; Mori, Masayuki; Qian, Jinze; Wang, Yaoyong; Higuchi, Keiichi

    2015-03-01

    Mouse senile amyloidosis is a disorder in which apolipoprotein A-II deposits extracellularly in many organs as amyloid fibrils (AApoAII). In this study, we intravenously injected 1??g of isolated AApoAII fibrils into R1.P1-Apoa2(c) mice, to induce AApoAII amyloidosis. We observed that the unfolded protein response was induced by deposition of AApoAII amyloid. We found that the mRNA and the protein expression levels of heat shock protein A5 (HSPA5; also known as glucose-regulated protein 78) were increased in the liver with AApoAII amyloid deposits. Immunohistochemistry showed that HSPA5 was only detected in hepatocytes close to AApoAII amyloid deposits. Furthermore, gene transcription of several endoplasmic reticulum (ER) stress-related proteins increased, including eukaryotic translation initiation factor 2 alpha kinase 3 (Eif2ak3), activating transcription factor 6 (Atf6), activating transcription factor 4 (Atf4), X-box-binding protein 1 splicing (Xbp1s), DNA-damage inducible transcript 3 (Ddit3), and autophagy protein 5 (Atg5). Moreover, apoptosis-positive cells were increased in the liver. Similar results were seen in the kidney but not in the heart. Our study indicates that ER stress responses differed among tissues with extracellular AApoAII amyloid fibril deposition. Although upregulated HSPA5 and the activated unfolded protein response might have roles in protecting tissues against aggregated extracellular AApoAII amyloid deposition, prolonged ER stress induced apoptosis in the liver and the kidney. PMID:25545477

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

    SciTech Connect

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

    2010-03-08

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

  9. Knockdown of glucose-regulated protein 78 abrogates chemoresistance of hypopharyngeal carcinoma cells to cisplatin induced by unfolded protein in response to severe hypoxia

    PubMed Central

    PI, LIHONG; LI, XIAOMING; SONG, QI; SHEN, YUPENG; LU, XIUYING; DI, BIN

    2014-01-01

    Hypoxia renders tumor cells with reduced sensitivity and increased resistance to chemotherapeutic agents. One of the possible mechanisms underlying this unfavorable status is activation of the unfolded protein response (UPR) under hypoxic conditions, due to the upregulation of glucose-regulated protein 78 (GRP78) expression. GRP78, an endoplasmic reticulum chaperone protein and a key regulator of the UPR, has been reported to be overexpressed in various types of cancer. However, the role of GRP78 in regulating the cell growth and apoptosis of hypopharyngeal carcinoma cells, with regard to the severity of hypoxia, remains unclear. Therefore, the aim of the present study was to investigate whether, and under what circumstances, GRP78 is associated with hypoxia-induced chemoresistance in hypopharyngeal carcinoma. For this purpose, cells from the FaDu human hypopharyngeal carcinoma cell line were cultured under normoxic and hypoxic conditions for different time periods. No significant changes in GRP78 and C/EBP homology protein (CHOP) protein expression levels were revealed under moderately hypoxic conditions (oxygen concentration, 1%), but these levels were changed over time under severely hypoxic conditions (oxygen concentration, <0.02%). This indicated that severe hypoxia, rather than moderate hypoxia, leads to UPR activation in hypopharyngeal carcinoma cells. Knockdown of GRP78 with short hairpin RNA inhibited cell proliferation and promoted apoptosis under severely hypoxic conditions, even with cisplatin treatment, indicating that GRP78 confers FaDu cells resistant to chemotherapy in response to severe hypoxia. Furthermore, knockdown of GRP78 resulted in a significant increase in CHOP and Bax expression levels and a decrease in Bcl-2 expression levels with simultaneous increase in the levels of apoptosis under severely hypoxic conditions. It was concluded that severe hypoxia leads to UPR activation and elevation of GRP78 expression, promoting cell survival and inducing chemoresistance. Silencing of GRP78 may block the pro-survival arm of UPR, simultaneously promoting proapoptotic signaling through induction of CHOP. Downregulation of GRP78 may be a promising strategy for overcoming the resistance of hypopharyngeal cancer to chemotherapy. PMID:24527073

  10. Charge effects on folded and unfolded proteins.

    PubMed

    Stigter, D; Dill, K A

    1990-02-01

    We develop a theory for the effects of charge on the stabilization of globular proteins. The folding process is modeled as occurring through a fictitious intermediate state along a two-part thermodynamic pathway in which the molecule (i) increases its density and then (ii) rearranges its ionic groups to the protein surface. The equilibrium for the binding of protons in salt solutions is assumed to be driven by the electrical potential due to the charge distribution, in addition to the intrinsic binding affinity and bulk proton concentration. The potential is calculated for inside and outside a porous sphere model of the protein using the Poisson-Boltzmann relation, wherein the interior dielectric constant is taken to be a linear function of the chain density. The model predicts the slope of the titration curves for native myoglobin in agreement with experiments by Breslow and Gurd (1962). From the similar experiments on the unfolded state, and from the experiments of Privalov et al. (1986) on the intrinsic viscosity of the unfolded molecules, the theory shows that the unfolded state has a much higher density than a chain in a theta solvent and that the density increases with ionic strength. In addition, from the free energy of proton binding to the protein, we also calculate the electrostatic contributions to protein stability, a major contribution deriving from changes in ionization. We consider the example of the stability of myoglobin as a function of pH, ionic strength, and ionic groups buried in the native protein structure. We show that although maximum stability of most proteins should occur at their isoelectric point, the burial of nontitratable groups should lead to maximum stabilities at pH values other than the isoelectric point. PMID:2322561

  11. Ask1 gene deletion blocks maternal diabetes-induced endoplasmic reticulum stress in the developing embryo by disrupting the unfolded protein response signalosome.

    PubMed

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

    2015-03-01

    Apoptosis signal-regulating kinase 1 (ASK1) is activated by various stresses. The link between ASK1 activation and endoplasmic reticulum (ER) stress, two causal events in diabetic embryopathy, has not been determined. We sought to investigate whether ASK1 is involved in the unfolded protein response (UPR) that leads to ER stress. Deleting Ask1 abrogated diabetes-induced UPR by suppressing phosphorylation of inositol-requiring enzyme 1? (IRE1?), and double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK) blocked the mitochondrial translocation of proapoptotic Bcl-2 members and ER stress. ASK1 participated in the IRE1? signalosome, and removing ASK1 abrogated the proapoptotic kinase activity of IRE1?. Ask1 deletion suppressed diabetes-induced IRE1? endoriboneclease activities, which led to X-box binding protein 1 mRNA cleavage, an ER stress marker, decreased expression of microRNAs, and increased expression of a miR-17 target, thioredoxin-interacting protein (Txnip), a thioredoxin binding protein, which enhanced ASK1 activation by disrupting the thioredoxin-ASK1 complexes. ASK1 is essential for the assembly and function of the IRE1? signalosome, which forms a positive feedback loop with ASK1 through Txnip. ASK1 knockdown in C17.2 neural stem cells diminished high glucose- or tunicamycin-induced IRE1? activation, which further supports our hypothesis that ASK1 plays a causal role in diabetes-induced ER stress and apoptosis. PMID:25249581

  12. What is the Origin of Internal Friction in Unfolded Proteins?

    NASA Astrophysics Data System (ADS)

    Papoian, Garegin; Echeverria, Ignacia

    2014-03-01

    The unfolded state is being increasingly recognized as critical to many biological processes. There are many proteins that are found only transiently in the unfolded state, eventually folding into globular structures. Other proteins, called intrinsically disordered proteins (IDPs), may be unfolded even when carrying out important biological functions. Despite its ubiquitousness, the unfolded ensemble is not fully understood. In this work, we have investigated the origin of friction for the unfolded proteins undergoing conformational diffusion. We used extensive all-atom molecular dynamics simulations to study the dynamics of the unfolded cold-shock protein (CSP) from Thermotoga maritima at different solvent viscosities and at different denaturant concentrations. We systematically analyzed the reconfiguration dynamics of relevant structural features such as dihedral angle rotations, hydrogen bonds and hydrophobic contacts forming and breaking. The results of our calculations are broadly consistent with the corresponding experimental measurements. Our findings have important implications for the folding kinetics of proteins, especially under physiological conditions.

  13. Characterization of Protein Unfolding with Solid-state Nanopores

    PubMed Central

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

    2014-01-01

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

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

    PubMed

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

    2014-09-15

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

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

    PubMed Central

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

    2014-01-01

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

  16. Stimulation of surface IgM of chronic lymphocytic leukemia cells induces an unfolded protein response dependent on BTK and SYK

    PubMed Central

    Steele, Andrew J.; Coelho, Vania; Linley, Adam; Sanchez Hidalgo, Marina; Carter, Matthew; Potter, Kathleen N.; Kennedy, Benjamin; Duncombe, Andrew S.; Ashton-Key, Margaret; Forconi, Francesco; Stevenson, Freda K.; Packham, Graham

    2014-01-01

    B-cell receptor (BCR) signaling plays a key role in the behavior of chronic lymphocytic leukemia (CLL). However, cellular consequences of signaling are incompletely defined. Here we explored possible links between BCR signaling and the unfolded protein response (UPR), a stress response pathway that can promote survival of normal and malignant cells. Compared with normal B cells, circulating CLL cells expressed increased, but variable, levels of UPR components. Higher expression of CHOP and XBP1 RNAs was associated with more aggressive disease. UPR activation appeared due to prior tissue-based antigenic stimulation because elevated expression of UPR components was detected within lymph node proliferation centers. Basal UPR activation also correlated closely with surface immunoglobulin M (sIgM) signaling capacity in vitro in both IGHV unmutated CLL and within mutated CLL. sIgM signaling increased UPR activation in vitro with responders showing increased expression of CHOP and XBP1 RNAs, and PERK and BIP proteins, but not XBP1 splicing. Inhibitors of BCR-associated kinases effectively prevented sIgM-induced UPR activation. Overall, this study demonstrates that sIgM signaling results in activation of some components the UPR in CLL cells. Modulation of the UPR may contribute to variable clinical behavior, and its inhibition may contribute to clinical responses to BCR-associated kinase inhibitors. PMID:25170122

  17. 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

  18. 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

  19. 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.

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

    PubMed

    Rojas-Rivera, D; Armisén, R; Colombo, A; Martínez, G; Eguiguren, A L; Díaz, A; Kiviluoto, S; Rodríguez, D; Patron, M; Rizzuto, R; Bultynck, G; Concha, M L; Sierralta, J; Stutzin, A; Hetz, C

    2012-06-01

    Transmembrane BAX inhibitor motif-containing (TMBIM)-6, also known as BAX-inhibitor 1 (BI-1), is an anti-apoptotic protein that belongs to a putative family of highly conserved and poorly characterized genes. Here we report the function of TMBIM3/GRINA in the control of cell death by endoplasmic reticulum (ER) stress. Tmbim3 mRNA levels are strongly upregulated in cellular and animal models of ER stress, controlled by the PERK signaling branch of the unfolded protein response. TMBIM3/GRINA synergies with TMBIM6/BI-1 in the modulation of ER calcium homeostasis and apoptosis, associated with physical interactions with inositol trisphosphate receptors. Loss-of-function studies in D. melanogaster demonstrated that TMBIM3/GRINA and TMBIM6/BI-1 have synergistic activities against ER stress in vivo. Similarly, manipulation of TMBIM3/GRINA levels in zebrafish embryos revealed an essential role in the control of apoptosis during neuronal development and in experimental models of ER stress. These findings suggest the existence of a conserved group of functionally related cell death regulators across species beyond the BCL-2 family of proteins operating at the ER membrane. PMID:22240901

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

    PubMed

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

    2013-10-01

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

  2. 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

  3. Fasudil inhibits ER stress-induced VCAM-1 expression by modulating unfolded protein response in endothelial cells.

    PubMed

    Kawanami, Daiji; Matoba, Keiichiro; Okada, Rina; Tsukamoto, Masami; Kinoshita, Jun; Ishizawa, Sho; Kanazawa, Yasushi; Yokota, Tamotsu; Utsunomiya, Kazunori

    2013-05-31

    The process of atherosclerosis is affected by interactions among numerous biological pathways. Accumulating evidence shows that endoplasmic reticulum (ER) stress plays a crucial role in the development of atherosclerosis. Rho-kinase is an effector of small GTP-binding protein Rho, and has been implicated as an atherogenic factor. Previous studies demonstrated that fasudil, a specific Rho-kinase inhibitor, exerts a cardioprotective effect by downregulating ER stress signaling. However, the molecular link between ER stress and Rho-kinase in endothelial cells has not been elucidated. In this study, we investigated the mechanisms by which fasudil regulates endothelial inflammation during ER stress. Tunicamycin, an established ER stress inducer, increased vascular cellular adhesion molecule (VCAM)-1 expression in endothelial cells. Intriguingly, fasudil inhibited VCAM-1 induction. From a mechanistic stand point, fasudil inhibited expression of activating transcription factor (ATF)4 and subsequent C/EBP homologous protein (CHOP) induction by tunicamycin. Furthermore, fasudil attenuated tunicamycin-induced phophorylation of p38MAPK that is crucial for the atherogenic response during ER stress. These findings indicate that Rho-kinase regulates ER stress-mediated VCAM-1 induction by ATF4- and p38MAPK-dependent signaling pathways. Rho-kinase inhibition by fasudil would be an important therapeutic approach against atherosclerosis, in particular, under conditions of ER stress. PMID:23665024

  4. 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

  5. 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…

  6. Yip1A, a novel host factor for the activation of the IRE1 pathway of the unfolded protein response during Brucella infection.

    PubMed

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

    2015-03-01

    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

  7. 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

  8. Taxol-induced unfolded protein response activation in breast cancer cells exposed to hypoxia: ATF4 activation regulates autophagy and inhibits apoptosis.

    PubMed

    Notte, Annick; Rebucci, Magali; Fransolet, Maude; Roegiers, Edith; Genin, Marie; Tellier, Celine; Watillon, Kassandra; Fattaccioli, Antoine; Arnould, Thierry; Michiels, Carine

    2015-05-01

    Understanding the mechanisms responsible for the resistance against chemotherapy-induced cell death is still of great interest since the number of patients with cancer increases and relapse is commonly observed. Indeed, the development of hypoxic regions as well as UPR (unfolded protein response) activation is known to promote cancer cell adaptive responses to the stressful tumor microenvironment and resistance against anticancer therapies. Therefore, the impact of UPR combined to hypoxia on autophagy and apoptosis activation during taxol exposure was investigated in MDA-MB-231 and T47D breast cancer cells. The results showed that taxol rapidly induced UPR activation and that hypoxia modulated taxol-induced UPR activation differently according to the different UPR pathways (PERK, ATF6, and IRE1?). The putative involvement of these signaling pathways in autophagy or in apoptosis regulation in response to taxol exposure was investigated. However, while no link between the activation of these three ER stress sensors and autophagy or apoptosis regulation could be evidenced, results showed that ATF4 activation, which occurs independently of UPR activation, was involved in taxol-induced autophagy completion. In addition, an ATF4-dependent mechanism leading to cancer cell adaptation and resistance against taxol-induced cell death was evidenced. Finally, our results demonstrate that expression of ATF4, in association with hypoxia-induced genes, can be used as a biomarker of a poor prognosis for human breast cancer patients supporting the conclusion that ATF4 might play an important role in adaptation and resistance of breast cancer cells to chemotherapy in hypoxic tumors. PMID:25724736

  9. AtBAG7, an Arabidopsis Bcl-2–associated athanogene, resides in the endoplasmic reticulum and is involved in the unfolded protein response

    PubMed Central

    Williams, Brett; Kabbage, Mehdi; Britt, Robert; Dickman, Martin B.

    2010-01-01

    The Bcl-2–associated athanogene (BAG) family is an evolutionarily conserved, multifunctional group of cochaperones that perform diverse cellular functions ranging from proliferation to growth arrest and cell death in yeast, in mammals, and, as recently observed, in plants. The Arabidopsis genome contains seven homologs of the BAG family, including four with domain organization similar to animal BAGs. In the present study we show that an Arabidopsis BAG, AtBAG7, is a uniquely localized endoplasmic reticulum (ER) BAG that is necessary for the proper maintenance of the unfolded protein response (UPR). AtBAG7 was shown to interact directly in vivo with the molecular chaperone, AtBiP2, by bimolecular fluorescence complementation assays, and the interaction was confirmed by yeast two-hybrid assay. Treatment with an inducer of UPR, tunicamycin, resulted in accelerated cell death of AtBAG7-null mutants. Furthermore, AtBAG7 knockouts were sensitive to known ER stress stimuli, heat and cold. In these knockouts heat sensitivity was reverted successfully to the wild-type phenotype with the addition of the chemical chaperone, tauroursodexycholic acid (TUDCA). Real-time PCR of ER stress proteins indicated that the expression of the heat-shock protein, AtBiP3, is selectively up-regulated in AtBAG7-null mutants upon heat and cold stress. Our results reveal an unexpected diversity of the plant's BAG gene family and suggest that AtBAG7 is an essential component of the UPR during heat and cold tolerance, thus confirming the cytoprotective role of plant BAGs. PMID:20231441

  10. A Closer Look on Protein Unfolding Simulations through Hierarchical Clustering

    Microsoft Academic Search

    Pedro Gabriel Ferreira; Candida G. Silvay; Rui M. M. Britoy; Paulo J. Azevedo

    Understanding protein folding and unfolding mech- anisms are a central problem in molecular biology. Data obtained from molecular dynamics unfolding simulations may provide valuable insights for a better understanding of these mechanisms. Here, we propose the application of an augmented version of hierarchical clustering analysis to detect clusters of amino- acid residues with similar bahavior. These clusters hold similar global

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

    PubMed Central

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

    2014-01-01

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

  12. Alzheimer's disease-related peptide PS2V plays ancient, conserved roles in suppression of the unfolded protein response under hypoxia and stimulation of ?-secretase activity.

    PubMed

    Moussavi Nik, Seyyed Hani; Newman, Morgan; Wilson, Lachlan; Ebrahimie, Esmaeil; Wells, Simon; Musgrave, Ian; Verdile, Giuseppe; Martins, Ralph N; Lardelli, Michael

    2015-07-01

    The PRESENILIN1 and PRESENILIN2 genes encode structurally related proteases essential for ?-secretase activity. Of nearly 200 PRESENILIN mutations causing early onset, familial Alzheimer's disease (FAD) only the K115Efx10 mutation of PSEN2 causes truncation of the open reading frame. If translated, the truncated product would resemble a naturally occurring isoform of PSEN2 named PS2V that is induced by hypoxia and found at elevated levels in late onset Alzheimer's disease (AD) brains. The function of PS2V is largely unexplored. We show that zebrafish possess a PS2V-like isoform, PS1IV, produced from the fish's PSEN1 rather than PSEN2 orthologous gene. The molecular mechanism controlling formation of PS2V/PS1IV was probably present in the ancient common ancestor of the PSEN1 and PSEN2 genes. Human PS2V and zebrafish PS1IV have highly divergent structures but conserved abilities to stimulate ?-secretase activity and to suppress the unfolded protein response (UPR) under hypoxia. The putative protein truncation caused by K115Efx10 resembles PS2V in its ability to increase ?-secretase activity and suppress the UPR. This supports increased A? levels as a common link between K115Efx10 early onset AD and sporadic, late onset AD. The ability of mutant variants of PS2V to stimulate ?-secretase activity partially correlates with their ability to suppress the UPR. The cytosolic, transmembrane and luminal domains of PS2V are all critical to its ?-secretase and UPR-suppression activities. Our data support a model in which chronic hypoxia in aged brains promotes excessive Notch signalling and accumulation of A? that contribute to AD pathogenesis. PMID:25814654

  13. Order Statistics Theory of Unfolding of Multimeric Proteins

    PubMed Central

    Zhmurov, A.; Dima, R.I.; Barsegov, V.

    2010-01-01

    Dynamic force spectroscopy has become indispensable for the exploration of the mechanical properties of proteins. In force-ramp experiments, performed by utilizing a time-dependent pulling force, the peak forces for unfolding transitions in a multimeric protein (D)N are used to map the free energy landscape for unfolding for a protein domain D. We show that theoretical modeling of unfolding transitions based on combining the observed first (f1), second (f2), …, Nth (fN) unfolding forces for a protein tandem of fixed length N, and pooling the force data for tandems of different length, n1 < n2 < … < N, leads to an inaccurate estimation of the distribution of unfolding forces for the protein D, ?D(f). This problem can be overcome by using Order statistics theory, which, in conjunction with analytically tractable models, can be used to resolve the molecular characteristics that determine the unfolding micromechanics. We present a simple method of estimation of the parent distribution, ?D(f), based on analyzing the force data for a tandem (D)n of arbitrary length n. Order statistics theory is exemplified through a detailed analysis and modeling of the unfolding forces obtained from pulling simulations of the monomer and oligomers of the all-?-sheet WW domain. PMID:20858442

  14. Employing Multiple Spectroscopic Techniques Simultaneously to Observe Protein Unfolding

    NASA Astrophysics Data System (ADS)

    Crowe, Michael; Kelty, Ben; Link, Justin

    2015-03-01

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

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

    PubMed

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

    2015-06-17

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

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

    PubMed Central

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

    2014-01-01

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

  17. An alpha 2 collagen VIII transgenic knock-in mouse model of Fuchs endothelial corneal dystrophy shows early endothelial cell unfolded protein response and apoptosis

    PubMed Central

    Jun, Albert S.; Meng, Huan; Ramanan, Naren; Matthaei, Mario; Chakravarti, Shukti; Bonshek, Richard; Black, Graeme C.M.; Grebe, Rhonda; Kimos, Martha

    2012-01-01

    Fuchs endothelial corneal dystrophy (FECD) is a leading indication for corneal transplantation. FECD is characterized by progressive alterations in endothelial cell morphology, excrescences (guttae) and thickening of the endothelial basement membrane and cell death. Ultimately, these changes lead to corneal edema and vision loss. Due to the lack of vision loss in early disease stages and the decades long disease course, early pathophysiology in FECD is virtually unknown as studies of pathologic tissues have been limited to end-stage tissues obtained at transplant. The first genetic defect shown to cause FECD was a point mutation causing a glutamine to lysine substitution at amino acid position 455 (Q455K) in the alpha 2 collagen 8 gene (COL8A2) which results in an early onset form of the disease. Homozygous mutant knock-in mice with this mutation (Col8a2Q455K/Q455K) show features strikingly similar to human disease, including progressive alterations in endothelial cell morphology, cell loss and basement membrane guttae. Ultrastructural analysis shows the predominant defect as dilated endoplasmic reticulum (ER), suggesting ER stress and unfolded protein response (UPR) activation. Immunohistochemistry, western blotting, quantitative reverse transcriptase polymerase chain reaction and terminal deoxynucleotidyl transferase 2?-deoxyuridine, 5?-triphosphate nick end-labeling analyses support UPR activation and UPR-associated apoptosis in the Col8a2Q455K/Q455K mutant corneal endothelium. This study confirms the Q455K substitution in the COL8A2 gene as being sufficient to cause FECD in the first mouse model of this disease and supports the role of the UPR and UPR-associated apoptosis in the pathogenesis of FECD caused by COL8A2 mutations. PMID:22002996

  18. 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

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

    PubMed

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

    2012-10-01

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

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

    PubMed Central

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

    2013-01-01

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

  1. Differential activation of placental unfolded protein response pathways implies heterogeneity in causation of early- and late-onset pre-eclampsia.

    PubMed

    Yung, Hong Wa; Atkinson, Daniel; Campion-Smith, Tim; Olovsson, Matts; Charnock-Jones, D Stephen; Burton, Graham J

    2014-10-01

    Based on gestational age at diagnosis and/or delivery, pre-eclampsia (PE) is commonly divided into early-onset (<34 weeks) and late-onset (?34 weeks) forms. Recently, the distinction between 'placental' and 'maternal' causation has been proposed, with 'placental' cases being more frequently associated with early-onset and intrauterine growth restriction. To test whether molecular placental pathology varies according to clinical presentation, we investigated stress-signalling pathways, including unfolded protein response (UPR) pathways, MAPK stress pathways, heat-shock proteins and AMPK? in placentae delivered by caesarean section for clinical indications at different gestational ages. Controls included second-trimester, pre-term and normal-term placentae. BeWo cells were used to investigate how these pathways react to different severities of hypoxia-reoxygenation (H/R) and pro-inflammatory cytokines. Activation of placental UPR and stress-response pathways, including P-IRE1?, ATF6, XBP-1, GRP78 and GRP94, P-p38/p38 and HSP70, was higher in early-onset PE than in both late-onset PE and normotensive controls (NTCs), with a clear inflection around 34 weeks. Placentae from ? 34 weeks PE and NTC were indistinguishable. Levels of UPR signalling were similar between second-trimester and term controls, but were significantly higher in pre-term 'controls' delivered vaginally for chorioamnionitis and other conditions. Severe H/R (1/20% O2 ) induced equivalent activation of UPR pathways, including P-eIF2?, ATF6, P-IRE1?, GRP78 and GRP94, in BeWo cells. By contrast, the pro-inflammatory cytokines TNF? and IL-1? induced only mild activation of P-eIF2? and GRP78. AKT, a central regulator of cell proliferation, was reduced in the < 34 weeks PE placentae and severe H/R-treated cells, but not in other conditions. These findings provide the first molecular evidence that placental stress may contribute to the pathophysiology of early-onset pre-eclampsia, whereas that is unlikely to be the case in the late-onset form of the syndrome. PMID:24931423

  2. Differential activation of placental unfolded protein response pathways implies heterogeneity in causation of early- and late-onset pre-eclampsia

    PubMed Central

    Yung, Hong Wa; Atkinson, Daniel; Campion-Smith, Tim; Olovsson, Matts; Charnock-Jones, D Stephen; Burton, Graham J

    2014-01-01

    Based on gestational age at diagnosis and/or delivery, pre-eclampsia (PE) is commonly divided into early-onset (<34 weeks) and late-onset (?34 weeks) forms. Recently, the distinction between ‘placental’ and ‘maternal’ causation has been proposed, with ‘placental’ cases being more frequently associated with early-onset and intrauterine growth restriction. To test whether molecular placental pathology varies according to clinical presentation, we investigated stress-signalling pathways, including unfolded protein response (UPR) pathways, MAPK stress pathways, heat-shock proteins and AMPK? in placentae delivered by caesarean section for clinical indications at different gestational ages. Controls included second-trimester, pre-term and normal-term placentae. BeWo cells were used to investigate how these pathways react to different severities of hypoxia–reoxygenation (H/R) and pro-inflammatory cytokines. Activation of placental UPR and stress-response pathways, including P-IRE1?, ATF6, XBP-1, GRP78 and GRP94, P-p38/p38 and HSP70, was higher in early-onset PE than in both late-onset PE and normotensive controls (NTCs), with a clear inflection around 34 weeks. Placentae from ? 34 weeks PE and NTC were indistinguishable. Levels of UPR signalling were similar between second-trimester and term controls, but were significantly higher in pre-term ‘controls’ delivered vaginally for chorioamnionitis and other conditions. Severe H/R (1/20% O2) induced equivalent activation of UPR pathways, including P-eIF2?, ATF6, P-IRE1?, GRP78 and GRP94, in BeWo cells. By contrast, the pro-inflammatory cytokines TNF? and IL-1? induced only mild activation of P-eIF2? and GRP78. AKT, a central regulator of cell proliferation, was reduced in the < 34 weeks PE placentae and severe H/R-treated cells, but not in other conditions. These findings provide the first molecular evidence that placental stress may contribute to the pathophysiology of early-onset pre-eclampsia, whereas that is unlikely to be the case in the late-onset form of the syndrome. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. PMID:24931423

  3. Signature of protein unfolding in chemical exchange saturation transfer imaging.

    PubMed

    Goerke, Steffen; Zaiss, Moritz; Kunz, Patrick; Klika, Karel D; Windschuh, Johannes D; Mogk, Axel; Bukau, Bernd; Ladd, Mark E; Bachert, Peter

    2015-07-01

    Chemical exchange saturation transfer (CEST) allows the detection of metabolites of low concentration in tissue with nearly the sensitivity of MRI with water protons. With this spectroscopic imaging approach, several tissue-specific CEST effects have been observed in vivo. Some of these originate from exchanging sites of proteins, such as backbone amide protons, or from aliphatic protons within the hydrophobic protein core. In this work, we employed CEST experiments to detect global protein unfolding. Spectral evaluation revealed exchange- and NOE-mediated CEST effects that varied in a highly characteristic manner with protein unfolding tracked by fluorescence spectroscopy. We suggest the use of this comprehensive spectral signature for the detection of protein unfolding by CEST, as it relies on several spectral hallmarks. As proof of principle, we demonstrate that the presented signature is readily detectable using a whole-body MR tomograph (B0 ?=?7?T), not only in denatured aqueous protein solutions, but also in heat-shocked yeast cells. A CEST imaging contrast with the potential to detect global protein unfolding would be of particular interest regarding protein unfolding as a marker for stress, ageing, and disease. Copyright © 2015 John Wiley & Sons, Ltd. PMID:26010522

  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. [Centre for Biomedical Sciences, Cardiff School of Health Sciences, University of Wales Institute Cardiff, Cardiff CF5 2YB (United Kingdom)] [Centre for Biomedical Sciences, Cardiff School of Health Sciences, University of Wales Institute Cardiff, Cardiff CF5 2YB (United Kingdom); Adam, E. [Howest University College, Campus Graaf Karel de Goedelaan 5, Marksesteenweg 58 - 8500 Kortrijk, West Flanders (Belgium)] [Howest University College, Campus Graaf Karel de Goedelaan 5, Marksesteenweg 58 - 8500 Kortrijk, West Flanders (Belgium); Roberts, A.; Lang, D. [School of Medicine, Cardiff University, Heath Park Campus, Cardiff CF14 4XN (United Kingdom)] [School of Medicine, Cardiff University, Heath Park Campus, Cardiff CF14 4XN (United Kingdom); Morris, R.H.K.; Thomas, A.W. [Centre for Biomedical Sciences, Cardiff School of Health Sciences, University of Wales Institute Cardiff, Cardiff CF5 2YB (United Kingdom)] [Centre for Biomedical Sciences, Cardiff School of Health Sciences, University of Wales Institute Cardiff, Cardiff CF5 2YB (United Kingdom); Webb, R., E-mail: rwebb@uwic.ac.uk [Centre for Biomedical Sciences, Cardiff School of Health Sciences, University of Wales Institute Cardiff, Cardiff CF5 2YB (United Kingdom)

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

    PubMed

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

    2013-02-01

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

  6. 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

  7. Detector response unfolding using artificial neural networks

    NASA Astrophysics Data System (ADS)

    Avdic, Senada; Pozzi, Sara A.; Protopopescu, Vladimir

    2006-09-01

    We present new results on the identification and unfolding of neutron spectra from the pulse height distribution measured with liquid scintillators. The novelty of the method consists of the dual use of linear and nonlinear artificial neural networks (ANNs). The linear networks solve the superposition problem in the general unfolding problem, whereas the nonlinear networks provide greater accuracy in the neutron source identification problem. Two additional new aspects of the present approach are (i) the use of a very accurate Monte Carlo code for the simulations needed in the training phase of the ANNs and (ii) the ability of the network to respond to short-time and therefore very noisy experimental measurements. This approach ensures sufficient accuracy, timeliness, and robustness to make it a candidate of choice for the heretofore unaddressed nuclear nonproliferation and safeguards applications in which both identification and unfolding are needed.

  8. Dry Molten Globule Intermediates and the Mechanism of Protein Unfolding

    PubMed Central

    Baldwin, Robert L.; Frieden, Carl; Rose, George D.

    2010-01-01

    New experimental results show that either gain or loss of close packing can be observed as a discrete step in protein folding or unfolding reactions. This finding poses a significant challenge to the conventional two-state model of protein folding. Results of interest involve dry molten globule intermediates, an expanded form of the protein that lacks appreciable solvent. When an unfolding protein expands to the dry molten globule state, side chains unlock and gain conformational entropy, while liquid-like van der Waals interactions persist. Four unrelated proteins are now known to form dry molten globules as the first step of unfolding, suggesting that such an intermediate may well be commonplace in both folding and unfolding. Data from the literature show that peptide amide protons are protected in the dry molten globule, indicating that backbone structure is intact despite loss of side chain close packing. Other complementary evidence shows that secondary structure formation provides a major source of compaction during folding. In our model, the major free-energy barrier separating unfolded from native states usually occurs during the transition between the unfolded state and the dry molten globule. The absence of close packing at this barrier provides an explanation for why ?-values, derived from a Brønsted-Leffler plot, depend primarily on structure at the mutational site and not on specific side chain interactions. The conventional two-state folding model breaks down when there are dry molten globule intermediates, a realization that has major implications for future experimental work on the mechanism of protein folding. PMID:20635344

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

    PubMed

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

    2014-12-16

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

  10. Single-molecule Protein Unfolding in Solid State Nanopores

    PubMed Central

    Talaga, David S.; Li, Jiali

    2009-01-01

    We use single silicon nitride nanopores to study folded, partially folded and unfolded single proteins by measuring their excluded volumes. The DNA-calibrated translocation signals of ?-lactoglobulin and histidine-containing phosphocarrier protein match quantitatively with that predicted by a simple sum of the partial volumes of the amino acids in the polypeptide segment inside the pore when translocation stalls due to the primary charge sequence. Our analysis suggests that the majority of the protein molecules were linear or looped during translocation and that the electrical forces present under physiologically relevant potentials can unfold proteins. Our results show that the nanopore translocation signals are sensitive enough to distinguish the folding state of a protein and distinguish between proteins based on the excluded volume of a local segment of the polypeptide chain that transiently stalls in the nanopore due to the primary sequence of charges. PMID:19530678

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

    NASA Astrophysics Data System (ADS)

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

    2008-02-01

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

  12. 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

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

    NASA Astrophysics Data System (ADS)

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

    2015-03-01

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

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

    NASA Astrophysics Data System (ADS)

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

    2010-03-01

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

  15. 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

  16. 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.

  17. Self-association of unfolded outer membrane proteins.

    PubMed

    Ebie Tan, Alexandra; Burgess, Nancy K; DeAndrade, Diana S; Marold, Jacob D; Fleming, Karen G

    2010-07-01

    We have investigated self-association propensities of aqueous unfolded (U(AQ)) forms of eight outer membrane proteins (OMPs), OmpA, OmpW, OmpX, PagP, OmpT, OmpLa, FadL, and Omp85. We found that high urea concentrations maintain all of these OMPs as monomers and that OmpA and OmpX remain monomeric upon dilution to 1 M urea. A pH screen showed that basic pH supports the least amount of U(AQ) OMP self-association, consistent with earlier studies showing that basic pH was optimal for better folding efficiencies. The addition of KCl increased U(AQ) OMP self-association, although the magnitudes of the responses were varied. These studies showed that urea can be used to tune the amount of U(AQ) OMP self-association and indicate that the presence of some urea may be useful in optimizing folding conditions because it diminishes aggregation. PMID:20491126

  18. Human replication protein A unfolds telomeric G-quadruplexes

    Microsoft Academic Search

    Tonatiuh Romero Salas; Irina Petruseva; Olga Lavrik; Anne Bourdoncle; Jean-Louis Mergny; Alain Favre; Carole Saintome ´

    2006-01-01

    G-quadruplex structures inhibit telomerase activity and must be disrupted for telomere elongation during S phase. It has been suggested that the replication protein A (RPA) could unwind and maintain single-stranded DNA in a state amenable to the binding of telomeric components. We show here that under near-physiological in vitro condi- tions, human RPA is able to bind and unfold G-quadruplex

  19. 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.

  20. Unfolding individual Als5p adhesion proteins on live cells

    PubMed Central

    Alsteens, David; Dupres, Vincent; Klotz, Stephen A.; Gaur, Nand K.; Lipke, Peter N.; Dufrêne, Yves F.

    2009-01-01

    Elucidating the molecular mechanisms behind the strength and mechanics of cell adhesion proteins is of central importance in cell biology, and offers exciting avenues for the identification of potential drug targets. Here we use single-molecule force spectroscopy to investigate the adhesive and mechanical properties of the widely expressed Als5p cell adhesion protein from the opportunistic pathogen Candida albicans. We show that the forces required to unfold individual tandem repeats of the protein are in the 150–250 pN range, both on isolated molecules and on live cells. We also find that the unfolding probability increases with the number of tandem repeats and correlates with the level of cell adherence. We suggest that the modular and flexible nature of Als5p conveys both strength and toughness to the protein, making it ideally-suited for cell adhesion. The single molecule measurements presented here open new avenues for understanding the mechanical properties of adhesion molecules from mammalian and microbial cells, and may help us to elucidate their potential implications in diseases like inflammation, cancer and infection. PMID:19534503

  1. Protein unfolding and refolding as transitions through virtual states

    NASA Astrophysics Data System (ADS)

    Bonilla, L. L.; Carpio, A.; Prados, A.

    2014-10-01

    Single-molecule atomic force spectroscopy probes elastic properties of titin, ubiquitin and other relevant proteins. We explain bioprotein folding dynamics under both length- and force-clamp by modeling polyprotein modules as particles in a bistable potential, weakly connected by harmonic spring linkers. Multistability of equilibrium extensions provides the characteristic sawtooth force-extension curve. We show that abrupt or stepwise unfolding and refolding under force-clamp conditions involve transitions through virtual states (which are quasi-stationary domain configurations) modified by thermal noise. These predictions agree with experimental observations.

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

    Microsoft Academic Search

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

    1999-01-01

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

  3. 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.

  4. 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.

    PubMed

    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. PMID:19846189

  5. 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…

  6. Experimental study of single protein mechanics and protein rates of unfolding

    NASA Astrophysics Data System (ADS)

    Hermans, Rodolfo I.

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

  7. Valosin-containing protein (VCP/p97) is capable of unfolding polyubiquitinated proteins through its ATPase domains.

    PubMed

    Song, Changcheng; Wang, Qing; Song, Changzheng; Rogers, Thomas J

    2015-07-31

    Valosin-containing protein (VCP or p97) is required for the proteasomal degradation of polyubiquitinated proteins. However, the molecular mechanism for VCP to process the polyubiquitinated proteins remains unclear. Here, we show that VCP can unfold polyubiquitinated proteins. It preferably unfolds the pentaubiquitin-over monoubiquin-conjugated dihydrofolate reductase (Ub5-DHFR or Ub-DHFR) in a dose dependent manner. In addition, the unfolding activity of VCP does not depend on its ATPase activity, on the contrary, ATP and its non-hydrolysable analogs suppress the unfolding of Ub5-DHFR. The structural and functional analysis showed that either D1 or D2 domain of VCP is sufficient to carry out this unfolding activity. The structure of the substrates also affects its unfolding by VCP. VCP is unable to unfold Ub5-DHFR in a tight structure when it binds with methotrexate, a folate analog with high affinity to DHFR. Thus, these results support that VCP is capable of unfolding polyubiquitinated proteins and suggest that VCP may facilitate the proteasomal degradation of polyubiquitinated proteins through its unfolding activity. PMID:26043696

  8. 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.

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

    PubMed

    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. PMID:25679645

  10. Lipid peroxidation end product 4-hydroxy-trans-2-nonenal triggers unfolded protein response and heme oxygenase-1 expression in PC12 cells: Roles of ROS and MAPK pathways.

    PubMed

    Lin, Meng-Han; Yen, Jui-Hung; Weng, Ching-Yi; Wang, Lisu; Ha, Choi-Lan; Wu, Ming-Jiuan

    2014-01-01

    This study investigates the roles of ROS overproduction and MAPK signaling pathways in the induction of unfolded protein response (UPR) and the expression of Phase II enzymes in response to 4-hydroxy-trans-2-nonenal (4-HNE) in a neuronal-like catecholaminergic PC12 cells. Our results showed that 4-HNE triggered three canonical pathways of UPR, namely IRE1-XBP1, PERK-eIF2?-ATF4 and ATF6, and induced the expression of UPR-targeted genes, GRP78, CHOP, TRB3, PUMA, and GADD34, as well as Phase II enzymes, HO-1 and GCLC. 4-HNE also induced apoptosis, intracellular calcium accumulation, caspase-3 activation, and G0/G1 cell cycle arrest, which was correlated with the increased expression of GADD45?. The addition of tiron, a cellular permeable superoxide scavenger, scavenged 4-HNE-mediated ROS formation, but did not alleviate cytotoxicity, or the expression of UPR-targeted genes or Phase II enzymes, indicating that ROS overproduction per se did not play a major role in 4-HNE-caused deleterious effects. HO-1 expression was attenuated by Nrf2 siRNA and chemical chaperone 4-phenylbutyrate (4-PBA), suggesting HO-1 expression was regulated by Nrf2-ARE, which may work downstream of ER stress. 4-HNE treatment promptly induced ERK, JNK and p38 MAPK activation. Addition of p38 MAPK specific inhibitor SB203580 attenuated HO-1 upregulation, but enhanced expression of CHOP, PUMA and TRB3, and cytotoxicity. These results indicate that 4-HNE-induced transient p38 MAPK activation may serve as an upstream negative regulator of ER stress and confer adaptive cytoprotection against 4-HNE-mediated cell injury. PMID:24291486

  11. Targeted Molecular Dynamics Simulations of Protein Unfolding Philippe Ferrara, Joannis Apostolakis, and Amedeo Caflisch*

    E-print Network

    Caflisch, Amedeo

    Targeted Molecular Dynamics Simulations of Protein Unfolding Philippe Ferrara, Joannis Apostolakis simulated by conventional, i.e., unrestrained, molecular dynamics at 375 and 475 K. In all simulations high-temperature molecular dynamics simulations of CI2 unfolding were performed with an implicit

  12. Is the Unfolded State the Rosetta Stone of the Protein Folding Problem?

    Microsoft Academic Search

    Per Hammarström; Uno Carlsson

    2000-01-01

    Solving the protein folding problem is one of the most challenging tasks in the post genomic era. Identification of folding-initiation sites is very important in order to understand the protein folding mechanism. Detection of residual structure in unfolded proteins can yield important clues to the initiation sites in protein folding. A substantial number of studied proteins possess residual structure in

  13. 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

  14. Probing the contribution of internal cavities to the volume change of protein unfolding under pressure.

    PubMed Central

    Frye, K. J.; Royer, C. A.

    1998-01-01

    The structural origin of the decrease in system volume upon protein denaturation by pressure has remained a puzzle for decades. This negative volume change upon unfolding is assumed to arise globally from more intimate interactions between the polypeptide chain and water, including electrostriction of buried charges that become exposed upon unfolding, hydration of the polypeptide backbone and amino acid side chains and elimination of packing defects and internal void volumes upon unfolding of the chain. However, the relative signs and magnitudes of each of these contributing factors have not been experimentally determined. Our laboratory has probed the fundamental basis for the volume change upon unfolding of staphylococcal nuclease (Snase) using variable solution conditions and point mutants of Snase (Royer CA et al., 1993, Biochemistry 32:5222-5232; Frye KJ et al., 1996, Biochemistry 35:10234-10239). Our prior results indicate that for Snase, neither electrostriction nor polar or nonpolar hydration contributes significantly to the value of the volume change of unfolding. In the present work, we investigate the pressure induced unfolding of three point mutants of Snase in which internal cavity size is altered. The experimentally determined volume changes of unfolding for the mutants suggest that loss of internal void volume upon unfolding represents the major contributing factor to the value of the volume change of Snase unfolding. PMID:9792110

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

    PubMed

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

    2013-05-01

    We aimed to investigate the role of insulin in the bladder and its relevance for the development of overactive bladder (OAB) in insulin-resistant obese mice. Bladders from male individuals who were involved in multiple organ donations were used. C57BL6/J mice were fed with a high-fat diet for 10 weeks to induce insulin-resistant obesity. Concentration-response curves to insulin were performed in human and mouse isolated mucosa-intact and mucosa-denuded bladders. Cystometric study was performed in terminally anaesthetized mice. Western blot was performed in bladders to detect phosphorylated endothelial NO synthase (eNOS) (Ser1177) and the phosphorylated protein kinase AKT (Ser473), as well as the unfolded protein response (UPR) markers TRIB3, CHOP and ATF4. Insulin (1-100 nm) produced concentration-dependent mouse and human bladder relaxations that were markedly reduced by mucosal removal or inhibition of the PI3K/AKT/eNOS pathway. In mouse bladders, insulin produced a 3.0-fold increase in cGMP levels (P < 0.05) that was prevented by PI3K/AKT/eNOS pathway inhibition. Phosphoinositide 3-kinase (PI3K) inhibition abolished insulin-induced phosphorylation of AKT and eNOS in bladder mucosa. Obese mice showed greater voiding frequency and non-voiding contractions, indicating overactive detrusor smooth muscle. Insulin failed to relax the bladder or to increase cGMP in the obese group. Insulin-stimulated AKT and eNOS phosphorylation in mucosa was also impaired in obese mice. The UPR markers TRIB3, CHOP and ATF4 were increased in the mucosa of obese mice. The UPR inhibitor 4-phenyl butyric acid normalized all the functional and molecular parameters in obese mice. Our data show that insulin relaxes human and mouse bladder via activation of the PI3K/AKT/eNOS pathway in the bladder mucosa. Endoplasmic reticulum stress-dependent insulin resistance in bladder contributes to OAB in obese mice. PMID:23478138

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

    E-print Network

    Hagen, Stephen J.

    - tration on the rate of intrachain diffusion in an unfolded protein. After photodissociating a ligand from@u¯.edu Abbreviations used: GdnHCl, guanidine hydrochloride; SVD, singular value decomposition; kB, Boltzmann's constant

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

    Microsoft Academic Search

    Georg Fehrenbacher; Reinhard Meckbach; Peter Jacob

    1996-01-01

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

  18. 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…

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

    SciTech Connect

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

    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.

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

    NASA Astrophysics Data System (ADS)

    Fitter, Jörg

    2003-08-01

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

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

    PubMed Central

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

    2012-01-01

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

  2. Unfolding the multi-length scale domain structure of silk fibroin protein

    Microsoft Academic Search

    Hennady Shulha; Cheryl Wong Po Foo; David L. Kaplan; Vladimir V. Tsukruk

    2006-01-01

    Multi-scale force spectroscopy was applied to measure unfolding properties and the internal domain structure of Bombyx mori silk fibroin. We demonstrated that the complex multi-domain sequence and block design in this protein can be directly related to multi-stage unfolding behavior of the specific regions through the use of force extension measurements. These new findings suggest relationships between polymer block chemistry

  3. Diffusional Barrier in the Unfolding of a Small Protein Lovy Pradeep and Jayant B. Udgaonkar

    E-print Network

    . is found to have a value of -0.7 cP in xylose and -0.5 cP in glycerol, in the case of unfolding is lower than the bulk solvent viscosity by either 0.7 cP or 0.5 cP. A second important result as the polypeptide chain surmounts the largely entropic barrier that slows down a protein folding or unfolding

  4. A simple method for probing the mechanical unfolding pathway of proteins in detail

    NASA Astrophysics Data System (ADS)

    Best, Robert B.; Fowler, Susan B.; Toca-Herrera, José L.; Clarke, Jane

    2002-09-01

    Atomic force microscopy is an exciting new single-molecule technique to add to the toolbox of protein (un)folding methods. However, detailed analysis of the unfolding of proteins on application of force has, to date, relied on protein molecular dynamics simulations or a qualitative interpretation of mutant data. Here we describe how protein engineering value analysis can be adapted to characterize the transition states for mechanical unfolding of proteins. Single-molecule studies also have an advantage over bulk experiments, in that partial values arising from partial structure in the transition state can be clearly distinguished from those averaged over alternate pathways. We show that unfolding rate constants derived in the standard way by using Monte Carlo simulations are not reliable because of the errors involved. However, it is possible to circumvent these problems, providing the unfolding mechanism is not changed by mutation, either by a modification of the Monte Carlo procedure or by comparing mutant and wild-type data directly. The applicability of the method is tested on simulated data sets and experimental data for mutants of titin I27.

  5. Mechanical Unfolding of a Simple Model Protein Goes Beyond the Reach of One-Dimensional Descriptions

    E-print Network

    Tapia-Rojo, Rafael; Mazo, Juan José; Falo, Fernando

    2015-01-01

    We study the mechanical unfolding of a simple model protein. The Langevin dynamics results are analyzed using Markov-model methods which allow to describe completely the configurational space of the system. Using transition path theory we also provide a quantitative description of the unfolding pathways followed by the system. Our study shows a complex dynamical scenario. In particular, we see that the usual one-dimensional picture: free-energy vs end-to-end distance representation, gives a misleading description of the process. Unfolding can occur following different pathways and configurations which seem to play a central role in one-dimensional pictures are not the intermediate states of the unfolding dynamics.

  6. Monitoring equilibria and kinetics of protein folding/unfolding reactions by capillary zone electrophoresis.

    PubMed

    Verzola, B; Chiti, F; Manao, G; Righetti, P G

    2000-07-01

    A method is described here for studying conformational transitions of proteins due to denaturing agents: capillary zone electrophoresis (CZE) in acidic, isoelectric buffers. The sample is run in 50 mM isoelectric glutamic acid (pH = pI = 3.2) added with 1 mM oligoamine (tetraethylene pentamine) for quenching protein interaction to the capillary wall (final pH = 3.3). Muscle acylphosphatase (AcP), in this buffer, exhibited a free solution mobility of 2.63 x 10(-4) cm(2) V(-1) s(-1). By studying the unfolding kinetics, as a function of time of incubation in 7 M urea, it was possible to measure the rate constant of the unfolding reaction, estimated to be 0.00030+/-0.00006 s(-1). The same measurements, when repeated via spectroscopic monitoring of intrinsic fluorescence, gave a value of 0.00034+/-0.00002 s(-1), thus in excellent agreement with CZE data. By equilibrium unfolding CZE studies, it was possible to construct the typical sigmoidal transition of unfolding vs urea molarity: the midpoint of this transition, at which the folded and unfolded states should be equally populated, was estimated to be at 4.56 M urea. Similar experiments by fluorometric analysis gave a value of 4.60 M urea as midpoint of the unfolding curve. PMID:10873279

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

    E-print Network

    Schuler, Ben

    of cold denaturation are still under debate, but the most common interpretation of the originSingle-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

  8. Free energy for blue copper protein unfolding determined by electrospray ionisation mass spectrometry.

    PubMed

    Cunsolo, V; Foti, S; La Rosa, C; Saletti, R; Canters, G W; Verbeet, M P

    2001-01-01

    An electrospray ionisation (ESI) mass spectrometric method for the determination of the free energy (DeltaG) of unfolding of proteins is described. The method was tested using three blue copper proteins: wild type azurin, Cys-3Ala/Cys-26Ala (C3A/C26A) azurin mutant and wild-type amicyanin. The time course of the denaturation process of the proteins dissolved in methanol/water (50:50, v/v, pH 3.5) was followed by recording ESI mass spectra at time intervals. The spectra showed two series of peaks, corresponding to the native holo-protein and the unfolded apo-protein. From the intensity ratio of these two series of peaks at increasing time and at equilibrium, the free energy for the unfolding process for the three proteins could be determined. To evaluate the reliability of the thermodynamic data obtained by the ESI mass spectrometric approach, the denaturation process was followed by UV-VIS spectroscopy. The two sets of data obtained by these independent methods were in good agreement indicating that the ESI-MS approach can be used to obtain reliable quantitative information about the protein unfolding process. In principle, this approach can be applied to other proteins and requires very low amounts of sample, due to the intrinsic sensitivity of mass spectrometry. This may prove particularly useful when the amount of sample available prevents the use of current methods. PMID:11565099

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

    Microsoft Academic Search

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

    2002-01-01

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

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. 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.

  15. Role of unfolded state heterogeneity and en-route ruggedness in protein folding kinetics

    PubMed Central

    Ellison, Paul A.; Cavagnero, Silvia

    2006-01-01

    In order to improve our understanding of the physical bases of protein folding, there is a compelling need for better connections between experimental and computational approaches. This work addresses the role of unfolded state conformational heterogeneity and en-route intermediates, as an aid for planning and interpreting protein folding experiments. The expected kinetics were modeled for different types of energy landscapes, including multiple parallel folding routes, preferential paths dominated by one primary folding route, and distributed paths with a wide spectrum of microscopic folding rate constants. In the presence of one or more preferential routes, conformational exchange among unfolded state populations slows down the observed rates for native protein formation. We find this to be a general phenomenon, taking place even when unfolded conformations interconvert much faster than the “escape” rate constants to folding. Dramatic kinetic deceleration is expected in the presence of an increasing number of folding-incompetent unfolded conformations. This argues for the existence of parallel folding paths involving several folding-competent unfolded conformations, during the early stages of protein folding. Deviations from single-exponential behavior are observed for unfolded conformations exchanging at comparable rates or more slowly than folding events. Analysis of the effect of en-route (on-path) intermediate formation and landscape ruggedness on folding kinetics leads to the following unexpected conclusions: (1) intermediates, which often retard native state formation, may in some cases accelerate folding, and (2) rugged landscapes, usually associated with stretched exponentials, display single-exponential behavior in the presence of late high-friction paths. PMID:16501227

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

    SciTech Connect

    Hsu, P. J.; Lai, S. K., E-mail: sklai@coll.phy.ncu.edu.tw [Complex Liquids Laboratory, Department of Physics, National Central University, Chungli 320 Taiwan (China); Molecular Science and Technology Program, Taiwan International Graduate Program, Academia Sinica, Taipei 115, Taiwan (China); Cheong, S. A. [Division of Physics and Applied Physics, School of Physical and Mathematical Sciences, Nanyang Technological University, 21 Nanyang Link, Singapore 637371 (Singapore)

    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.

  17. 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 secondary structure. In contrast, the 229-nm Raman spectra are dominated by the Tyr and Trp Raman bands that the A helix melts. The Tyr Raman bands are pH independent, which indicates that the G and H helices around

  18. Native topology determines force-induced unfolding pathways in globular proteins

    E-print Network

    Thirumalai, Devarajan

    ) and two fibronectin III type domains (9FnIII and 10FnIII). The calculated unfolding path- ways basis for understanding elastic behavior in titin (a muscle protein), tenascin (an extracellular matrix-tooth pattern in the force-extension profiles. The near constant spacing between the saw-tooth peaks gives

  19. 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

  20. Modest influence of FRET chromophores on the properties of unfolded proteins.

    PubMed

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

    2014-10-01

    Single-molecule Förster resonance energy transfer (FRET) experiments are often used to study the properties of unfolded and intrinsically disordered proteins. Because of their large extinction coefficients and quantum yields, synthetic heteroaromatic chromophores covalently linked to the protein are often used as donor and acceptor fluorophores. A key issue in the interpretation of such experiments is the extent to which the properties of the unfolded chain may be affected by the presence of these chromophores. In this article, we investigate this question using all-atom explicit solvent replica exchange molecular dynamics simulations of three different unfolded or intrinsically disordered proteins. We find that the secondary structure and long-range contacts are largely the same in the presence or absence of the fluorophores, and that the dimensions of the chain with and without chromophores are similar. This suggests that, at least in the cases studied, extrinsic fluorophores have little effect on the structural properties of unfolded or disordered proteins. We also find that the critical FRET orientational factor ?(2), has an average value and equilibrium distribution very close to that expected for isotropic orientations, which supports one of the assumptions frequently made when interpreting FRET efficiency in terms of distances. PMID:25296318

  1. Polymer scaling laws of unfolded and intrinsically disordered proteins quantified with single-molecule spectroscopy

    PubMed Central

    Hofmann, Hagen; Soranno, Andrea; Borgia, Alessandro; Gast, Klaus; Nettels, Daniel; Schuler, Benjamin

    2012-01-01

    The dimensions of unfolded and intrinsically disordered proteins are highly dependent on their amino acid composition and solution conditions, especially salt and denaturant concentration. However, the quantitative implications of this behavior have remained unclear, largely because the effective theta-state, the central reference point for the underlying polymer collapse transition, has eluded experimental determination. Here, we used single-molecule fluorescence spectroscopy and two-focus correlation spectroscopy to determine the theta points for six different proteins. While the scaling exponents of all proteins converge to 0.62 ± 0.03 at high denaturant concentrations, as expected for a polymer in good solvent, the scaling regime in water strongly depends on sequence composition. The resulting average scaling exponent of 0.46 ± 0.05 for the four foldable protein sequences in our study suggests that the aqueous cellular milieu is close to effective theta conditions for unfolded proteins. In contrast, two intrinsically disordered proteins do not reach the ?-point under any of our solvent conditions, which may reflect the optimization of their expanded state for the interactions with cellular partners. Sequence analyses based on our results imply that foldable sequences with more compact unfolded states are a more recent result of protein evolution. PMID:22984159

  2. Continuous dissolution of structure during the unfolding of a small protein

    PubMed Central

    Jha, Santosh Kumar; Dhar, Deepak; Krishnamoorthy, Guruswamy; Udgaonkar, Jayant B.

    2009-01-01

    The unfolding kinetics of many small proteins appears to be first order, when measured by ensemble-averaging probes such as fluorescence and circular dichroism. For one such protein, monellin, it is shown here that hidden behind this deceptive simplicity is a complexity that becomes evident with the use of experimental probes that are able to discriminate between different conformations in an ensemble of structures. In this study, the unfolding of monellin has been probed by measurement of the changes in the distributions of 4 different intramolecular distances, using a multisite, time-resolved fluorescence resonance energy transfer methodology. During the course of unfolding, the protein molecules are seen to undergo slow and continuous, diffusive swelling. The swelling process can be modeled as the slow diffusive swelling of a Rouse-like chain with some additional noncovalent, intramolecular interactions. Here, we show that specific structure is lost during the swelling process gradually, and not in an all-or-none manner, during unfolding. PMID:19553216

  3. Different effects of l-arginine on the heat-induced unfolding and aggregation of proteins

    Microsoft Academic Search

    Andrejus Cirkovas; Jolanta Sereikaite

    2011-01-01

    Circular dichroism spectroscopy was used to study the effect of l-arginine on the temperature related unfolding and aggregation of three growth hormones, i.e. human, porcine and mink growth hormones, and human interferon-?2b. l-arginine can stabilize some proteins and suppress their aggregation as it was exemplified by porcine and mink growth hormones. For some other proteins, on the contrary, the effect

  4. Unfolded cholera toxin is transferred to the ER membrane and released from protein disulfide isomerase upon oxidation by Ero1

    Microsoft Academic Search

    Billy Tsai; Tom A. Rapoport

    2002-01-01

    he toxic effect of cholera toxin (CT) on target cells is caused by its A1 chain. This polypeptide is released from the holotoxin and unfolded in the lumen of the ER by the action of protein disulfide isomerase (PDI), before being retrotranslocated into the cytosol. The polypeptide is initially unfolded by binding to the reduced form of PDI. We show

  5. Chaperone activation by unfolding

    PubMed Central

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

    2013-01-01

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

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

    PubMed Central

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

    2012-01-01

    We study the unbiased folding/unfolding thermodynamics of the Trp-cage miniprotein using detailed molecular dynamics simulations of an all-atom model of the protein in explicit solvent, using the Amberff99SB force field. Replica-exchange molecular dynamics (REMD) simulations are used to sample the protein ensembles over a broad range of temperatures covering the folded and unfolded states, and at two densities. The obtained ensembles are shown to reach equilibrium in the 1 ?s per replica timescale. The total simulation time employed in the calculations exceeds 100 ?s. Ensemble averages of the fraction folded, pressure, and energy differences between the folded and unfolded states as a function of temperature are used to model the free energy of the folding transition, ?G(P,T), over the whole region of temperature and pressures sampled in the simulations. The ?G(P,T) diagram describes an ellipse over the range of temperatures and pressures sampled, predicting that the system can undergo pressure induced unfolding and cold denaturation at low temperatures and high pressures, and unfolding at low pressures and high temperatures. The calculated free energy function exhibits remarkably good agreement with the experimental folding transition temperature (Tf = 321 K), free energy and specific heat changes. However, changes in enthalpy and entropy are significantly different than the experimental values. We speculate that these differences may be due to the simplicity of the semi-empirical force field used in the simulations and that more elaborate force fields may be required to describe appropriately the thermodynamics of proteins. PMID:20408169

  7. 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

  8. Dynamical properties of ?-amylase in the folded and unfolded state: the role of thermal equilibrium fluctuations for conformational entropy and protein stabilisation

    NASA Astrophysics Data System (ADS)

    Fitter, J.; Herrmann, R.; Hauß, T.; Lechner, R. E.; Dencher, N. A.

    2001-07-01

    A comparative analysis of thermal equilibrium fluctuations occurring in a mesophilic and in a thermophilic ?-amylase was performed to study the effect of structural fluctuations on thermostability. The thermal fluctuations determining the conformational entropy of both enzymes have been characterised for the folded (at 30°C and 60°C) and for the unfolded state by applying neutron spectroscopy (at 30°C). The folded state shows a higher structural flexibility for the thermophilic protein as compared to the mesophilic homologue. In contrast, the unfolded state of both enzymes is rather similar with respect to the structural fluctuations. On the basis of this result, a mechanism characterised by entropic stabilisation (i.e., smaller ? S for the unfolding transition of thermophilic ?-amylase) can be assumed to be responsible for the higher thermostability of the thermophilic enzyme.

  9. Sequential unfolding of the two-domain protein Pseudomonas stutzeri cytochrome c 4

    Microsoft Academic Search

    Niels H Andersen; Allan Nørgaard; Thomas J Jensen; Jens Ulstrup

    2002-01-01

    P. stutzeri cytochrome c4 is a di-haem protein, composed of two globular domains each with His–Met coordinated haem, and a hydrogen bond network between the domains. The domain foldings are highly symmetric but with specific differences including structural differences of ligand coordination, and different spin states of the oxidised haem groups. We have studied unfolding of oxidised P. stutzeri cyt

  10. Does deamidation cause protein unfolding? A top-down tandem mass spectrometry study.

    PubMed

    Soulby, Andrew J; Heal, Jack W; Barrow, Mark P; Roemer, Rudolf A; O'Connor, Peter B

    2015-05-01

    Deamidation is a nonenzymatic post-translational modification of asparagine to aspartic acid or glutamine to glutamic acid, converting an uncharged amino acid to a negatively charged residue. It is plausible that deamidation of asparagine and glutamine residues would result in disruption of a proteins' hydrogen bonding network and thus lead to protein unfolding. To test this hypothesis Calmodulin and B2M were deamidated and analyzed using tandem mass spectrometry on a Fourier transform ion cyclotron resonance mass spectrometer (FTICR-MS). The gas phase hydrogen bonding networks of deamidated and nondeamidated protein isoforms were probed by varying the infra-red multi-photon dissociation laser power in a linear fashion and plotting the resulting electron capture dissociation fragment intensities as a melting curve at each amino acid residue. Analysis of the unfolding maps highlighted increased fragmentation at lower laser powers localized around heavily deamidated regions of the proteins. In addition fragment intensities were decreased across the rest of the proteins which we propose is because of the formation of salt-bridges strengthening the intramolecular interactions of the central regions. These results were supported by a computational flexibility analysis of the mutant and unmodified proteins, which would suggest that deamidation can affect the global structure of a protein via modification of the hydrogen bonding network near the deamidation site and that top down FTICR-MS is an appropriate technique for studying protein folding. PMID:25653127

  11. Melting points of lysozyme and ribonuclease A crystals correlated with protein unfolding: a Raman spectroscopic study.

    PubMed

    Jacob, J; Krafft, C; Welfle, K; Welfle, H; Saenger, W

    1998-01-01

    The effects of a temperature increase on monoclinic and tetragonal lysozyme single crystals were investigated by polarizing microscopy, X-ray diffraction and laser Raman spectroscopy. To prevent dissolution, the mother liquor was removed, and the crystals were covered by the oil poly-(chlorotrifluoroethylene). Upon heating, their macroscopic shape was stable beyond 453 K but a change (or loss) of birefringence was observed around 352 and 367 K for the tetragonal and monoclinic crystal forms, respectively, which is associated with tighter packing and higher crystal forces in monoclinic lysozyme. Raman spectral changes in the amide I and amide III regions indicated denaturation of the protein within the crystalline environment at temperature where birefringence changes, and differences in the S-S band suggest that in monoclinic lysozyme, denaturation is accompanied with disruption of some S-S bonds. Comparison with thermal denaturation and gel formation (beta-aggregation) of lysozyme in solution indicates that intermolecular interactions are mainly involved in the stabilization of the denatured lysozyme crystals. The behavior of ribonuclease A is very different. This protein unfolds and refolds reversibly in solution and its crystals melt at the unfolding temperature at 333 K, i.e. loss of structure induces breakdown of crystal lattice and macroscopic shape. Although the crystal lattice of proteins is stabilized by only few intermolecular contacts, its breakdown with increasing temperature is primarily a result of thermal unfolding of the polypeptide chains. PMID:9761818

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

    PubMed Central

    Purohit, Prashant K.; Litvinov, Rustem I.; Brown, Andre E. X.; Discher, Dennis E.; Weisel, John W.

    2011-01-01

    We describe the mechanical behavior of isotropic fibrin networks at the macroscopic scale in terms of the nanoscale force response of fibrin molecules that are its basic building blocks. We show that the remarkable extensibility and compressibility of fibrin networks have their origins in the unfolding of fibrin molecules. The force-stretch behavior of a single fibrin fiber is described using a two-state model in which the fiber has a linear force-stretch relation in the folded phase and behaves like a worm-like-chain in the unfolded phase. The nanoscale force-stretch response is connected to the macro-scale stress-stretch response by means of the eight-chain model. This model is able to capture the macroscopic response of a fibrin network in uniaxial tension and appears remarkably simple given the molecular complexity. We use the eight-chain model to explain why fibrin networks have negative compressibility and Poisson’s ratio greater than one due to unfolding of fibrin molecules. PMID:21342665

  13. Solvent Sensitivity of Protein Unfolding: Study of Chicken Villin Headpiece Subdomain in Water-Ethanol and Water-DMSO Mixtures

    E-print Network

    Rikhia Ghosh; Susmita Roy; Biman Bagchi

    2013-07-10

    In the present work we study and compare unfolding of a small protein, chicken villin headpiece (HP-36) in two different aqueous binary mixtures, namely water-ethanol (EtOH) and water-dimethyl sulphoxide (DMSO). In both the binary mixtures, HP-36 is found to unfold (fully or partially, depending on the mixture) under ambient conditions, that otherwise requires temperature as high as ~600 K to denature in pure aqueous solvent. In all the cases, first step of unfolding is found to be similar, i.e. separation of the cluster formed by three hydrophobic (phenylalanine) residues, namely Phe-7, Phe-11 and Phe-18, which constitute the hydrophobic core, thereby initiating melting of helix-2 of the protein. Subsequent unfolding steps follow different paths in different chemical environments. As both water-DMSO and water-ethanol show composition dependent anomalies, so do the details of unfolding dynamics. With an increase of co-solvent concentration different partially unfolded intermediates are found to be formed in both the cases. This is reflected in a remarkable non-monotonic composition dependence of several order parameters, including fraction of native contacts and protein-solvent interaction energy. The emergence of such partially unfolded states is particularly attributed to the preferential solvation of the hydrophobic residues by the ethyl groups of ethanol and methyl groups of DMSO. While in DMSO the protein gradually attains a completely unfolded state at xDMSO=0.30, unfolding in water-ethanol appears to be more complex and sensitive to solvent composition.

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

    PubMed Central

    Schanda, Paul; Forge, Vincent; Brutscher, Bernhard

    2007-01-01

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

  15. Simulation studies of protein folding/unfolding equilibrium under polar and nonpolar confinement.

    PubMed

    Tian, Jianhui; Garcia, Angel E

    2011-09-28

    We study the equilibrium folding/unfolding thermodynamics of a small globular miniprotein, the Trp cage, that is confined to the interior of a 2 nm radius fullerene ball. The interactions of the fullerene surface are changed from nonpolar to polar to mimic the interior of the GroEL/ES chaperonin that assists proteins to fold in vivo. We find that nonpolar confinement stabilizes the folded state of the protein due to the effects of volume reduction that destabilize the unfolded state and also due to interactions with the fullerene surface. For the Trp cage, polar confinement has a net destabilizing effect that results from the stabilizing confinement and the competitive exclusion effect that keeps the protein away from the surface hydration shell and stronger interactions between charged side chains in the protein and the polar surface that compete against the formation of an ion pair that stabilizes the protein folded state. We show that confinement effects due to volume reduction can be overcome by sequence-specific interactions of the protein side chains with the encapsulating surface. This study shows that there is a complex balance among many competing effects that determine the mechanism of GroEL chaperonin in enhancing the folding rate of polypeptide inside its cavity. PMID:21854029

  16. 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

  17. Mechanical Unfolding of a Titin Ig Domain: Structure of Transition State Revealed by Combining Atomic Force Microscopy, Protein Engineering and Molecular Dynamics Simulations

    Microsoft Academic Search

    Robert B. Best; Susan B. Fowler; José L. Toca Herrera; Annette Steward; Emanuele Paci; Jane Clarke

    2003-01-01

    Titin I27 shows a high resistance to unfolding when subject to external force. To investigate the molecular basis of this mechanical stability, protein engineering ?-value analysis has been combined with atomic force microscopy to investigate the structure of the barrier to forced unfolding. The results indicate that the transition state for forced unfolding is significantly structured, since highly destabilising mutations

  18. Unfolding and translocation pathway of substrate protein controlled by structure in repetitive allosteric cycles of the ClpY ATPase

    PubMed Central

    Kravats, Andrea; Jayasinghe, Manori; Stan, George

    2011-01-01

    Clp ATPases are ring-shaped AAA+ motors in the degradation pathway that perform critical actions of unfolding and translocating substrate proteins (SPs) through narrow pores to deliver them to peptidase components. These actions are effected by conserved diaphragm-forming loops found in the central channel of the Clp ATPase hexamer. Conformational changes, that take place in the course of repetitive ATP-driven cycles, result in mechanical forces applied by the central channel loops onto the SP. We use coarse-grained simulations to elucidate allostery-driven mechanisms of unfolding and translocation of a tagged four-helix bundle protein by the ClpY ATPase. Unfolding is initiated at the tagged C-terminal region via an obligatory intermediate. The resulting nonnative conformation is competent for translocation, which proceeds on a different time scale than unfolding and involves sharp stepped transitions. Completion of the translocation process requires assistance from the ClpQ peptidase. These mechanisms contrast nonallosteric mechanical unfolding of the SP. In atomic force microscopy experiments, multiple unfolding pathways are available and large mechanical forces are required to unravel the SP relative to those exerted by the central channel loops of ClpY. SP threading through a nonallosteric ClpY nanopore involves simultaneous unfolding and translocation effected by strong pulling forces. PMID:21266546

  19. Polyelectrolyte and unfolded protein pore entrance depends on the pore geometry.

    PubMed

    Pastoriza-Gallego, Manuela; Gibrat, Gabriel; Thiebot, Bénédicte; Betton, Jean-Michel; Pelta, Juan

    2009-06-01

    We determined the ability of Maltose Binding Protein and the polyelectrolyte dextran sulfate to enter into and interact with channels formed by Staphylococcus aureus alpha-hemolysin. The entry of either macromolecule in the channel pore causes transient, but well-defined decreases in the single-channel ionic current. The protein and polyelectrolyte were more likely to enter the pore mouth at the channel's cap domain than at the stem side. When the cap domain was denatured in the presence of 4 M urea, the probability that either the denatured protein or polyelectrolyte entered the pore from the cap-domain side decreased. For channels in their native conformation, the polyelectrolyte-induced current blockades were characterized by two mean residence times that were independent of the side of entry. For channels with a denaturated cap domain, the mean polyelectrolyte residence times for relatively long-lived blockades decreased, while that for short-lived blockades were unchanged. For denatured protein, we also observed 2 characteristic residence times that were relatively fast. Only the relatively short-lived blockades were observed with native channels. When the alpha-hemolysin monomers in aqueous solution were incubated in 4 M urea before channel formation, the two characteristic residence times were greater than those for pre-formed pores that were subsequently perturbed by urea. These times might correspond to the interactions between the unfolded protein and the partially unfolded channel. PMID:19328774

  20. Resolution of the unfolded state

    Microsoft Academic Search

    Gregory Beaucage

    2008-01-01

    The unfolded states in proteins and nucleic acids remain weakly understood despite their importance to protein folding; misfolding diseases (Parkinson's & Alzheimer's); natively unfolded proteins (˜ 30% of eukaryotic proteins); and to understanding ribozymes. Research has been hindered by the inability to quantify the residual (native) structure present in an unfolded protein or nucleic acid. Here, a scaling model is

  1. A structural model for unfolded proteins from residual dipolar couplings and small-angle x-ray scattering

    PubMed Central

    Bernadó, Pau; Blanchard, Laurence; Timmins, Peter; Marion, Dominique; Ruigrok, Rob W. H.; Blackledge, Martin

    2005-01-01

    Natively unfolded proteins play key roles in normal and pathological biochemical processes. Despite their importance for function, this category of proteins remains beyond the reach of classical structural biology because of their inherent conformational heterogeneity. We present a description of the intrinsic conformational sampling of unfolded proteins based on residue-specific ?/? propensities from loop regions of a folded protein database and simple volume exclusion. This approach is used to propose a structural model of the 57-aa, natively disordered region of the nucleocapsid-binding domain of Sendai virus phosphoprotein. Structural ensembles obeying these simple rules of conformational sampling are used to simulate averaged residual dipolar couplings (RDCs) and small-angle x-ray scattering data. This protein is particularly informative because RDC data from the equally sized folded and unfolded domains both report on the unstructured region, allowing a quantitative analysis of the degree of order present in this part of the protein. Close agreement between experimental and simulated RDC and small-angle x-ray scattering data validates this simple model of conformational sampling, providing a precise description of local structure and dynamics and average dimensions of the ensemble of sampled structures. RDC data from two urea-unfolded systems are also closely reproduced. The demonstration that conformational behavior of unfolded proteins can be accurately predicted from the primary sequence by using a simple set of rules has important consequences for our understanding of the structure and dynamics of the unstructured state. PMID:16284250

  2. Unfolding and Refolding Occur Much Faster for a Proline-Free Protein than for Most Proline-Containing Proteins

    Microsoft Academic Search

    John F. Brandts; Maureen Brennan; Lung-Nan Lin

    1977-01-01

    The kinetics for unfolding and refolding of a parvalbumin (band 5) have been examined as a function of pH near the transition region, using stopped-flow techniques. This protein is rather unusual in that it has no proline residues, and therefore serves as a good example to test the hypothesis that the rate-limiting step seen in denaturation reactions is due to

  3. Sulfatide-hsp70 interaction promotes hsp70 clustering and stabilizes binding to unfolded protein.

    PubMed

    Harada, Yoichiro; Sato, Chihiro; Kitajima, Ken

    2015-01-01

    The 70-kDa heat shock protein (Hsp70), one of the major stress-inducible molecular chaperones, is localized not only in the cytosol, but also in extracellular milieu in mammals. Hsp70 interacts with various cell surface glycolipids including sulfatide (3'-sulfogalactosphingolipid). However, the molecular mechanism, as well as the biological relevance, underlying the glycolipid-Hsp70 interaction is unknown. Here we report that sulfatide promotes Hsp70 oligomerization through the N-terminal ATPase domain, which stabilizes the binding of Hsp70 to unfolded protein in vitro. We find that the Hsp70 oligomer has apparent molecular masses ranging from 440 kDa to greater than 669 kDa. The C-terminal peptide-binding domain is dispensable for the sulfatide-induced oligomer formation. The oligomer formation is impaired in the presence of ATP, while the Hsp70 oligomer, once formed, is unable to bind to ATP. These results suggest that sulfatide locks Hsp70 in a high-affinity state to unfolded proteins by clustering the peptide-binding domain and blocking the binding to ATP that induces the dissociation of Hsp70 from protein substrates. PMID:25989600

  4. Sulfatide-Hsp70 Interaction Promotes Hsp70 Clustering and Stabilizes Binding to Unfolded Protein

    PubMed Central

    Harada, Yoichiro; Sato, Chihiro; Kitajima, Ken

    2015-01-01

    The 70-kDa heat shock protein (Hsp70), one of the major stress-inducible molecular chaperones, is localized not only in the cytosol, but also in extracellular milieu in mammals. Hsp70 interacts with various cell surface glycolipids including sulfatide (3'-sulfogalactosphingolipid). However, the molecular mechanism, as well as the biological relevance, underlying the glycolipid-Hsp70 interaction is unknown. Here we report that sulfatide promotes Hsp70 oligomerization through the N-terminal ATPase domain, which stabilizes the binding of Hsp70 to unfolded protein in vitro. We find that the Hsp70 oligomer has apparent molecular masses ranging from 440 kDa to greater than 669 kDa. The C-terminal peptide-binding domain is dispensable for the sulfatide-induced oligomer formation. The oligomer formation is impaired in the presence of ATP, while the Hsp70 oligomer, once formed, is unable to bind to ATP. These results suggest that sulfatide locks Hsp70 in a high-affinity state to unfolded proteins by clustering the peptide-binding domain and blocking the binding to ATP that induces the dissociation of Hsp70 from protein substrates. PMID:25989600

  5. Temperature dependent equilibrium native to unfolded protein dynamics and properties observed with IR absorption and 2D IR vibrational echo experiments.

    PubMed

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

    2011-05-01

    Dynamic and structural properties of carbonmonoxy (CO)-coordinated cytochrome c(552) from Hydrogenobacter thermophilus (Ht-M61A) at different temperatures under thermal equilibrium conditions were studied with infrared absorption spectroscopy and ultrafast two-dimensional infrared (2D IR) vibrational echo experiments using the heme-bound CO as the vibrational probe. Depending on the temperature, the stretching mode of CO shows two distinct bands corresponding to the native and unfolded proteins. As the temperature is increased from low temperature, a new absorption band for the unfolded protein grows in and the native band decreases in amplitude. Both the temperature-dependent circular dichroism and the IR absorption area ratio R(A)(T), defined as the ratio of the area under the unfolded band to the sum of the areas of the native and unfolded bands, suggest a two-state transition from the native to the unfolded protein. However, it is found that the absorption spectrum of the unfolded protein increases its inhomogeneous line width and the center frequency shifts as the temperature is increased. The changes in line width and center frequency demonstrate that the unfolding does not follow simple two-state behavior. The temperature-dependent 2D IR vibrational echo experiments show that the fast dynamics of the native protein are virtually temperature independent. In contrast, the fast dynamics of the unfolded protein are slower than those of the native protein, and the unfolded protein fast dynamics and at least a portion of the slower dynamics of the unfolded protein change significantly, becoming faster as the temperature is raised. The temperature dependence of the absorption spectrum and the changes in dynamics measured with the 2D IR experiments confirm that the unfolded ensemble of conformers continuously changes its nature as unfolding proceeds, in contrast to the native state, which displays a temperature-independent distribution of structures. PMID:21469666

  6. 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

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

    Microsoft Academic Search

    Yong-Bin Yan; Q. Wang; Hua-Wei He; Hai-Meng Zhou

    2004-01-01

    Protein thermal aggregation plays a crucial role in protein science and engineering. Despite its biological importance, little is known about the mechanism and pathway(s) involved in the formation of aggregates. In this report, the sequential events occurring during thermal unfolding and aggregation process of hemoglobin were studied by two-dimensional infrared correlation spectroscopy. Analysis of the infrared spectra recorded at different

  8. Escherichia coli Ribosomal Protein S1 Unfolds Structured mRNAs Onto the Ribosome for Active Translation Initiation

    PubMed Central

    Duval, Mélodie; Korepanov, Alexey; Fuchsbauer, Olivier; Fechter, Pierre; Haller, Andrea; Fabbretti, Attilio; Choulier, Laurence; Micura, Ronald; Klaholz, Bruno P.; Romby, Pascale; Springer, Mathias; Marzi, Stefano

    2013-01-01

    Regulation of translation initiation is well appropriate to adapt cell growth in response to stress and environmental changes. Many bacterial mRNAs adopt structures in their 5? untranslated regions that modulate the accessibility of the 30S ribosomal subunit. Structured mRNAs interact with the 30S in a two-step process where the docking of a folded mRNA precedes an accommodation step. Here, we used a combination of experimental approaches in vitro (kinetic of mRNA unfolding and binding experiments to analyze mRNA–protein or mRNA–ribosome complexes, toeprinting assays to follow the formation of ribosomal initiation complexes) and in vivo (genetic) to monitor the action of ribosomal protein S1 on the initiation of structured and regulated mRNAs. We demonstrate that r-protein S1 endows the 30S with an RNA chaperone activity that is essential for the docking and the unfolding of structured mRNAs, and for the correct positioning of the initiation codon inside the decoding channel. The first three OB-fold domains of S1 retain all its activities (mRNA and 30S binding, RNA melting activity) on the 30S subunit. S1 is not required for all mRNAs and acts differently on mRNAs according to the signals present at their 5? ends. This work shows that S1 confers to the ribosome dynamic properties to initiate translation of a large set of mRNAs with diverse structural features. PMID:24339747

  9. Escherichia coli ribosomal protein S1 unfolds structured mRNAs onto the ribosome for active translation initiation.

    PubMed

    Duval, Mélodie; Korepanov, Alexey; Fuchsbauer, Olivier; Fechter, Pierre; Haller, Andrea; Fabbretti, Attilio; Choulier, Laurence; Micura, Ronald; Klaholz, Bruno P; Romby, Pascale; Springer, Mathias; Marzi, Stefano

    2013-12-01

    Regulation of translation initiation is well appropriate to adapt cell growth in response to stress and environmental changes. Many bacterial mRNAs adopt structures in their 5' untranslated regions that modulate the accessibility of the 30S ribosomal subunit. Structured mRNAs interact with the 30S in a two-step process where the docking of a folded mRNA precedes an accommodation step. Here, we used a combination of experimental approaches in vitro (kinetic of mRNA unfolding and binding experiments to analyze mRNA-protein or mRNA-ribosome complexes, toeprinting assays to follow the formation of ribosomal initiation complexes) and in vivo (genetic) to monitor the action of ribosomal protein S1 on the initiation of structured and regulated mRNAs. We demonstrate that r-protein S1 endows the 30S with an RNA chaperone activity that is essential for the docking and the unfolding of structured mRNAs, and for the correct positioning of the initiation codon inside the decoding channel. The first three OB-fold domains of S1 retain all its activities (mRNA and 30S binding, RNA melting activity) on the 30S subunit. S1 is not required for all mRNAs and acts differently on mRNAs according to the signals present at their 5' ends. This work shows that S1 confers to the ribosome dynamic properties to initiate translation of a large set of mRNAs with diverse structural features. PMID:24339747

  10. Effect of osmolytes on pressure-induced unfolding of proteins: a high-pressure SAXS study.

    PubMed

    Krywka, Christina; Sternemann, Christian; Paulus, Michael; Tolan, Metin; Royer, Catherine; Winter, Roland

    2008-12-22

    Herein, we explore the effect of different types of osmolytes on the high-pressure stability and tertiary structure of a well-characterized monomeric protein, staphylococcal nuclease (SNase). Changes in the denaturation pressure and the radius of gyration are obtained in the presence of different concentrations of trimethylamine N-oxide (TMAO), glycerol and urea. To reveal structural changes in the protein upon compression at various osmolyte conditions, small-angle X-ray scattering (SAXS) experiments were carried out. To this end, a new high-pressure cell suitable for high-precision SAXS studies at synchrotron sources was built, which allows one to carry out scattering experiments up to maximum pressures of about 7 kbar. Our data clearly indicate that the osmolytes that stabilize proteins against temperature-induced unfolding drastically increase their pressure stability and that the elliptically shaped curve of the pressure-temperature-stability diagram of proteins is shifted to higher temperatures and pressures with increasing osmolyte concentration. A drastic stabilization is observed for the osmolyte TMAO, which exhibits not only a significant stabilization against temperature-induced unfolding, but also a particularly strong stabilization of the protein against pressure. In fact, such findings are in accordance with in vivo studies (for example P. J. Yancey, J. Exp. Biol. 2005, 208, 2819-2830), where unusually high TMAO concentrations in some deep-sea animals were found. Conversely, chaotropic agents such as urea have a strong destabilizing effect on both the temperature and pressure stability of the protein. Our data also indicate that sufficiently high TMAO concentrations might be able to largely offset the destabilizing effect of urea. The different scenarios observed are discussed in the context of recent experimental and theoretical studies. PMID:18924198

  11. The Janus face of the archaeal Cdc48/p97 homologue VAT: protein folding versus unfolding.

    PubMed

    Golbik, R; Lupas, A N; Koretke, K K; Baumeister, W; Peters, J

    1999-09-01

    Members of the AAA family of ATPases have been implicated in chaperone-like activities. We used the archaeal Cdc48/p97 homologue VAT as a model system to investigate the effect of an AAA protein on the folding and unfolding of two well-studied, heterologous substrates, cyclophilin and penicillinase. We found that, depending on the Mg2+ concentration, VAT assumes two states with maximum rates of ATP hydrolysis that differ by an order of magnitude. In the low-activity state, VAT accelerated the refolding of penicillinase, whereas in the high-activity state, it accelerated its unfolding. Both reactions were ATP-dependent. In its interaction with cyclophilin, VAT was ATP-independent and only promoted refolding. The N-terminal domain of VAT, which lacks ATPase activity, also accelerated the refolding of cyclophilin but showed no effect on penicillinase. VAT appears to be structurally equivalent over its entire length to Sec18/NSF, suggesting that these results apply more broadly to group II AAA proteins. PMID:10543442

  12. Comparison of intra-organellar chaperone capacity for dealing with stress-induced protein unfolding.

    PubMed

    Hageman, Jurre; Vos, Michel J; van Waarde, Maria A W H; Kampinga, Harm H

    2007-11-23

    Molecular chaperones are essential for cells to prevent that partially unfolded proteins form non-functional, toxic aggregates. This requirement is increased when cells experience protein unfolding stresses and such could affect all compartments in the eukaryotic cell. Whether all organelles are equipped with comparable chaperone capacities is largely unknown, mainly due to the lack of suitable reporters that allow such a comparison. Here we describe the development of fluorescent luciferase reporters that are sorted to various cellular locations (nucleus, cytoplasm, endoplasmic reticulum, and peroxisomes) and that differ minimally in their intrinsic thermal stability properties. When heating living cells, the rate of inactivation was most rapid for the nuclear-targeted luciferase, indicating that the nucleus is the most sensitive organelle toward heat-induced denaturing stress. Post-heat re-activation, however, occurred at equal kinetics irrespective of luciferase localization. Also, induction of thermotolerance by a priming heat treatment, that coordinately up-regulates all heat-inducible chaperones, resulted in a transient heat resistance of the luciferase in all organelles in a comparable manner. Overexpression of the main heat-inducible Hsp70 family member, HspA1A, protected only the cytosolic and nuclear, but not the other luciferases. Together, our data suggest that in each compartment investigated, including the peroxisome in which so far no chaperones could be detected, chaperone machines are present and can be induced with activities similar to those present in the cytosolic/nuclear compartment. PMID:17875648

  13. 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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:25882479

  15. Core-Shell Model of Folding-Unfolding Transitions (UFT) in Proteins

    NASA Astrophysics Data System (ADS)

    Aroutiounian, Svetlana

    2008-03-01

    There are ˜10^N conformations for a protein of length N to sort out randomly in search of lowest free energy state. Can protein folding be simple and fast? Core-shell model introduces principles, proposes mechanisms and scores residues of fast, reversible UFT in protein. According to it, during UFT the realm of intra-residual interactions leads the residue motion. The scaffold of hydrophilic residues forms external shell of unstructured, tube-like protein in unfolded state, just as the hydrophobic residues form internal scaffold -- core, of the protein in folded state. As UFT proceeds, residue slides into lowest-score position permitted by its structure. Model accounts for experimentally observed features of UFT. It is based on three principles: 1) During UFT protein is virtual - its features or structure are inferred only statistically and with limited precision; 2) Mechanism of UFT memory is not longitudinal, but transverse; 3) Native design overrides specific features of residues - the alphabet of amino acids assumes an intrinsic score-function. Per-residue mechanism of UFT is proposed and score-function is described. Difference graphs of transitional score-function and average genome-wide abundance index show that our score-function is the order parameter of UFT in protein and by virtue of being it, reveals transitional key residues. It echoes the multiple-tier and funnel concepts of FEL perspective. Monte Carlo simulations of UFT in myoglobin illustrate the idea.

  16. 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

  17. Thermal protein unfolding in photo-activated adenylate cyclase nano-clusters from the amoeboflagellate Naegleria gruberi NEG-M strain

    Microsoft Academic Search

    A. Penzkofer; M. Stierl; P. Hegemann; S. Kateriya

    2011-01-01

    The photo-activated adenylate cyclase (nPAC) protein from the amoeboflagellate Naegleria gruberi NEG-M strain consists of a BLUF domain (sensor of blue light using flavin) and a cyclase homology domain (CHD). The nPAC thermal stability is determined by its protein unfolding behavior which is quantified by the protein melting temperature and protein melting time. The protein unfolding in nPAC nano-clusters in

  18. LDL phospholipid hydrolysis produces modified electronegative particles with an unfolded apoB-100 protein

    PubMed Central

    Asatryan, Liana; Hamilton, Ryan T.; Isas, J. Mario; Hwang, Juliana; Kayed, Rakez; Sevanian, Alex

    2012-01-01

    Electronegative low density lipoprotein (LDL? formation that structurally resembles LDL? isolated from plasma was evaluated after LDL treatment with snake venom phospholipase A2 (PLA2). PLA2 treatment of LDL increased its electrophoretic mobility in proportion to the amount of LDL? formed without evidence of lipid peroxidation. These changes dose-dependently correlated with the degree of phospholipid hydrolysis. Strong immunoreactivity of LDL? subfraction from plasma and PLA2-treated LDL (PLA2-LDL) to amyloid oligomer-specific antibody was observed. Higher ?-strand structural content and unfolding proportionate to the loss of ?-helical structure of apolipoprotein B-100 (apoB-100) of LDL? isolated from both native and PLA2-LDLs was demonstrated by circular dichroism (CD) spectropolarimetry. These structural changes resembled the characteristics of some oxidatively modified LDLs and soluble oligomeric aggregates of amyloidogenic proteins. PLA2-LDL was also more susceptible to nitration by peroxynitrite, likely because of exposure of otherwise inaccessible hydrophilic and hydrophobic domains arising from apoB-100 unfolding. This was also demonstrated for plasma LDL?. In contrast, PLA2-LDL was more resistant to copper-mediated oxidation that was reversed upon the addition of small amounts of unsaturated fatty acids. PMID:15489541

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

    PubMed Central

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

    2013-01-01

    Biogenesis of ?-barrel membrane proteins is a complex, multi-step, and as yet incompletely characterized process. The bacterial porin family is perhaps the best studied protein family among the ?-barrel membrane proteins that allows diffusion of small solutes across the bacterial outer membrane. In this study, we have identified residues that contribute significantly to the protein-protein interaction (PPI) interface between the chains of Outer Membrane Protein F (OmpF), a trimeric porin, using an empirical energy function in conjunction with an evolutionary analysis. By replacing these residues through site-directed mutagenesis, either with energetically favorable residues or substitutions that do not occur in natural bacterial outer membrane proteins, we succeeded in engineering OmpF mutants with dimeric and monomeric instead of trimeric oligomerization state. Moreover, our results suggest that the oligomerization of OmpF proceeds through a series of interactions involving two distinct regions of the extensive PPI interface: Two monomers interact to form a dimer through the PPI interface near G19. This dimer than interacts with another monomer through the PPI interface near G135 to form a trimer. We have found that perturbing the PPI interface near G19 results in the formation of the monomeric OmpF only. Thermal de-naturation of the designed dimeric OmpF mutant suggests that the oligomer dissociation can be separated from the process of protein unfolding. Furthermore, the conserved site near G57, G59 is important for the PPI interface and might provide the essential scaffold for protein-protein interactions. PMID:22391420

  20. 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

  1. Hydration-responsive folding and unfolding in graphene oxide liquid crystal phases.

    PubMed

    Guo, Fei; Kim, Franklin; Han, Tae Hee; Shenoy, Vivek B; Huang, Jiaxing; Hurt, Robert H

    2011-10-25

    Graphene oxide is promising as a plate-like giant molecular building block for the assembly of new carbon materials. Its water dispersibility, liquid crystallinity, and ease of reduction offer advantages over other carbon precursors if its fundamental assembly rules can be identified. This article shows that graphene oxide sheets of known lateral dimension form nematic liquid crystal phases with transition points in agreement with the Onsager hard-plate theory. The liquid crystal phases can be systematically ordered into defined supramolecular patterns using surface anchoring, complex fluid flow, and microconfinement. Graphene oxide is seen to exhibit homeotropic surface anchoring at interfaces driven by excluded volume entropy and by adsorption enthalpy associated with its partially hydrophobic basal planes. Surprisingly, some of the surface-ordered graphene oxide phases dry into graphene oxide solids that undergo a dramatic anisotropic swelling upon rehydration to recover their initial size and shape. This behavior is shown to be a unique hydration-responsive folding and unfolding transition. During drying, surface tension forces acting parallel to the layer planes cause a buckling instability that stores elastic energy in accordion-folded structures in the dry solid. Subsequent water infiltration reduces interlayer frictional forces and triggers release of the stored elastic energy in the form of dramatic unidirectional expansion. We explain the folding/unfolding phenomena by quantitative nanomechanics and introduce the potential of liquid crystal-derived graphene oxide phases as new stimuli-response materials. PMID:21877716

  2. Active conformation control of unfolded proteins by hyperthermal collision with a metal surface.

    PubMed

    Rinke, Gordon; Rauschenbach, Stephan; Harnau, Ludger; Albarghash, Alyazan; Pauly, Matthias; Kern, Klaus

    2014-10-01

    The physical and chemical properties of macromolecules like proteins are strongly dependent on their conformation. The degrees of freedom of their chemical bonds generate a huge conformational space, of which, however, only a small fraction is accessible in thermal equilibrium. Here we show that soft-landing electrospray ion beam deposition (ES-IBD) of unfolded proteins allows to control their conformation. The dynamics and result of the deposition process can be actively steered by selecting the molecular ion beam's charge state or tuning the incident energy. Using these parameters, protein conformations ranging from fully extended to completely compact can be prepared selectively on a surface, as evidenced on the subnanometer/amino acid resolution level by scanning tunneling microscopy (STM). Supported by molecular dynamics (MD) simulations, our results demonstrate that the final conformation on the surface is reached through a mechanical deformation during the hyperthermal ion surface collision. Our experimental results independently confirm the findings of ion mobility spectrometry (IMS) studies of protein gas phase conformations. Moreover, we establish a new route for the processing of macromolecular materials, with the potential to reach conformations that would be inaccessible otherwise. PMID:25198655

  3. Inactivation and Unfolding of Protein Tyrosine Phosphatase from Thermus thermophilus HB27 during Urea and Guanidine Hydrochloride Denaturation

    PubMed Central

    Liu, Lina; Gao, Chunyan; Xu, Shui; Zhao, Ping; Xia, Qingyou

    2014-01-01

    The effects of urea and guanidine hydrochloride (GdnHCl) on the activity, conformation and unfolding process of protein tyrosine phosphatase (PTPase), a thermostable low molecular weight protein from Thermus thermophilus HB27, have been studied. Enzymatic activity assays showed both urea and GdnHCl resulted in the inactivation of PTPase in a concentration and time-dependent manner. Inactivation kinetics analysis suggested that the inactivation of PTPase induced by urea and GdnHCl were both monophasic and reversible processes, and the effects of urea and GdnHCl on PTPase were similar to that of mixed-type reversible inhibitors. Far-ultraviolet (UV) circular dichroism (CD), Tryptophan and 1-anilinonaphthalene -8-sulfonic acid (ANS) fluorescence spectral analyses indicated the existence of a partially active and an inactive molten globule-like intermediate during the unfolding processes induced by urea and GdnHCl, respectively. Based on the sequence alignment and the homolog Tt1001 protein structure, we discussed the possible conformational transitions of PTPase induced by urea and GdnHCl and compared the conformations of these unfolding intermediates with the transient states in bovine PTPase and its complex structures in detail. Our results may be able to provide some valuable clues to reveal the relationship between the structure and enzymatic activity, and the unfolding pathway and mechanism of PTPase. PMID:25255086

  4. Effect of temperature on the conformation of natively unfolded protein 4E-BP1 in aqueous and mixed solutions containing trifluoroethanol and hexafluoroisopropanol.

    PubMed

    Hackl, Ellen V

    2015-02-01

    Natively unfolded (intrinsically disordered) proteins have attracted growing attention due to their high abundance in nature, involvement in various signalling and regulatory pathways and direct association with many diseases. In the present work the combined effect of temperature and alcohols, trifluoroethanol (TFE) and hexafluoroisopropanol (HFIP), on the natively unfolded 4E-BP1 protein was studied to elucidate the balance between temperature-induced folding and unfolding in intrinsically disordered proteins. It was shown that elevated temperatures induce reversible partial folding of 4E-BP1 both in buffer and in the mixed solutions containing denaturants. In the mixed solutions containing TFE (HFIP) 4E-BP1 adopts a partially folded helical conformation. As the temperature increases, the initial temperature-induced protein folding is replaced by irreversible unfolding/melting only after a certain level of the protein helicity has been reached. Onset unfolding temperature decreases with TFE (HFIP) concentration in solution. It was shown that an increase in the temperature induces two divergent processes in a natively unfolded protein--hydrophobicity-driven folding and unfolding. Balance between these two processes determines thermal behaviour of a protein. The correlation between heat-induced protein unfolding and the amount of helical content in a protein is revealed. Heat-induced secondary structure formation can be a valuable test to characterise minor changes in the conformations of natively unfolded proteins as a result of site-directed mutagenesis. Mutants with an increased propensity to fold into a structured form reveal different temperature behaviour. PMID:25503819

  5. Interactions of main chain in folding and self assembly of unfolded protein structure: Enquiries with a serine solubilized nonapeptide

    NASA Astrophysics Data System (ADS)

    Srivastava, Kinshuk Raj; Durani, Susheel

    2014-06-01

    Interactions of the protein main chain are probed for their role in folding and self-assembly. The interactions are assessed with serine nonapeptide Ac-(Ser-Ala)4-Ser-NH2 in poly-L and alternating-L,D structure variations. Being a neutral molecule, Serine nonapeptide has been found to display not only folding-unfolding equilibrium, but also association-dissociation equilibrium as a function of solvent and concentration. Thus scrutiny of intra- and inter-molecular interactions have been undertaken in water, methanol, and DMSO solvents. In water, poly-L peptide displays a PPII-helix conformation which unfolds to extended ?-conformation with increase of temperature, apparently in a two-state equilibrium. Poly-L peptide at high concentration and on transfer to the low polarity solvent, methanol, displays ordering as a ?-hairpin. This implies folding of the peptide by self assembly. Self assembly and ordering possibly as double-stranded ?-helix is also evidence for alternating-L,D peptide. Both isomers were observed to be unfolded in high polarity solvent DMSO. Dynamic light scattering suggests that assembly in both isomers may involve large size aggregates. The results have established that folding and self-assembly can be coupled equilibria dependent upon solute structure, concentration, and solvent. The interactions of the protein main chain involved in folding and self assembly of unfolded structure are illuminated and have been discussed.

  6. Nanosecond laser temperature-jump optical rotatory dispersion: Application to early events in protein folding\\/unfolding

    Microsoft Academic Search

    Eefei Chen; Youxian Wen; James W. Lewis; Robert A. Goldbeck; David S. Kliger; Charlie E. M. Strauss

    2005-01-01

    Nanosecond time-resolved optical rotatory dispersion (TRORD) techniques are coupled with laser temperature-jump (T-jump) triggering in an instrument that measures ultrafast protein folding-unfolding dynamics with high specificity to secondary structure. Far-ultraviolet (UV) ORD can be measured with this instrument over a wide wavelength range at times as early as 35 ns after a 3 ns laser T-jump pulse. The fundamental of

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

    Microsoft Academic Search

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

    2005-01-01

    Summary: An easy-to-use, versatile and freely available graphic web server, FoldIndex© is described: it predicts if a given protein sequence is intrinsically unfolded implementing the algorithm of Uversky and co-workers, which is based on the average residue hydrophobi- city and net charge of the sequence. FoldIndex© has an error rate comparable to that of more sophisticated fold prediction methods. Sliding

  8. The Unfolded Protein Response (UPR) & The Endoplasmic Reticulum

    E-print Network

    Bedwell, David M.

    diabetes (insulin production) · Type II diabetes (insulin receptor signaling) · Necrotizing enterocolitis mutation prevents oxidative insulin folding ­ UPR - diabetes · Environmental ­ High cholesterol, alcohol

  9. 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.

  10. 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.

  11. Speeding up protein folding: mutations that increase the rate at which Rop folds and unfolds by over four orders of magnitude

    E-print Network

    Mochrie, Simon

    Speeding up protein folding: mutations that increase the rate at which Rop folds and unfolds. Introduction When a protein folds, the backbone and sidechain atoms organize from the extensive number protein folding usually occurs on the order of milliseconds to seconds, it is gener- ally accepted

  12. Ion Mobility Spectrometry-Mass Spectrometry of Intrinsically Unfolded Proteins: Trying to Put Order into Disorder.

    PubMed

    Knapman, T W; Valette, N M; Warriner, S L; Ashcroft, A E

    2013-04-01

    Intrinsically disordered proteins do not adopt well-defined native structures and therefore present an intriguing challenge in terms of structural elucidation as they are relatively inaccessible to traditional approaches such as NMR and X-ray crystallography. Many members of this important group of proteins have a distinct biological function and frequently undergo a conformational change on binding to their physiological targets which can in turn modulate their function. Furthermore, many intrinsically unstructured proteins are associated with a wide range of major diseases including cancer and amyloid-related disorders. Here, electrospray ionisation-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) has been used to probe the conformational characteristics of two intrinsically disordered proteins: apo-cytochrome c and apo-osteocalcin. Both proteins are structured in their holo-states when bound to their respective substrates, but disordered in their apo-states. Here, the conformational properties of the holo- and the apo-protein forms for both species have been analysed and their mass spectral data and ion mobility spectrometry-derived collision cross-sectional areas, indicative of their physical size, compared to study the relationship between substrate binding and tertiary structure. In both cases, the intrinsically unstructured apo-states populated multiple conformations with larger cross-sectional areas than their holo-analogues, suggesting that intrinsic disorder in proteins does not preclude the formation of preferred conformations. Additionally, analysis of truncated analogues of osteocalcin has located the region of the protein responsible for the conformational changes detected upon metal cation binding. Together, the data illustrate the scope and utility of ESI-IMS-MS for studying the characteristics and properties of intrinsically disordered proteins whose analysis by other techniques is limited. PMID:23885220

  13. Ion Mobility Spectrometry-Mass Spectrometry of Intrinsically Unfolded Proteins: Trying to Put Order into Disorder

    PubMed Central

    Knapman, T. W; Valette, N. M; Warriner, S. L; Ashcroft, A. E

    2013-01-01

    Intrinsically disordered proteins do not adopt well-defined native structures and therefore present an intriguing challenge in terms of structural elucidation as they are relatively inaccessible to traditional approaches such as NMR and X-ray crystallography. Many members of this important group of proteins have a distinct biological function and frequently undergo a conformational change on binding to their physiological targets which can in turn modulate their function. Furthermore, many intrinsically unstructured proteins are associated with a wide range of major diseases including cancer and amyloid-related disorders. Here, electrospray ionisation-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS) has been used to probe the conformational characteristics of two intrinsically disordered proteins: apo-cytochrome c and apo-osteocalcin. Both proteins are structured in their holo-states when bound to their respective substrates, but disordered in their apo-states. Here, the conformational properties of the holo- and the apo-protein forms for both species have been analysed and their mass spectral data and ion mobility spectrometry-derived collision cross-sectional areas, indicative of their physical size, compared to study the relationship between substrate binding and tertiary structure. In both cases, the intrinsically unstructured apo-states populated multiple conformations with larger cross-sectional areas than their holo-analogues, suggesting that intrinsic disorder in proteins does not preclude the formation of preferred conformations. Additionally, analysis of truncated analogues of osteocalcin has located the region of the protein responsible for the conformational changes detected upon metal cation binding. Together, the data illustrate the scope and utility of ESI-IMS-MS for studying the characteristics and properties of intrinsically disordered proteins whose analysis by other techniques is limited. PMID:23885220

  14. Unfolding stabilities of two paralogous proteins from Naja naja naja (Indian cobra) as probed by molecular dynamics simulations.

    PubMed

    Gorai, Biswajit; Sivaraman, Thirunavukkarasu

    2013-09-01

    Structurally similar but functionally different two paralogous proteins, CTX1 (a cardiotoxin) and LNTX2 (an alpha-neurotoxin), from venom of Naja naja naja have been homology modeled and subjected to molecular dynamics (MD) simulations at four different temperatures (298 K, 310 K, 373 K & 473 K) under close quarters of physiological conditions. Each MD simulation was performed for 25 ns and trajectory structures stored at every 25 ps were used to probe various structural events occurring in the temperature-induced unfolding of the proteins. Notwithstanding their similar scaffolds, the two proteins are drastically differing in their unfolding stabilities from each other. The structural orders of flexibilities for the CTX1 and LNTX2 were found to be loop II > loop III > loop I > C-terminal and C-terminal > loop I > loop III > loop II, respectively. Based on the comprehensive analyses of the simulation data and studies on the various structural interactions of all cardiotoxins (CTXs) and alpha-neurotoxins (NTXs) for which three-dimensional structures determined by experimental techniques are available to date, we have herein proposed a hypothesis ('CN network') rationalizing the differential stabilities of the CTXs and NTXs belonging to a three-finger toxin superfamily of snake venoms. PMID:23791667

  15. 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

  16. Near-infrared spectroscopy for in-line monitoring of protein unfolding and its interactions with lyoprotectants during freeze-drying.

    PubMed

    Pieters, Sigrid; De Beer, Thomas; Kasper, Julia Christina; Boulpaep, Dorien; Waszkiewicz, Oliwia; Goodarzi, Mohammad; Tistaert, Christophe; Friess, Wolfgang; Remon, Jean-Paul; Vervaet, Chris; Vander Heyden, Yvan

    2012-01-17

    This work presents near-infrared spectroscopy (NIRS) as an in-line process analyzer for monitoring protein unfolding and protein-lyoprotectant hydrogen bond interactions during freeze-drying. By implementing a noncontact NIR probe in the freeze-drying chamber, spectra of formulations containing a model protein immunoglobulin G (IgG) were collected each process minute. When sublimation was completed in the cake region illuminated by the NIR probe, the frequency of the amide A/II band (near 4850 cm(-1)) was monitored as a function of water elimination. These two features were well correlated during protein dehydration in the absence of protein unfolding (desired process course), whereas consistent deviations from this trend to higher amide A/II frequencies were shown to be related to protein unfolding. In formulations with increased sucrose concentrations, the markedly decreased amide A/II frequencies seen immediately after sublimation indicated an increased extent of hydrogen bond interaction between the protein's backbone and surrounding molecules. At the end of drying, there was evidence of nearly complete water substitution for formulations with 1%, 5%, and 10% sucrose. The presented approach shows promising perspectives for early fault detection of protein unfolding and for obtaining mechanistic process information on actions of lyoprotectants. PMID:22122716

  17. Folding studies of purified LamB protein, the maltoporin from the Escherichia coli outer membrane: Trimer dissociation can be separated from unfolding

    Microsoft Academic Search

    Valerie Baldwin; Mandeep Bhatia; Mary Luckey

    2011-01-01

    The folding mechanisms for ?-barrel membrane proteins present unique challenges because acquisition of both secondary and tertiary structure is coupled with insertion into the bilayer. For the porins in Escherichia coli outer membrane, the assembly pathway also includes association into homotrimers. We study the folding pathway for purified LamB protein in detergent and observe extreme hysteresis in unfolding and refolding,

  18. Unfolding Simulations Reveal the Mechanism of Extreme Unfolding Cooperativity in the Kinetically Stable alpha-Lytic Protease

    Microsoft Academic Search

    Neema L. Salimi; Bosco Ho; David A. Agard; B. Montgomery Pettitt

    2010-01-01

    Kinetically stable proteins, those whose stability is derived from their slow unfolding kinetics and not thermodynamics, are examples of evolution's best attempts at suppressing unfolding. Especially in highly proteolytic environments, both partially and fully unfolded proteins face potential inactivation through degradation and\\/or aggregation, hence, slowing unfolding can greatly extend a protein's functional lifetime. The prokaryotic serine protease ?-lytic protease (?LP)

  19. Programs in C for parameterizing measured 5? × 5? NaI gamma response functions and unfolding of continuous gamma spectra

    NASA Astrophysics Data System (ADS)

    Nguyen, H. V.; Campbell, J. M.; Couchell, G. P.; Li, S.; Pullen, D. J.; Schier, W. A.; Seabury, E. H.; Tipnis, S. V.

    1996-02-01

    A 5? × 5? NaI(Tl) detector has been used to measure gamma-ray spectra resulting from the decay of aggregate fission products. In order to extract the true gamma-ray energy distribution from the measured spectra, the detector response functions for monoenergetic gamma rays spanning the energy range of the measurements must be determined. At present we have measured 13 such response functions in the energy range 0.081-6.13 MeV. NGRC is a program in C written to implement an interpolation scheme for estimating the response function at any other intermediate energy. This program takes a library of response function tails and constructs a response function matrix which is used as input to a second program CRSUP written for obtaining gamma-ray energy distributions. It assumes the measured spectrum consists of a superposition of a specified number of response functions placed at energies determined by the program according to the detector resolution and spectrum end point energy. The program then computes the distribution of the strength of the response functions in a least-squares fashion. This program is designed to maximize the number of response functions that can be used in modeling the measured spectrum without reducing the number of bins used in each response function. The response functions constructed by the interpolation procedure have been used in the program SPEC-FIT to fit in a least-squares fashion the gamma-ray spectrum of 152Eu. The fit is an excellent reproduction of both the photopeak and continuous regions of the entire measured spectrum. Finally the validity of the least-square method implemented by CRSUP has also been tested by using this program to unfold an analytically constructed continuous spectrum. The results obtained were in excellent agreement with the assumed distribution function, illustrating the applicability of CRSUP for unfolding other types of continuous spectra as encountered in beta, neutron-time-of-flight and Rutherford-backscattering spectroscopy.

  20. Folding studies of Purified LamB Protein, the Maltoporin from the Escherichia coli Outer Membrane: Trimer Dissociation can be separated from Unfolding

    PubMed Central

    Baldwin, Valerie; Bhatia, Mandeep; Luckey, Mary

    2011-01-01

    The folding mechanisms for ?-barrel membrane proteins present unique challenges because acquisition of both secondary and tertiary structure is coupled with insertion into the bilayer. For the porins in Escherichia coli outer membrane, the assembly pathway also includes association into homotrimers. We study the folding pathway for purified LamB protein in detergent and observe extreme hysteresis in unfolding and refolding, as indicated by the shift in intrinsic fluorescence. The strong hysteresis is not seen in unfolding and refolding a mutant LamB protein lacking the disulfide bond, as it unfolds at much lower denaturant concentrations than wild type LamB protein. The disulfide bond is proposed to stabilize the structure of LamB protein by clasping together the two sides of Loop 1 as it lines the inner cavity of the barrel. In addition we find that low pH promotes dissociation of the LamB trimer to folded monomers, which run at about one third the size of the native trimer during SDS PAGE and are much more resistant to trypsin than the unfolded protein. We postulate the loss at low pH of two salt bridges between Loop 2 of the neighboring subunit and the inner wall of the monomer barrel destabilizes the quaternary structure. PMID:21640073

  1. Influence of the sample-solvent on protein retention, mass transfer and unfolding kinetics in hydrophobic interaction chromatography.

    PubMed

    Muca, Renata; Marek, Wojciech; Piatkowski, Wojciech; Antos, Dorota

    2010-04-23

    Typical mobile phase employed in hydrophobic interaction chromatography contains cosmotropic salts, which promote retention and simultaneously reduce the protein solubility in the mobile phase. To increase mass overloading in the separation process the protein can be dissolved in a sample-solvent with concentration of salt lower than that in the mobile phase or in salt free solutions. However, this methodology may cause band splitting and band deformation, which results in yield losses. In this study, these phenomena were analyzed based on the retention behavior of two model proteins, i.e., lysozyme and bovine serum albumin. Retention of these proteins was accompanied by strong band broadening originated from slow rates of mass transfer and/or of adsorption-desorption process involving the protein conformational changes. The mass transport resistances and unfolding kinetics were found to contribute to the sample-solvent effects. To avoid band deformations the process variables such as the salt concentration and temperature were adjusted in such a way that complete resolution between band profile of the sample-solvent and the protein was achieved. For the process simulation a dynamic model, which accounted for underlying kinetics was used. General guidelines of the process design were developed. PMID:20236645

  2. "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

  3. “Invisible” Conformers of an Antifungal Disulfide Protein Revealed by Constrained Cold and Heat Unfolding, CEST-NMR Experiments, and Molecular Dynamics Calculations

    PubMed Central

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

    2015-01-01

    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 15N-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

  4. An Aggregation-Prone Intermediate Species Is Present in the Unfolding Pathway of the Monomeric Portal Protein of Bacteriophage P22:  Implications for Portal Assembly †

    Microsoft Academic Search

    Carolina A. C. A. Braga; Danielle Carvalho; Flávio Alves Lara; Juliana Reis Cortines; Sean D. Moore; Debora Foguel

    2007-01-01

    The head of the P22 bacteriophage is interrupted by a unique dodecameric portal vertex that serves as a conduit for the entrance and exit of the DNA. Here, the in vitro unfolding\\/refolding processes of the portal protein of P22 were investigated at different temperatures (1, 25, and 37 °C) through the use of urea and high hydrostatic pressure (HHP) combined

  5. Dewetting-induced globule-coil transitions of model polymers and possible implications high-temperature and low-pressure unfolding of proteins.

    PubMed

    Sumi, Tomonari; Imazaki, Nobuyuki; Sekino, Hideo

    2010-04-28

    A thermodynamic analysis of high-temperature and low-pressure unfolding of proteins using a coarse-grained multiscale simulation combined with a liquid-state density-functional theory is presented. In this study, a hydrophobic polymer chain is employed as a probe molecule for investigating qualitative changes in a hydration free energy surface acting on proteins with changes in temperature and pressure. When water is heated so that its vapor pressure is equal to the atmospheric pressure, it boils. Long-ranged dewetting or drying caused by a hydrophobic planar wall and a large hydrophobic solute surface is significantly enhanced as it approaches the liquid-vapor coexistence curve of water. In this study, we demonstrate that high-temperature and low-pressure unfolding of the polymer chain is interpreted as dewetting-induced unfolding that occurs as it approaches the liquid-vapor coexistence. The unfolding of proteins due to high-temperature and low-pressure denaturation enhances the long-ranged dewetting or drying around them. The long-ranged dewetting phenomenon is considered to be originating from positive changes in both volume and entropy due to the high-temperature and low-pressure denaturation of the proteins. PMID:20441309

  6. Characterizing the Structures and Folding of Free Proteins Using 2-D Gas-Phase Separations: Observation of Multiple Unfolded Conformers

    SciTech Connect

    Shvartsburg, Alexandre A.; Li, Fumin; Tang, Keqi; Smith, Richard D.

    2006-05-15

    Understanding the 3-D structure and dynamics of proteins and other biological macromolecules in various environments is among the central challenges of chemistry. Electrospray ionization (ESI) can transfer ions from solution to gas phase with only limited structural distortion, allowing their profiling using mass spectrometry and other gas phase approaches. Ion mobility spectrometry (IMS) can be used to separate and characterize macroion conformations with high sensitivity and speed. However, IMS separation power has proven insufficient for full resolution of major structural variants of protein ions and elucidation of their interconversion dynamics. Here we report characterization of macromolecular conformations using field asymmetric waveform IMS (FAIMS) coupled to conventional IMS in conjunction with mass spectrometry. The controlled activation of ions in the electrodynamic funnel trap between FAIMS and IMS stages enables investigating the structural evolution of particular isomeric precursors as a function of the extent and duration of activation that can be varied over a large range. These new capabilities are demonstrated for bovine ubiquitin, a common model for study of structure and folding of gas-phase proteins. For nearly all charge states, two-dimensional FAIMS/IMS separations distinguish many more conformations than either FAIMS or IMS alone, including some species with very low abundance. The unfolding of specific ubiquitin conformers has been studied employing ion heating in the FAIMS/IMS interface.

  7. Slow Proton Transfer Coupled to Unfolding Explains the Puzzling Results of Single-Molecule Experiments on BBL, a Paradigmatic Downhill Folding Protein

    PubMed Central

    Cerminara, Michele; Campos, Luis A.; Ramanathan, Ravishankar; Muñoz, Victor

    2013-01-01

    A battery of thermodynamic, kinetic, and structural approaches has indicated that the small ?-helical protein BBL folds-unfolds via the one-state downhill scenario. Yet, single-molecule fluorescence spectroscopy offers a more conflicting view. Single-molecule experiments at pH 6 show a unique half-unfolded conformational ensemble at mid denaturation, whereas other experiments performed at higher pH show a bimodal distribution, as expected for two-state folding. Here we use thermodynamic and laser T-jump kinetic experiments combined with theoretical modeling to investigate the pH dependence of BBL stability, folding kinetics and mechanism within the pH 6–11 range. We find that BBL unfolding is tightly coupled to the protonation of one of its residues with an apparent pKa of ?7. Therefore, in chemical denaturation experiments around neutral pH BBL unfolds gradually, and also converts in binary fashion to the protonated species. Moreover, under the single-molecule experimental conditions (denaturant midpoint and 279 K), we observe that proton transfer is much slower than the ?15 microseconds folding-unfolding kinetics of BBL. The relaxation kinetics is distinctly biphasic, and the overall relaxation time (i.e. 0.2–0.5 ms) becomes controlled by the proton transfer step. We then show that a simple theoretical model of protein folding coupled to proton transfer explains quantitatively all these results as well as the two sets of single-molecule experiments, including their more puzzling features. Interestingly, this analysis suggests that BBL unfolds following a one-state downhill folding mechanism at all conditions. Accordingly, the source of the bimodal distributions observed during denaturation at pH 7–8 is the splitting of the unique conformational ensemble of BBL onto two slowly inter-converting protonation species. Both, the unprotonated and protonated species unfold gradually (one-state downhill), but they exhibit different degree of unfolding at any given condition because the native structure is less stable for the protonated form. PMID:24205082

  8. Oligomerization, Conformational Stability and Thermal Unfolding of Harpin, HrpZPss and Its Hypersensitive Response-Inducing C-Terminal Fragment, C-214-HrpZPss

    PubMed Central

    Tarafdar, Pradip K.; Vedantam, Lakshmi Vasudev; Sankhala, Rajeshwer S.; Purushotham, Pallinti; Podile, Appa Rao; Swamy, Musti J.

    2014-01-01

    HrpZ—a harpin from Pseudomonas syringae—is a highly thermostable protein that exhibits multifunctional abilities e.g., it elicits hypersensitive response (HR), enhances plant growth, acts as a virulence factor, and forms pores in plant plasma membranes as well as artificial membranes. However, the molecular mechanism of its biological activity and high thermal stability remained poorly understood. HR inducing abilities of non-overlapping short deletion mutants of harpins put further constraints on the ability to establish structure-activity relationships. We characterized HrpZPss from Pseudomonas syringae pv. syringae and its HR inducing C-terminal fragment with 214 amino acids (C-214-HrpZPss) using calorimetric, spectroscopic and microscopic approaches. Both C-214-HrpZPss and HrpZPss were found to form oligomers. We propose that leucine-zipper-like motifs may take part in the formation of oligomeric aggregates, and oligomerization could be related to HR elicitation. CD, DSC and fluorescence studies showed that the thermal unfolding of these proteins is complex and involves multiple steps. The comparable conformational stability at 25°C (?10.0 kcal/mol) of HrpZPss and C-214-HrpZPss further suggest that their structures are flexible, and the flexibility allows them to adopt proper conformation for multifunctional abilities. PMID:25502017

  9. An Unfolded Putative Transmembrane Polypeptide, which Can Lead to Endoplasmic Reticulum Stress, Is a Substrate of Parkin

    Microsoft Academic Search

    Yuzuru Imai; Mariko Soda; Haruhisa Inoue; Nobutaka Hattori; Yoshikuni Mizuno; Ryosuke Takahashi

    2001-01-01

    A putative G protein-coupled transmembrane polypeptide, named Pael receptor, was identified as an interacting protein with Parkin, a gene product responsible for autosomal recessive juvenile Parkinsonism (AR-JP). When overexpressed in cells, this receptor tends to become unfolded, insoluble, and ubiquitinated in vivo. The insoluble Pael receptor leads to unfolded protein-induced cell death. Parkin specifically ubiquitinates this receptor in the presence

  10. Life in the pressure cooker: The thermal unfolding of proteins from hyperthermophiles

    Microsoft Academic Search

    H. H. Klump; M. W. W. Adams; F. T. Robb

    1994-01-01

    Xieported here are the first results of a qcantitative ther- modynamic st-dy involvixg a series of proteins isolated from the anaerobic sulfur-reducixg archaebacterium Pyrococcus furiosus which grows near submrize geotherml vents on the deep ocean floor. The proteins isolated and investigated here are the two non-henie iron proteins rubredoxin (Kid 5400), and ferredoxin (Mw 7500), and the er-zyine glutamate dehydrogeriase

  11. Unfolding and aggregation of a glycosylated monoclonal antibody on a cation exchange column. Part II. Protein structure effects by hydrogen deuterium exchange mass spectrometry.

    PubMed

    Guo, Jing; Carta, Giorgio

    2014-08-22

    Hydrogen-deuterium exchange mass spectrometry (HX-MS) with proteolytic fragmentation is used to determine patterns of unfolding, as measured by increased solvent exposure, with peptide-level resolution for a glycosylated monoclonal antibody both when eluted from a tentacle-type cation exchange column (Fractogel EMD SO3-) and while bound to the resin. Two peaks are obtained when the bound protein is eluted with either a NaCl gradient or with two NaCl steps at increasing concentration. The first, early eluting peak contains only monomeric species whose structure is consistent with the native monomer. The second, late eluting peak contains a mixture of monomeric and aggregated species. The monomeric species in this mixture is also found to have a structure consistent with that of the native mAb, showing no evidence of increased solvent exposure. The aggregated species show instead significant unfolding in areas of the protein structure contained within the Fc region. The same peptides that exhibit the greatest level of solvent exposure in the aggregated species are also found in the fraction of protein that elutes from the resin only at high salt concentration, indicating that the aggregates are formed when the strongly-bound unfolded intermediate is desorbed at high salt. There is no evidence that the unfolded intermediate, formed while the protein is bound on the resin, is present in any of the eluted fractions indicating that, upon desorption from the resin, the intermediate either quickly refolds or forms aggregates which end-up co-eluting with the refolded protein at high salt concentrations. PMID:25011681

  12. 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

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

    PubMed

    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 PMID:26046628

  14. Monitoring Equilibria and Kinetics of Protein Folding\\/Unfolding Reactions by Capillary Zone Electrophoresis

    Microsoft Academic Search

    Barbara Verzola; Fabrizio Chiti; Giampaolo Manao; Pier Giorgio Righetti

    2000-01-01

    A method is described here for studying conformational transitions of proteins due to denaturing agents: capillary zone electrophoresis (CZE) in acidic, isoelectric buffers. The sample is run in 50 mM isoelectric glutamic acid (pH = pI = 3.2) added with 1 mM oligoamine (tetraethylene pentamine) for quenching protein interaction to the capillary wall (final pH = 3.3). Muscle acylphosphatase (AcP),

  15. Studies on the degradation pathway of iron-sulfur centers during unfolding of a hyperstable ferredoxin: cluster dissociation, iron release and protein stability

    Microsoft Academic Search

    Sónia S. Leal; Miguel Teixeira; Cláudio M. Gomes

    2004-01-01

    The ferredoxin from the thermoacidophile Acidianus ambivalens is a representative of the archaeal family of di-cluster [3Fe-4S][4Fe-4S] ferredoxins. Previous studies have shown that these ferredoxins are intrinsically very stable and led to the suggestion that upon protein unfolding the iron-sulfur clusters degraded via linear three-iron sulfur center species, with 610 and 520 nm absorption bands, resembling those observed in purple aconitase.

  16. Natively unfolded tubulin polymerization promoting protein TPPP/p25 is a common marker of alpha-synucleinopathies.

    PubMed

    Kovács, Gábor G; László, Lajos; Kovács, János; Jensen, Poul Henning; Lindersson, Evo; Botond, Gergo; Molnár, Tamás; Perczel, András; Hudecz, Ferenc; Mezo, Gábor; Erdei, Anna; Tirián, László; Lehotzky, Attila; Gelpi, Ellen; Budka, Herbert; Ovádi, Judit

    2004-11-01

    The novel basic, heat-stable tubulin polymerization promoting protein TPPP/p25 is associated with microtubules in vitro and can induce the formation of aberrant microtubule assemblies. We show by 1H-NMR spectroscopy that TPPP/p25 is natively unfolded. Antisera against peptide 186GKGKAGRVDLVDESG200NH2 (186-200) are highly specific to TPPP/p25. Immunohistochemistry and confocal microscopy demonstrates that TPPP/p25 is enriched in filamentous alpha-synuclein bearing Lewy bodies of Parkinson's (PD) and diffuse Lewy body disease (DLBD), as well as glial inclusions of multiple system atrophy (MSA). There is a correlation between TPPP/p25 and alpha-synuclein immunoreactivity in Western blot. In contrast, TPPP/p25 is not associated with abnormally phosphorylated tau in various inclusions of Pick's disease (PiD), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD). However, electron microscopy confirms clusters of TPPP/p25 immunoreactivity along filaments of unstructured but not compact neurofibrillary tangles in Alzheimer's disease (AD). TPPP/p25 seems to be a novel marker of alpha-synucleinopathies. PMID:15474353

  17. 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 ...

  18. Proteins in frozen solutions: evidence of ice-induced partial unfolding.

    PubMed Central

    Strambini, G B; Gabellieri, E

    1996-01-01

    From a drastic decrease in the phosphorescence lifetime of tryptophan residues buried in compact rigid cores of globular proteins, it was possible to demonstrate that freezing of aqueous solutions is invariably accompanied by a marked loosening of the native fold, an alteration that entails considerable loss of secondary and tertiary structure. The phenomenon is largely reversible on ice melting although, in some cases, a small fraction of macromolecules recovers neither the initial phosphorescence properties nor the catalytic activity. The variation in the lifetime parameter was found to be a smooth function of the residual volume of liquid water in equilibrium with ice and to depend on the morphology of ice. The addition of cryoprotectants such as glycerol and sucrose profoundly attenuates or even eliminates the perturbation. These results are interpreted in terms of adsorption of protein molecules onto the surface of ice. PMID:8789114

  19. Circular dichroism analysis for multidomain proteins: studies of the irreversible unfolding of Hepatitis C virus helicase

    Microsoft Academic Search

    Agnieszka Gozdek; Anna Stankiewicz-Drogo?; Anna M. Boguszewska-Chachulska

    2008-01-01

    The non-structural protein 3 (NS3) of Hepatitis C virus (HCV) is a bifunctional enzyme with RNA-dependent NTPase\\/RNA helicase and serine protease activities, and thus represents a prom- ising target for anti-HCV therapy. These functions are performed by two distinct moieties; the N-terminal protease domain and the C-terminal helicase domain that further folds into three structural subdomains. To obtain lower molecular

  20. Thermodynamic Characterization of the Unfolding of the Prion Protein Roumita Moulick and Jayant B. Udgaonkar*

    E-print Network

    of the mouse prion protein (moPrP), the full-length moPrP (23­231) and the structured C-terminal domain, mo conformation. Three-dimensional NMR-derived structures of the re- combinant mouse (8), human (9), and Syrian-terminal domain (NTD), and a structured C-terminal domain (CTD) comprised of three a-helices and two short b

  1. 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

  2. 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

  3. Low energy of activation for amide hydrogen exchange reactions in proteins supports a local unfolding model.

    PubMed

    Thomsen, N K; Poulsen, F M

    1993-11-01

    Hydrogen exchange reactions of amides in hen egg white lysozyme that are pH dependent and have a low energy of activation have been shown to be in accordance with a reaction mechanism in two steps, an equilibrium step and an exchange step. These results are not in agreement with the model, proposed by C.K. Woodward & B.D. Hilton, known as the penetration model. Therefore our results suggest that this model should be revised. The amide hydrogen/deuterium exchange rates in hen egg white lysozyme were measured at 4 degrees C, 10 degrees C, 15 degrees C and 25 degrees C at pH 7.0 by 1H nuclear magnetic resonance spectroscopy. Activation energies of the exchange reactions in the range from 20 kJ mol-1 to 333 kJ mol-1 were obtained for 32 of the 129 residues in the protein. The amides of lysozyme studied here could be divided into two groups, one group of amides are characterized by an observed amide exchange rate (ko) in the range 10(-4) to 10(-6) s-1, an equilibrium constant k1/k2 close to 10(-5), a low energy of activation (20 to 50 kJ mol-1) and a distance less than 6 A from solvent. The other group of amides are characterized by a ko less than 10(-6) s-1, a k1/k2 close to 10(-7), higher energies of activation (40 to 330 kJ mol-1) and a distance more than 4 A from solvent. In terms of structure the amides of the last group are from the core of the protein. They are typically involved in a hydrogen bond and form part of the secondary structure either as interior alpha-helices or central strands of beta-sheets. The first group consists of amides that are in the shell of the protein between the core and the surface. These amides are typically hydrogen bonded and involved in secondary structure such as external alpha-helices or outer strands of beta-sheets and turns. PMID:8230202

  4. Guanidine hydrochloride stabilization of a partially unfolded intermediate during the reversible denaturation of protein disulfide isomerase.

    PubMed Central

    Morjana, N A; McKeone, B J; Gilbert, H F

    1993-01-01

    The reversible denaturation of protein disulfide isomerase proceeds through intermediates that are stabilized by interaction with guanidine hydrochloride. At pH 7.5, the equilibrium denaturation by urea is completely reversible and the transition can be reasonably well-described by a two-state model involving only native and denatured forms. In comparison, the equilibrium denaturation by guanidine hydrochloride occurs in two distinct steps. In the presence of a low constant amount of guanidine hydrochloride (0.5-1.4 M), urea denaturation also becomes biphasic, suggesting the accumulation of an intermediate species that is stabilized by specific interaction with guanidine hydrochloride but not by high concentrations of other salts or other denaturants. PMID:8460117

  5. A nanobody binding to non-amyloidogenic regions of the protein human lysozyme enhances partial unfolding but inhibits amyloid fibril formation.

    PubMed

    De Genst, Erwin; Chan, Pak-Ho; Pardon, Els; Hsu, Shang-Te D; Kumita, Janet R; Christodoulou, John; Menzer, Linda; Chirgadze, Dimitri Y; Robinson, Carol V; Muyldermans, Serge; Matagne, André; Wyns, Lode; Dobson, Christopher M; Dumoulin, Mireille

    2013-10-24

    We report the effects of the interaction of two camelid antibody fragments, generally called nanobodies, namely cAb-HuL5 and a stabilized and more aggregation-resistant variant cAb-HuL5G obtained by protein engineering, on the properties of two amyloidogenic variants of human lysozyme, I56T and D67H, whose deposition in vital organs including the liver, kidney, and spleen is associated with a familial non-neuropathic systemic amyloidosis. Both NMR spectroscopy and X-ray crystallographic studies reveal that cAb-HuL5 binds to the ?-domain, one of the two lobes of the native lysozyme structure. The binding of cAb-HuL5/cAb-HuL5G strongly inhibits fibril formation by the amyloidogenic variants; it does not, however, suppress the locally transient cooperative unfolding transitions, characteristic of these variants, in which the ?-domain and the C-helix unfold and which represents key early intermediate species in the formation of amyloid fibrils. Therefore, unlike two other nanobodies previously described, cAb-HuL5/cAb-HuL5G does not inhibit fibril formation via the restoration of the global cooperativity of the native structure of the lysozyme variants to that characteristic of the wild-type protein. Instead, it inhibits a subsequent step in the assembly of the fibrils, involving the unfolding and structural reorganization of the ?-domain. These results show that nanobodies can protect against the formation of pathogenic aggregates at different stages in the structural transition of a protein from the soluble native state into amyloid fibrils, illustrating their value as structural probes to study the molecular mechanisms of amyloid fibril formation. Combined with their amenability to protein engineering techniques to improve their stability and solubility, these findings support the suggestion that nanobodies can potentially be developed as therapeutics to combat protein misfolding diseases. PMID:23919586

  6. Quantitative Determination of Site-Specific Conformational Distributions in an Unfolded Protein by Solid State Nuclear Magnetic Resonance

    PubMed Central

    Hu, Kan-Nian; Havlin, Robert H.; Yau, Wai-Ming; Tycko, Robert

    2009-01-01

    Summary Solid state nuclear magnetic resonance (NMR) techniques are used to investigate the structure of the 35-residue villin headpiece subdomain (HP35) in folded, partially denatured, and fully denatured states. Experiments are carried out in frozen glycerol/water solutions, with chemical denaturation by guanidine hydrochloride (GdnHCl). Without GdnHCl, two-dimensional solid state 13C NMR spectra of samples prepared with uniform 13C labeling of selected residues show relatively sharp crosspeaks at chemical shifts that are consistent with the known three-helix bundle structure of folded HP35. At high GdnHCl concentrations, most crosspeaks broaden and shift, qualitatively indicating disruption of the folded structure and development of static conformational disorder in the frozen denatured state. Conformational distributions at one residue in each helical segment are probed quantitatively with three solid state NMR techniques that provide independent constraints on backbone ? and ? torsion angles in samples with sequential pairs of carbonyl 13C labels. Without GdnHCl, the combined data are well fit by ?-helical conformations. At [GdnHCl] = 4.5 M, corresponding to the approximate denaturation midpoint, the combined data are well fit by a combination of ?-helical and partially extended conformations at each site, but with a site-dependent population ratio. At [GdnHCl] = 7.0 M, corresponding to the fully denatured state, the combined data are well fit by a combination of partially extended and polyproline II conformations, again with a site-dependent population ratio. Two entirely different models for conformational distributions lead to nearly the same best-fit distributions, demonstrating the robustness of these conclusions. This work represents the first quantitative investigation of site-specific conformational distributions in partially folded and unfolded states of a protein by solid state NMR. PMID:19647001

  7. The basic helix-loop-helix region of human neurogenin 1 is a monomeric natively unfolded protein which forms a "fuzzy" complex upon DNA binding.

    PubMed

    Aguado-Llera, David; Goormaghtigh, Erik; de Geest, Natalie; Quan, Xiao-Jiang; Prieto, Alicia; Hassan, Bassen A; Gómez, Javier; Neira, José L

    2010-03-01

    Neuronal specification is regulated by the activity of transcription factors containing the basic helix-loop-helix motif (bHLH); these regulating proteins include, among others, the neurogenin (Ngn) family, related to the atonal family of genes. Neurogenin 1 (NGN1) is a 237-residue protein that contains a bHLH domain and is involved in neuronal differentiation. In this work, we synthesized the bHLH region of NGN1 (bHLHN) comprising residues 90-150 of the full-length NGN1. The domain is a monomeric natively unfolded protein with a pH-dependent premolten globule conformation, as shown by several spectroscopic techniques (namely, NMR, fluorescence, FTIR, and circular dichroism). The unfolded character of the domain also explains, first, the impossibility of its overexpression in several Escherichia coli strains and, second, its insolubility in aqueous buffers. To the best of our knowledge, this is the first extensive study of the conformational preferences of a bHLH domain under different solution conditions. Upon binding to two DNA E-boxes, the protein forms "fuzzy" complexes (that is, the complexes were not fully folded). The affinities of bHLHN for both DNA boxes were smaller than those of other bHLH domains, which might explain why the protein-DNA complexes were not fully folded. PMID:20102160

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

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

    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.

  9. Gibbs Adsorption Isotherm Combined with Monte Carlo Sampling to See Action of Cosolutes on Protein Folding

    E-print Network

    Harries, Daniel

    Driven by conditions set by smaller solutes, proteins fold and unfold. Experimentally, these conditions stability. Proteins 2004;57:311­321. © 2004 Wiley-Liss, Inc. INTRODUCTION Inside cells, proteins fold poten- tials, to follow the process of protein folding or unfolding in response to its environment

  10. The direction of protein entry into the proteasome determines the variety of products and depends on the force needed to unfold its two termini.

    PubMed

    Berko, Dikla; Tabachnick-Cherny, Shira; Shental-Bechor, Dalit; Cascio, Paolo; Mioletti, Silvia; Levy, Yaakov; Admon, Arie; Ziv, Tamar; Tirosh, Boaz; Goldberg, Alfred L; Navon, Ami

    2012-11-30

    Poorly structured domains in proteins enhance their susceptibility to proteasomal degradation. To learn whether the presence of such a domain near either end of a protein determines its direction of entry into the proteasome, directional translocation was enforced on several proteasome substrates. Using archaeal PAN-20S complexes, mammalian 26S proteasomes, and cultured cells, we identified proteins that are degraded exclusively from either the C or N terminus and some showing no directional preference. This property results from interactions of the substrate's termini with the regulatory ATPase and could be predicted based on the calculated relative stabilities of the N and C termini. Surprisingly, the direction of entry into the proteasome affected markedly the spectrum of peptides released and consequently influenced the efficiency of MHC class I presentation. Thus, easily unfolded termini are translocated first, and the direction of translocation influences the peptides generated and presented to the immune system. PMID:23041283

  11. 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

  12. Steered Molecular Dynamics Studies of Titin I1 Domain Unfolding

    Microsoft Academic Search

    M. Gao; Matthias Wilmanns; Klaus Schulten

    2002-01-01

    The cardiac muscle protein titin, responsible for developing passive elasticity and extensibility of muscle, possesses about 40 immunoglobulin-like (Ig) domains in its I-band region. Atomic force microscopy (AFM) and steered molecular dynamics (SMD) have been successfully combined to investigate the reversible unfolding of individual Ig domains. However, previous SMD studies of titin I-band modules have been restricted to I27, the

  13. The unfolding story of anthrax toxin translocation.

    PubMed

    Thoren, Katie L; Krantz, Bryan A

    2011-05-01

    The essential cellular functions of secretion and protein degradation require a molecular machine to unfold and translocate proteins either across a membrane or into a proteolytic complex. Protein translocation is also critical for microbial pathogenesis, namely bacteria can use translocase channels to deliver toxic proteins into a target cell. Anthrax toxin (Atx), a key virulence factor secreted by Bacillus anthracis, provides a robust biophysical model to characterize transmembrane protein translocation. Atx is comprised of three proteins: the translocase component, protective antigen (PA) and two enzyme components, lethal factor (LF) and oedema factor (OF). Atx forms an active holotoxin complex containing a ring-shaped PA oligomer bound to multiple copies of LF and OF. These complexes are endocytosed into mammalian host cells, where PA forms a protein-conducting translocase channel. The proton motive force unfolds and translocates LF and OF through the channel. Recent structure and function studies have shown that LF unfolds during translocation in a force-dependent manner via a series of metastable intermediates. Polypeptide-binding clamps located throughout the PA channel catalyse substrate unfolding and translocation by stabilizing unfolding intermediates through the formation of a series of interactions with various chemical groups and ?-helical structure presented by the unfolding polypeptide during translocation. PMID:21443527

  14. 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. [School of Physical Sciences, Jawaharlal Nehru University, New Delhi - 110 067 (India); Bandyopadhyay, Pradipta [School of Computational and Integrative Sciences, Jawaharlal Nehru University, New Delhi - 110 067 (India)

    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.

  15. Novel unfolding sequence of banana lectin: Folded, unfolded and natively unfolded-like monomeric states in guanidine hydrochloride.

    PubMed

    Ghosh, Goutam; Mandal, Dipak K

    2014-04-01

    The sequence of unfolding events of dimeric banana lectin (Banlec), as induced by guanidine hydrochloride (GdnHCl), has been investigated by size-exclusion HPLC, fluorescence, far-UV CD, low temperature phosphorescence and selective chemical modification. 8-Anilino-1-naphthalenesulfonate (ANS) binding indicates a structured unfolding intermediate which has been characterized as dissociated monomer by size-exclusion chromatography. Interestingly, the unfolding elution pattern reveals two distinct unfolded states. One is a usual random coil. The other represents a novel species having elution behavior and structural compactness (Stokes radius) similar to dissociated monomer but showing no regular secondary structure as determined by far-UV CD, thus resembling a natively unfolded state. N-Bromosuccinimide (NBS) oxidation shows that single tryptophan residue remains unmodified in dissociated monomer intermediate while the same is oxidized in natively unfolded-like species. Such difference in tryptophan environment in these species is supported by acrylamide quenching studies, and phosphorescence results at 77 K which show a blue-shift of (0,0) band from 414.8 nm to 409.2 nm. The present results reveal subtlety of structural characteristics of unfolded states of Banlec in GdnHCl, which provide important insight in protein unfolding reaction. PMID:24316282

  16. A thermodynamic coupling mechanism for GroEL-mediated unfolding.

    PubMed Central

    Walter, S; Lorimer, G H; Schmid, F X

    1996-01-01

    Chaperonins prevent the aggregation of partially folded or misfolded forms of a protein and, thus, keep it competent for productive folding. It was suggested that GroEL, the chaperonin of Escherichia coli, exerts this function 1 unfolding such intermediates, presumably in a catalytic fashion. We investigated the kinetic mechanism of GroEL-induced protein unfolding by using a reduced and carbamidomethylated variant of RNase T1, RCAM-T1, as a substrate. RCAM-T1 cannot fold to completion, because the two disulfide bonds are missing, and it is, thus, a good model for long-lived folding intermediates. RCAM-T1 unfolds when GroEL is added, but GroEL does not change the microscopic rate constant of unfolding, ruling out that it catalyzes unfolding. GroEL unfolds RCAM-T1 because it binds with high affinity to the unfolded form of the protein and thereby shifts the overall equilibrium toward the unfolded state. GroEL can unfold a partially folded or misfolded intermediate by this thermodynamic coupling mechanism when the Gibbs free energy of the binding to GroEL is larger than the conformational stability of the intermediate and when the rate of its unfolding is high. PMID:8790346

  17. Investigations of effects of environmental factors in unfolding/refolding pathway of proteins on 8-anilino-1-naphthalene-sulfonic acid (ANS) fluorescence

    NASA Astrophysics Data System (ADS)

    Qu, Peng; Lu, Hua; Yan, Shancheng; Zhou, Dequan; Lu, Zuhong

    2009-11-01

    The effects of various environmental factors in unfolding/refolding pathway of proteins on ANS fluorescence were investigated. In phosphate buffer of pH 7.4, ANS bound cetyl trimethyl ammonium bromide (CTMAB) and resulted in significant increase of ANS fluorescence and a sudden blue-shift in emission maximum wavelength. Sodium dodecyl sulfate (SDS) caused less increase in ANS fluorescence and less blue-shift in emission maximum wavelength than CTMAB and Tween 20. Tween 20, urea, polyethylene glycol 8000 (PEG 8000) and glycerol which contains hydrophobic groups also enhanced ANS fluorescence and caused their emission maximum wavelength blue-shift. For guanidine hydrochloride (GdmCl), due to its shorter hydrophobic chain, it caused less increase in ANS fluorescence and less decrease in emission maximum wavelength than CTMAB although GdmCl also has a positive charge. The results demonstrated the need for caution in interpreting enhancement of ANS fluorescence as an evidence for the existence of partially folded protein intermediates.

  18. Oxidative Stress Impairs the Heat Stress Response and Delays Unfolded Protein Recovery

    Microsoft Academic Search

    Masaaki Adachi; Yaohua Liu; Kyoko Fujii; Stuart K. Calderwood; Akira Nakai; Kohzoh Imai; Yasuhisa Shinomura

    2009-01-01

    Background: Environmental changes, air pollution and ozone depletion are increasing oxidative stress, and global warming threatens health by heat stress. We now face a high risk of simultaneous exposure to heat and oxidative stress. However, there have been few studies investigating their combined adverse effects on cell viability. Principal Findings: Pretreatment of hydrogen peroxide (H2O2) specifically and highly sensitized cells

  19. Overexpression of Myocilin in the Drosophila Eye Activates the Unfolded Protein Response: Implications

    E-print Network

    Mackay, Trudy F.C.

    : Implications for Glaucoma Mary Anna Carbone1,3 , Julien F. Ayroles1,3 , Akihiko Yamamoto1,2,3 , Tatiana V Carolina, United States of America Abstract Background: Glaucoma is the world's second leading cause associated with congenital glaucoma and 2­4% of primary open angle glaucoma (POAG) cases, but the pathogenic

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

    E-print Network

    Nicchitta, Chris

    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 Cells Laura M. Epple1,2 , Rebecca D. Dodd3 , Andrea L. Merz4 , Anjelika M. Dechkovskaia5 , Matthew

  1. Roles of STAT3 in protein secretion pathways during the acute-phase response.

    PubMed

    Ahyi, Ayele-Nati N; Quinton, Lee J; Jones, Matthew R; Ferrari, Joseph D; Pepper-Cunningham, Zachary A; Mella, Juan R; Remick, Daniel G; Mizgerd, Joseph P

    2013-05-01

    The acute-phase response is characteristic of perhaps all infections, including bacterial pneumonia. In conjunction with the acute-phase response, additional biological pathways are induced in the liver and are dependent on the transcription factors STAT3 and NF-?B, but these responses are poorly understood. Here, we demonstrate that pneumococcal pneumonia and other severe infections increase expression of multiple components of the cellular secretory machinery in the mouse liver, including the endoplasmic reticulum (ER) translocon complex, which mediates protein translation into the ER, and the coat protein complexes (COPI and COPII), which mediate vesicular transport of proteins to and from the ER. Hepatocyte-specific mutation of STAT3 prevented the induction of these secretory pathways during pneumonia, with similar results observed following pharmacological activation of ER stress by using tunicamycin. These findings implicate STAT3 in the unfolded protein response and suggest that STAT3-dependent optimization of secretion may apply broadly. Pneumonia also stimulated the binding of phosphorylated STAT3 to promoter regions of secretion-related genes in the liver, supporting a direct role for STAT3 in their transcription. Altogether, these results identify a novel function of STAT3 during the acute-phase response, namely, the induction of secretory machinery in hepatocytes. This may facilitate the processing and delivery of newly synthesized loads of acute-phase proteins, enhancing innate immunity and preventing liver injury during infection. PMID:23460517

  2. Influence of the sample-solvent on protein retention, mass transfer and unfolding kinetics in hydrophobic interaction chromatography

    Microsoft Academic Search

    Renata Muca; Wojciech Marek; Wojciech Pi?tkowski; Dorota Antos

    2010-01-01

    Typical mobile phase employed in hydrophobic interaction chromatography contains cosmotropic salts, which promote retention and simultaneously reduce the protein solubility in the mobile phase. To increase mass overloading in the separation process the protein can be dissolved in a sample-solvent with concentration of salt lower than that in the mobile phase or in salt free solutions. However, this methodology may

  3. Dynamics of protein folding: Probing the kinetic network of folding–unfolding transitions with experiment and theory

    Microsoft Academic Search

    Ginka S. Buchner; Ronan D. Murphy; Nicolae-Viorel Buchete; Jan Kubelka

    2011-01-01

    The problem of spontaneous folding of amino acid chains into highly organized, biologically functional three-dimensional protein structures continues to challenge the modern science. Understanding how proteins fold requires characterization of the underlying energy landscapes as well as the dynamics of the polypeptide chains in all stages of the folding process. In recent years, important advances toward these goals have been

  4. Three-state thermal unfolding of onconase.

    PubMed

    Casares-Atienza, Salvador; Weininger, Ulrich; Cámara-Artigas, Ana; Balbach, Jochen; Garcia-Mira, Maria M

    2011-12-01

    Onconase is a member of the ribonuclease A superfamily currently in phase IIIb clinical trials as a treatment for malign mesothelioma due to its cytotoxic activity selective against tumor-cells. In this work, we have studied the equilibrium thermal unfolding of onconase using a combination of several structural and biophysical techniques. Our results indicate that at least one significantly populated intermediate, which implies the exposure of hydrophobic surface and significant changes in the environment around Trp3, occurs during the equilibrium unfolding process of this protein. The intermediate begins to populate at about 30° below the global unfolding temperature, reaching a maximum population of nearly 60%, 10° below the global unfolding temperature. PMID:21840114

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

    PubMed

    McMurray, Michael

    2014-09-01

    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

  6. The ER chaperone encoding b i p A gene of black Aspergilli is induced by heat shock and unfolded proteins

    Microsoft Academic Search

    I. A van Gemeren; P. J Punt; A Drint-Kuyvenhoven; M. P Broekhuijsen; A van't Hoog; A Beijersbergen; C. T Verrips; C. A. M. J. J van den Hondel

    1997-01-01

    We describe the cloning and characterisation of the BiP gene homologues of the filamentous fungi Aspergillus niger and Aspergillus awamori. The BiP genes of these black Aspergilli encode an identical protein of 672 amino acids, which has a high homology with the BiP protein from Saccharomyces cerevisiae and contains a putative signal sequence of 38 amino acids. The DNA sequences

  7. Comparative Fourier Transform Infrared Spectroscopy Study of Cold, Pressure, and Heat-Induced Unfolding and Aggregation of Myoglobin

    Microsoft Academic Search

    Filip Meersman; László Smeller; Karel Heremans

    2002-01-01

    We studied the cold unfolding of myoglobin with Fourier transform infrared spectroscopy and compared it with pressure and heat unfolding. Because protein aggregation is a phenomenon with medical as well as biotechnological implications, we were interested in both the structural changes as well as the aggregation behavior of the respective unfolded states. The cold- and pressure-induced unfolding both yield a

  8. Expression of three topologically distinct membrane proteins elicits unique stress response pathways in the yeast Saccharomyces cerevisiae.

    PubMed

    Buck, Teresa M; Jordan, Rick; Lyons-Weiler, James; Adelman, Joshua L; Needham, Patrick G; Kleyman, Thomas R; Brodsky, Jeffrey L

    2015-06-01

    Misfolded membrane proteins are retained in the endoplasmic reticulum (ER) and are subject to ER-associated degradation, which clears the secretory pathway of potentially toxic species. While the transcriptional response to environmental stressors has been extensively studied, limited data exist describing the cellular response to misfolded membrane proteins. To this end, we expressed and then compared the transcriptional profiles elicited by the synthesis of three ER retained, misfolded ion channels: The ?-subunit of the epithelial sodium channel, ENaC, the cystic fibrosis transmembrane conductance regulator, CFTR, and an inwardly rectifying potassium channel, Kir2.1, which vary in their mass, membrane topologies, and quaternary structures. To examine transcriptional profiles in a null background, the proteins were expressed in yeast, which was previously used to examine the degradation requirements for each substrate. Surprisingly, the proteins failed to induce a canonical unfolded protein response or heat shock response, although messages encoding several cytosolic and ER lumenal protein folding factors rose when ?ENaC or CFTR was expressed. In contrast, the levels of these genes were unaltered by Kir2.1 expression; instead, the yeast iron regulon was activated. Nevertheless, a significant number of genes that respond to various environmental stressors were upregulated by all three substrates, and compared with previous microarray data we deduced the existence of a group of genes that reflect a novel misfolded membrane protein response. These data indicate that aberrant proteins in the ER elicit profound yet unique cellular responses. PMID:25759377

  9. Protein Dynamical Transition in Terahertz Dielectric Response

    E-print Network

    Markelz, Andrea G; Chen, Jing Yin; He, Yunfen

    2007-01-01

    The 200 K protein dynamical transition is observed for the first time in the teraherz dielectric response. The complex dielectric permittivity $\\epsilon$ = $\\epsilon$' + i$\\epsilon$" is determined in the 0.2 - 2.0 THz and 80-294 K ranges. $\\epsilon$" has a linear temperature dependence up to 200 K then sharply increases. The low temperature linear dependence in $\\epsilon$" indicates anharmonicity for temperatures 80 K K, challenging the assumed harmonicity below 200K. The temperature dependence is consistent with beta relaxation response and shows the protein motions involved in the dynamical transition extend to subpicosecond time scales.

  10. Disorder and order in unfolded and disordered peptides and proteins: a view derived from tripeptide conformational analysis. I. Tripeptides with long and predominantly hydrophobic side chains.

    PubMed

    Schweitzer-Stenner, Reinhard; Hagarman, Andrew; Toal, Siobhan; Mathieu, Daniel; Schwalbe, Harald

    2013-06-01

    We performed a conformational analysis of the central residues of three tripeptides glycyl-L-isoleucyl-glycine (GIG), glycyl-L-tyrosyl-glycine (GYG) and glycyl-L-arginyl-glycine (GRG) in aqueous solution, based on a global analysis of amide I' band profiles and NMR J-coupling constants. The results are compared with recently reported distributions of GVG, GFG and GEG. For GIG and GYG, we found that even though the polyproline II (pPII) fraction is below 0.5, it is still the most populated conformation, whereas GVG and GFG show both a larger ?-strand fraction. For GRG, we observed a clear dominance of pPII over ?-strand, reminiscent of observations for GEG and GKG. This finding indicates that terminal charges on otherwise hydrophobic residue side chains stabilize pPII over ?-strand conformations. For all peptides investigated we found that a variety of compact and turn-like conformations constitute nearly 20 percent of their conformational distributions. Attempts to analyze our data with a simple two-state pPII-->/<--? model therefore do not yield any satisfactory reproduction of experimental results. A comparison of the obtained GxG ensembles with conformational distributions of GxG segments in truncated coil libraries (helices and sheets omitted) revealed a much larger fraction of type II ?(i+2) and type III ? like conformations for the latter. Thus, a comparison of conformational distributions of unfolded peptide segments in solution and in coil libraries reveal interesting information on how the interplay between intrinsic propensities of amino acid residues and non-local interactions in polypeptide chains determine the conformations of loop segments in proteins. PMID:23229832

  11. Mechanical Unfolding of a -Hairpin Using Molecular Dynamics Zev Bryant,* Vijay S. Pande,

    E-print Network

    Bryant, Zev

    Mechanical Unfolding of a -Hairpin Using Molecular Dynamics Zev Bryant,* Vijay S. Pande, and Daniel Laboratory, Berkeley, California 94720, USA ABSTRACT Single-molecule mechanical unfolding experiments have simulations of the mechanical unfolding of the C-terminal hairpin of protein G. We have studied the dependence

  12. Unfolding of Titin Domains Explains the Viscoelastic Behavior of Skeletal Myofibrils

    Microsoft Academic Search

    Ave Minajeva; Michael Kulke; Julio M. Fernandez; Wolfgang A. Linke

    2001-01-01

    The elastic section of the giant muscle protein titin contains many immunoglobulin-like domains, which have been shown by single-molecule mechanical studies to unfold and refold upon stretch-release. Here we asked whether the mechanical properties of Ig domains and\\/or other titin regions could be responsible for the viscoelasticity of nonactivated skeletal-muscle sarcomeres, particularly for stress relaxation and force hysteresis. We show

  13. A natively unfolded ??-crystallin domain from Hahella chejuensis.

    PubMed

    Srivastava, Atul K; Sharma, Yogendra; Chary, Kandala V R

    2010-11-16

    To date, very few ??-crystallins have been identified and structurally characterized. Several of them have been shown to bind Ca(2+) and thereby enhance their stability without any significant change in structure. Although Ca(2+)-induced conformational changes have been reported in two putative ??-crystallins from Caulobacter crescentus and Yersinia pestis, they are shown to be partially unstructured, and whether they acquire a ??-crystallin fold is not known. We describe here a ??-crystallin domain, hahellin, its Ca(2+) binding properties and NMR structure. Unlike any other ??-crystallin, hahellin is characterized as a pre-molten globule (PMG) type of natively unfolded protein domain. It undergoes drastic conformational change and acquires a typical ??-crystallin fold upon Ca(2+) binding and hence acts as a Ca(2+)-regulated conformational switch. However, it does not bind Mg(2+). The intrinsically disordered Ca(2+)-free state and the close structural similarity of Ca(2+)-bound hahellin to a microbial ??-crystallin homologue, Protein S, which shows Ca(2+)-dependent stress response, make it a potential candidate for the cellular functions. This study indicates the presence of a new class of natively unfolded ??-crystallins and therefore the commencement of the possible functional roles of such proteins in this superfamily. PMID:20929244

  14. Understanding beta-hairpin formation by molecular dynamics simulations of unfolding.

    PubMed Central

    Lee, J; Shin, S

    2001-01-01

    We have studied the mechanism of formation of a 16-residue beta-hairpin from the protein GB1 using molecular dynamics simulations in an aqueous environment. The analysis of unfolding trajectories at high temperatures suggests a refolding pathway consisting of several transient intermediates. The changes in the interaction energies of residues are related with the structural changes during the unfolding of the hairpin. The electrostatic energies of the residues in the turn region are found to be responsible for the transition between the folded state and the hydrophobic core state. The van der Waals interaction energies of the residues in the hydrophobic core reflect the behavior of the radius of gyration of the core region. We have examined the opposing influences of the protein-protein (PP) energy, which favors the native state, and the protein-solvent (PS) energy, which favors unfolding, in the formation of the beta-hairpin structure. It is found that the behavior of the electrostatic components of PP and PS energies reflects the structural changes associated with the loss of backbone hydrogen bonding. Relative changes in the PP and PS van der Waals interactions are related with the disruption of the hydrophobic core of a protein. The results of the simulations support the hydrophobic collapse mechanism of beta-hairpin folding. PMID:11606266

  15. Chaperone release and unfolding of substrates in type III secretion

    Microsoft Academic Search

    Yukihiro Akeda; Jorge E. Galán

    2005-01-01

    Type III protein secretion systems are essential virulence factors of many bacteria pathogenic to humans, animals and plants. These systems mediate the transfer of bacterial virulence proteins directly into the host cell cytoplasm. Proteins are thought to travel this pathway in a largely unfolded manner, and a family of customized cytoplasmic chaperones, which specifically bind cognate secreted proteins, are essential

  16. The muscle protein synthetic response to food ingestion.

    PubMed

    Gorissen, Stefan H M; Rémond, Didier; van Loon, Luc J C

    2015-11-01

    Preservation of skeletal muscle mass is of great importance for maintaining both metabolic health and functional capacity. Muscle mass maintenance is regulated by the balance between muscle protein breakdown and synthesis rates. Both muscle protein breakdown and synthesis rates have been shown to be highly responsive to physical activity and food intake. Food intake, and protein ingestion in particular, directly stimulates muscle protein synthesis rates. The postprandial muscle protein synthetic response to feeding is regulated on a number of levels, including dietary protein digestion and amino acid absorption, splanchnic amino acid retention, postprandial insulin release, skeletal muscle tissue perfusion, amino acid uptake by muscle, and intramyocellular signaling. The postprandial muscle protein synthetic response to feeding is blunted in many conditions characterized by skeletal muscle loss, such as aging and muscle disuse. Therefore, it is important to define food characteristics that modulate postprandial muscle protein synthesis. Previous work has shown that the muscle protein synthetic response to feeding can be modulated by changing the amount of protein ingested, the source of dietary protein, as well as the timing of protein consumption. Most of this work has studied the postprandial response to the ingestion of isolated protein sources. Only few studies have investigated the postprandial muscle protein synthetic response to the ingestion of protein dense foods, such as dairy and meat. The current review will focus on the capacity of proteins and protein dense food products to stimulate postprandial muscle protein synthesis and identifies food characteristics that may modulate the anabolic properties. PMID:26021783

  17. Unfolding CR Singularities Adam Coffman

    E-print Network

    Coffman, Adam

    Unfolding CR Singularities Adam Coffman Author address: Department of Mathematical Sciences-mail address: CoffmanA@ipfw.edu #12;Contents List of Figures vii Unfolding CR singularities 1 1. Introduction 1 construction 14 5. Real surfaces in C2 17 5.1. Normal forms 17 5.2. Unfolding CR singularities of surfaces 23 5

  18. Contemporary Mathematics Unfolding Orthogonal Polyhedra

    E-print Network

    O'Rourke, Joseph

    Contemporary Mathematics Unfolding Orthogonal Polyhedra Joseph O'Rourke Abstract. Recent progress nonconvex polyhedra cannot be edge-unfolded without overlap. However, no example is known of a nonconvex by specializing the class of polyhedra, or easing the stringency of the unfolding criteria. On one hand

  19. 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.

  20. 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.

  1. Unfolding polyhedral bands

    Microsoft Academic Search

    Greg Aloupis; Erik D. Demaine; Stefan Langerman; Pat Morin; Joseph O'rourke; Ileana Streinu; Godfried T. Toussaint

    2004-01-01

    Abstract: A band is dened as the intersection of the surface of aconvex polyhedron with the space between two parallelplanes, as long as this space does not contain any verticesof the polyhedron. An unfolding of a given bandis obtained by cutting along exactly one edge and placingall faces of the band into the plane, without causingintersections. We prove that for

  2. Thermal Unfolding Pathway of PHD2 Catalytic Domain in Three Different PHD2 Species: Computational Approaches

    PubMed Central

    Hadi-Alijanvand, Hamid; Proctor, Elizabeth A.; Goliaei, Bahram; Dokholyan, Nikolay V.; Moosavi-Movahedi, Ali A.

    2012-01-01

    Prolyl hydroxylase domain 2 containing protein (PHD2) is a key protein in regulation of angiogenesis and metastasis. In normoxic condition, PHD2 triggers the degradation of hypoxia-inducible factor 1 (HIF-1?) that induces the expression of hypoxia response genes. Therefore the correct function of PHD2 would inhibit angiogenesis and consequent metastasis of tumor cells in normoxic condition. PHD2 mutations were reported in some common cancers. However, high levels of HIF-1? protein were observed even in normoxic metastatic tumors with normal expression of wild type PHD2. PHD2 malfunctions due to protein misfolding may be the underlying reason of metastasis and invasion in such cases. In this study, we scrutinize the unfolding pathways of the PHD2 catalytic domain’s possible species and demonstrate the properties of their unfolding states by computational approaches. Our study introduces the possibility of aggregation disaster for the prominent species of PHD2 during its partial unfolding. This may justify PHD2 inability to regulate HIF-1? level in some normoxic tumor types. PMID:23077544

  3. Spectroscopic probe analysis for exploring probe-protein interaction: a mapping of native, unfolding and refolding of protein bovine serum albumin by extrinsic fluorescence probe.

    PubMed

    Samanta, Anuva; Paul, Bijan Kumar; Guchhait, Nikhil

    2011-07-01

    Steady state and dynamic fluorescence measurements have been used to investigate interaction between Bovine Serum Albumin (BSA) and fluorescence probe para-N,N-dimethylamino orthohydroxy benzaldehyde (PDOHBA), a structurally important molecule exhibiting excited state coupled proton transfer (PT) and charge transfer (CT) reaction. Fluorescence anisotropy, acrylamide quenching, and time resolved fluorescence measurements corroborate the binding nature of the probe with protein. The binding constant between BSA and PDOHBA has been determined by using Benesi-Hildebrand and Stern-Volmer equations. The negative value of ?G indicates the spontaneity of this probe-protein complexation process. Observations from synchronous, three dimensional fluorescence spectra and circular dichroism spectra point toward the fact that the hydrophobicity as well as ?-helix content of BSA are altered in presence of probe PDOHBA. The PT band of PDOHBA is found to be an excellent reporter for the mapping of destructive and protective behavior of SDS with variation of chaotrope concentration. PMID:21514035

  4. 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

  5. Role of low native state kinetic stability and interaction of partially unfolded states with molecular chaperones in the mitochondrial protein mistargeting associated with primary hyperoxaluria

    Microsoft Academic Search

    Angel L. Pey; Eduardo Salido; Jose M. Sanchez-Ruiz

    The G170R variant of the alanine:glyoxylate aminotransferase (AGT) is the most common pathogenic allele associated to primary\\u000a hyperoxaluria type I (PH1), leading to mitochondrial mistargeting when combined with the P11L and I340M polymorphisms (minor allele; AGTLM). In this work, we have performed a comparative analysis on the conformation, unfolding energetics and interaction with molecular\\u000a chaperones between AGTwt, AGTLM and AGTLRM

  6. Thermal unfolding of barstar and the properties of interfacial water around the unfolded forms

    SciTech Connect

    Pal, Somedatta; Bandyopadhyay, Sanjoy, E-mail: sanjoy@chem.iitkgp.ernet.in [Molecular Modeling Laboratory, Department of Chemistry, Indian Institute of Technology, Kharagpur - 721302 (India)] [Molecular Modeling Laboratory, Department of Chemistry, Indian Institute of Technology, Kharagpur - 721302 (India)

    2013-12-21

    Identification of the intermediates along the folding-unfolding pathways and probing their interactions with surrounding solvent are two important but relatively unexplored issues in protein folding. In this work, we have carried out atomistic molecular dynamics simulations to study the thermal unfolding of barstar in aqueous solution from its folded native form at two different temperatures (400 K and 450 K). The calculations at 400 K reveal partial unfolding of two ?-helices (helix-1 and helix-2) and their interconnecting loop. At 450 K, on the other hand, the entire protein attains an expanded flexible conformation due to disruption of a large fraction of tertiary contacts and breaking of almost all the secondary structures. These two disordered structures obtained at such high temperatures are then studied around room temperature to probe their influence on the properties of surrounding solvent. It is found that though the unfolding of the protein in general leads to increasingly hydrated interface, but new structural motifs with locally dehydrated interface may also form during the structural transition. Additionally, independent of the conformational state of the protein, its influence on surrounding solvent has been found to be restricted to the first hydration layer.

  7. The endoplasmic reticulum stress response in immunity and autoimmunity

    Microsoft Academic Search

    Derrick J. Todd; Ann-Hwee Lee; Laurie H. Glimcher

    2008-01-01

    Many exogenous sources of stress can lead to cell death. In recent years, endogenous cellular sources of stress have also been identified, including the stress that arises from the accumulation of unfolded proteins within a cell's endoplasmic reticulum (ER). To counterbalance this type of ER stress, higher eukaryotic cells possess a three-pronged signal-transduction pathway termed the unfolded-protein response (UPR). This

  8. 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

  9. Becoming a Peroxidase: Cardiolipin-Induced Unfolding of Cytochrome c

    PubMed Central

    Muenzner, Julia; Toffey, Jason R.; Hong, Yuning; Pletneva, Ekaterina V.

    2014-01-01

    Interactions of cytochrome c (cyt c) with a unique mitochondrial glycerophospholipid cardiolipin (CL) are relevant for the protein’s function in oxidative phosphorylation and apoptosis. Binding to CL-containing membranes promotes cyt c unfolding and dramatically enhances the protein’s peroxidase activity, which is critical in early stages of apoptosis. We have employed a collection of seven dansyl variants of horse heart cyt c to probe the sequence of steps in this functional transformation. Kinetic measurements have unraveled four distinct processes during CL-induced cyt c unfolding: rapid protein binding to CL liposomes; rearrangements of protein substructures with small unfolding energies; partial insertion of the protein into the lipid bilayer; and extensive protein restructuring leading to “open” extended structures. While early rearrangements depend on a hierarchy of foldons in the native structure, the later process of large-scale unfolding is influenced by protein interactions with the membrane surface. The opening of the cyt c structure exposes the heme group, which enhances the protein’s peroxidase activity and also frees the C-terminal helix to aid in the translocation of the protein through CL membranes. PMID:23713573

  10. 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

  11. Protein Structural Change Upon Ligand Binding: Linear Response Theory

    Microsoft Academic Search

    Mitsunori Ikeguchi; Jiro Ueno; Miwa Sato; Akinori Kidera

    2005-01-01

    A simple formula based on linear response theory is proposed to explain and predict the structural change of proteins upon ligand binding. By regarding ligand binding as an external perturbation, the structural change as a response is described by atomic fluctuations in the ligand-free form and the protein-ligand interactions. The results for three protein systems of various sizes are consistent

  12. Conformational distributions of unfolded polypeptides from novel NMR techniques

    NASA Astrophysics Data System (ADS)

    Meier, Sebastian; Blackledge, Martin; Grzesiek, Stephan

    2008-02-01

    How the information content of an unfolded polypeptide sequence directs a protein towards a well-formed three-dimensional structure during protein folding remains one of the fundamental questions in structural biology. Unfolded proteins have recently attracted further interest due to their surprising prevalence in the cellular milieu, where they fulfill not only central regulatory functions, but also are implicated in diseases involving protein aggregation. The understanding of both the protein folding transition and these often natively unfolded proteins hinges on a more detailed experimental characterization of the conformations and conformational transitions in the unfolded state. This description is intrinsically very difficult due to the very large size of the conformational space. In principle, solution NMR can monitor unfolded polypeptide conformations and their transitions at atomic resolution. However, traditional NMR parameters such as chemical shifts, J couplings, and nuclear Overhauser enhancements yield only rather limited and often qualitative descriptions. This situation has changed in recent years by the introduction of residual dipolar couplings and paramagnetic relaxation enhancements, which yield a high number of well-defined, quantitative parameters reporting on the averages of local conformations and long-range interactions even under strongly denaturing conditions. This information has been used to obtain plausible all-atom models of the unfolded state at increasing accuracy. Currently, the best working model is the coil model, which derives amino acid specific local conformations from the distribution of amino acid torsion angles in the nonsecondary structure conformations of the protein data bank. Deviations from the predictions of such models can often be interpreted as increased order resulting from long-range contacts within the unfolded ensemble.

  13. Pea lectin unfolding reveals a unique molten globule fragment chain

    Microsoft Academic Search

    Debasish Sen; Dipak K. Mandal

    2011-01-01

    Pea lectin (PSL) is a dimeric protein in which each subunit comprises two intertwined, post-translationally processed polypeptide chains -a long ?-fragment and a short ?-fragment. Using guanidine hydrochloride-induced denaturation, we have investigated and characterized the species obtained in the unfolding equilibrium of PSL by steady-state and time-resolved fluorescence, phosphorescence, and selective chemical modification. During unfolding, the fragment chains become separated,

  14. 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.

  15. 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)

  16. 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.

  17. Comparative Fourier transform infrared spectroscopy study of cold-, pressure-, and heat-induced unfolding and aggregation of myoglobin.

    PubMed Central

    Meersman, Filip; Smeller, László; Heremans, Karel

    2002-01-01

    We studied the cold unfolding of myoglobin with Fourier transform infrared spectroscopy and compared it with pressure and heat unfolding. Because protein aggregation is a phenomenon with medical as well as biotechnological implications, we were interested in both the structural changes as well as the aggregation behavior of the respective unfolded states. The cold- and pressure-induced unfolding both yield a partially unfolded state characterized by a persistent amount of secondary structure, in which a stable core of G and H helices is preserved. In this respect the cold- and pressure-unfolded states show a resemblance with an early folding intermediate of myoglobin. In contrast, the heat unfolding results in the formation of the infrared bands typical of intermolecular antiparallel beta-sheet aggregation. This implies a transformation of alpha-helix into intermolecular beta-sheet. H/2H-exchange data suggest that the helices are first unfolded and then form intermolecular beta-sheets. The pressure and cold unfolded states do not give rise to the intermolecular aggregation bands that are typical for the infrared spectra of many heat-unfolded proteins. This suggests that the pathways of the cold and pressure unfolding are substantially different from that of the heat unfolding. After return to ambient conditions the cold- or pressure-treated proteins adopt a partially refolded conformation. This aggregates at a lower temperature (32 degrees C) than the native state (74 degrees C). PMID:11964250

  18. 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

  19. Epsilon-Unfolding Orthogonal Polyhedra

    Microsoft Academic Search

    Mirela Damian; Robin Y. Flatland; Joseph O'rourke

    2006-01-01

    An unfolding of a polyhedron is produced by cutting the surface and flat- tening to a single, connected, planar piece without overlap (except possibly at boundary points). It is a long unsolved problem to determine whether every polyhedron may be unfolded. Here we prove, via an algorithm, that every orthogonal polyhedron (one whose faces meet at right angles) of genus

  20. Unfolding and Reconstructing Brendan Lucier

    E-print Network

    Toronto, University of

    Unfolding and Reconstructing Polyhedra by Brendan Lucier A thesis presented to the University work on two topics: unfolding polyhedra into the plane and reconstructing polyhedra from partial particular types of overlaps, and use the results to construct examples of polyhedra with interesting

  1. 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

  2. GGUM2004: AWindows-Based Program to Estimate Parameters in the Generalized Graded Unfolding Model

    ERIC Educational Resources Information Center

    Roberts, James S.; Fang, Haw-ren

    2006-01-01

    The GGUM2004 computer program estimates parameters for a family of unidimensional unfolding item response theory (IRT) models. These unfolding IRT models predict higher item scores to the extent that a respondent is located close to an item on an underlying latent continuum. This prediction is often consistent with responses to traditional…

  3. Ultrafast thermally induced unfolding of RNase A.

    PubMed Central

    Phillips, C M; Mizutani, Y; Hochstrasser, R M

    1995-01-01

    A temperature jump (T-jump) method capable of initiating thermally induced processes on the picosecond time scale in aqueous solutions is introduced. Protein solutions are heated by energy from a laser pulse that is absorbed by homogeneously dispersed molecules of the dye crystal violet. These act as transducers by releasing the energy as heat to cause a T-jump of up to 10 K with a time resolution of 70 ps. The method was applied to the unfolding of RNase A. At pH 5.7 and 59 degrees C, a T-jump of 3-6 K induced unfolding which was detected by picosecond transient infrared spectroscopy of the amide I region between 1600 and 1700 cm-1. The difference spectral profile at 3.5 ns closely resembled that found for the equilibrium (native-unfolded) states. The signal at 1633 cm-1, corresponding to the beta-sheet structure, achieved 15 +/- 2% of the decrease found at equilibrium, within 5.5 ns. However, no decrease in absorbance was detected until 1 ns after the T-ump. The disruption of beta-sheet therefore appears to be subject to a delay of approximately 1 ns. Prior to 1 ns after the T-jump, water might be accessing the intact hydrophobic regions. PMID:7638183

  4. Electrostatics of folded and unfolded bovine ?-lactoglobulin.

    PubMed

    Eberini, Ivano; Sensi, Cristina; Barbiroli, Alberto; Bonomi, Franco; Iametti, Stefania; Galliano, Monica; Gianazza, Elisabetta

    2012-05-01

    We report on electrophoretic, spectroscopic, and computational studies aimed at clarifying, at atomic resolution, the electrostatics of folded and unfolded bovine ?-lactoglobulin (BLG) with a detailed characterization of the specific aminoacids involved. The procedures we used involved denaturant gradient gel electrophoresis, isoelectric focusing, electrophoretic titration curves, circular dichroism and fluorescence spectra in the presence of increasing concentrations of urea (up to 8 M), electrostatics computations and low-mode molecular dynamics. Discrepancy between electrophoretic and spectroscopic evidence suggests that changes in mobility induced by urea are not just the result of changes in gyration radius upon unfolding. Electrophoretic titration curves run across a pH range of 3.5-9 in the presence of urea suggest that more than one aminoacid residue may have anomalous pKa value in native BLG. Detailed computational studies indicate a shift in pKa of Glu44, Glu89, and Glu114, mainly due to changes in global and local desolvation. For His161, the formation of hydrogen bond(s) could add up to desolvation contributions. However, since His161 is at the C terminus, the end-effect associated to the solvated form strongly influences its pKa value with extreme variation between crystal structures on one side and NMR or low-mode molecular dynamics structures on the other. The urea concentration effective in BLG unfolding depends on pH, with higher stability of the protein at lower pH. PMID:21614557

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

    E-print Network

    Bigelow, Stephen

    network that makes up the biological clock whose disruption may cause irregular sleep-wake patterns, impaired alertness/performance, jet lag, and depression disorders. She defined phase as a principle measure

  6. HIV-1 TAT IS A NATIVELY UNFOLDED PROTEIN: THE SOLUTION CONFORMATION AND DYNAMICS OF REDUCED HIV-1 TAT1-72 BY NMR

    E-print Network

    O'Neil, Joe

    @cc.umanitoba.ca. The Transactivator of transcription (Tat) is a small RNA-binding protein that plays a central role in the regulation, RNA- binding protein, the transactivator of transcription (Tat) (2), plays a central role that may include the Tat message (3). Following translation, Tat protein is transported from the cytoplasm

  7. Unfolding Simulations of Holomyoglobin from Four Mammals: Identification of Intermediates and ?-Sheet Formation from Partially Unfolded States

    PubMed Central

    Dasmeh, Pouria; Kepp, Kasper P.

    2013-01-01

    Myoglobin (Mb) is a centrally important, widely studied mammalian protein. While much work has investigated multi-step unfolding of apoMb using acid or denaturant, holomyoglobin unfolding is poorly understood despite its biological relevance. We present here the first systematic unfolding simulations of holoMb and the first comparative study of unfolding of protein orthologs from different species (sperm whale, pig, horse, and harbor seal). We also provide new interpretations of experimental mean molecular ellipticities of myoglobin intermediates, notably correcting for random coil and number of helices in intermediates. The simulated holoproteins at 310 K displayed structures and dynamics in agreement with crystal structures (Rg ?1.48–1.51 nm, helicity ?75%). At 400 K, heme was not lost, but some helix loss was observed in pig and horse, suggesting that these helices are less stable in terrestrial species. At 500 K, heme was lost within 1.0–3.7 ns. All four proteins displayed exponentially decaying helix structure within 20 ns. The C- and F-helices were lost quickly in all cases. Heme delayed helix loss, and sperm whale myoglobin exhibited highest retention of heme and D/E helices. Persistence of conformation (RMSD), secondary structure, and ellipticity between 2–11 ns was interpreted as intermediates of holoMb unfolding in all four species. The intermediates resemble those of apoMb notably in A and H helices, but differ substantially in the D-, E- and F-helices, which interact with heme. The identified mechanisms cast light on the role of metal/cofactor in poorly understood holoMb unfolding. We also observed ?-sheet formation of several myoglobins at 500 K as seen experimentally, occurring after disruption of helices to a partially unfolded, globally disordered state; heme reduced this tendency and sperm-whale did not display any sheet propensity during the simulations. PMID:24386077

  8. Responses of proteins to different ionic environment are linearly interrelated.

    PubMed

    Ferreira, Luisa A; Madeira, Pedro P; Uversky, Alexey V; Uversky, Vladimir N; Zaslavsky, Boris Y

    2015-03-27

    Protein partitioning in aqueous two-phase systems (ATPS) is widely used as a convenient, inexpensive, and readily scaled-up separation technique. Protein partition behavior in ATPS is known to be readily manipulated by ionic composition. However, the available data on the effects of salts and buffer concentrations on protein partitioning are very limited. To fill this gap, partitioning of 15 proteins was examined in dextran-poly(ethylene glycol) ATPSs with different salt additives (Na2SO4, NaClO4, NaSCN, CsCl) in 0.11 M sodium phosphate buffer, pH 7.4. This analysis reveals that there is a linear relationship between the logarithms of the protein partition coefficients determined in the presence of different salts. This relationship suggests that the protein response to ionic environment is determined by the protein structure and type and concentrations of the ions present. Analysis of the differences between protein structures (described in terms of proteins responses to different salts) and that of cytochrome c chosen as a reference showed that the peculiarities of the protein surface structure and B-factor used as a measure of the protein flexibility are the determining parameters. Our results provide better insight into the use of different salts in manipulating protein partitioning in aqueous two-phase systems. These data also demonstrate that the protein responses to different ionic environments are interrelated and are determined by the structural peculiarities of protein surface. It is suggested that changes in ionic microenvironment of proteins may regulate protein transport and behavior in biological systems. PMID:25708470

  9. Probing the Role of Hydration in the Unfolding Transitions of Carbonmonoxy Myoglobin and Apomyoglobin

    E-print Network

    Shorter, James

    Probing the Role of Hydration in the Unfolding Transitions of Carbonmonoxy Myoglobin unfolding transition of horse carbonmonoxy myoglobin monitored by the stretching vibration of the CO ligand conformational reporters. In addition, the denatured protein exhibits an A0- like CO band. We hypothesize

  10. Thermal Unfolding Simulations of Bacterial Flagellin: Insight into its Refolding Before Assembly

    Microsoft Academic Search

    Choon-Peng Chng; Akio Kitao

    2008-01-01

    Flagellin is the subunit of the bacterial filament, the micrometer-long propeller of a bacterial flagellum. The protein is believed to undergo unfolding for transport through the channel of the filament and to refold in a chamber at the end of the channel before being assembled into the growing filament. We report a thermal unfolding simulation study of S. typhimurium flagellin

  11. fluctuating structures A natively unfolded yeast prion monomer adopts an ensemble of collapsed and rapidly

    E-print Network

    Lindquist, Susan

    fluctuating structures A natively unfolded yeast prion monomer adopts an ensemble of collapsed reprints, see: Notes: #12;A natively unfolded yeast prion monomer adopts an ensemble of collapsed Lindquist, December 22, 2006 (sent for review December 5, 2006) The yeast prion protein Sup35

  12. 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. [Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, Av. da Republica, 2780-157 Oeiras (Portugal)] [Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, Av. da Republica, 2780-157 Oeiras (Portugal); Lopes, Carlos [Centre for Molecular and Structural Biomedicine, Institute for Biotechnology and Bioengineering, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro (Portugal)] [Centre for Molecular and Structural Biomedicine, Institute for Biotechnology and Bioengineering, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro (Portugal); Martins, Ligia O. [Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, Av. da Republica, 2780-157 Oeiras (Portugal)] [Instituto de Tecnologia Quimica e Biologica, Universidade Nova de Lisboa, Av. da Republica, 2780-157 Oeiras (Portugal); Melo, Eduardo Pinho, E-mail: emelo@ualg.pt [Centre for Molecular and Structural Biomedicine, Institute for Biotechnology and Bioengineering, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro (Portugal)

    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.

  13. 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.

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

    PubMed

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

    2015-05-28

    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. PMID:26026459

  15. Epsilon-Unfolding Orthogonal Polyhedra

    Microsoft Academic Search

    Mirela Damian; Robin Flatland; Joseph O’Rourke

    2007-01-01

    An unfolding of a polyhedron is produced by cutting the surface and flattening to a single, connected, planar piece without overlap (except\\u000a possibly at boundary points). It is a long unsolved problem to determine whether every polyhedron may be unfolded. Here we\\u000a prove, via an algorithm, that every orthogonal polyhedron (one whose faces meet at right angles) of genus zero

  16. Mechanical unfolding of titin domains at low temperatures

    NASA Astrophysics Data System (ADS)

    Liu, Ensheng; Yang, Guoliang

    2002-03-01

    Titin is a large protein composed of several hundred of globular domains and a number of still unknown structures, with total molecular weight of more than 3 MDa. Titin spans half the sarcomere in striated muscle, and plays critical roles to the proper function of the muscle. Recent studies using single-molecule techniques have revealed that the force-induced changes in the titin domains are intimately related to the mechanisms of titin’s biological functions. To further investigate the behaviors of titin under the influence of an external force, we have investigated the mechanical unfolding of individual titin domains under various temperatures using the atomic force microscopy. The sample used was a polymer composed of 12 identical titin I27 domains, which was synthesized by protein engineering (a gift from M. Carrion and J.Fernandez, Mayo Foundation). The force-induced domain unfolding has been characterized in the temperature range of 5.5 ºC to 30 ºC. From the temperature dependence of the unfolding forces and the unfolding/refolding rates, the force-induced unfolding/refolding pathways of titin domains can be probed in more details. The results can also provide information on the molecular basis of muscle elasticity at low temperatures.

  17. The role of the N-terminal domain of photoactive yellow protein in the transient partial unfolding during signalling state formation

    E-print Network

    van Stokkum, Ivo

    Ectothiorhodospira halophila. This protein is a small water-soluble protein, which functions as the blue light recep-isomerization of its anionic 4-hydroxy-cinnamyl chromophore, from the 7- trans,9-cis to the 7-cis,9-trans con-exponentially to a blue-shifted state (pB355), the tentative signalling state. In a few hundred milliseconds the ground

  18. Characteristics of antibody responses induced in mice by protein allergens

    Microsoft Academic Search

    J. Hilton; R. J. Dearman; N. Sattar; D. A. Basketter; I. Kimber

    1997-01-01

    Whereas many foreign proteins are immunogenic, only a proportion is also allergenic, having the capacity to induce the quality of immune response necessary to support the production of IgE antibody. We have demonstrated previously that intraperitoneal administration to mice of proteins such as ovalbumin (OVA) or the industrial enzyme A. oryzae lipase, which possess significant allergenic potential, stimulates the production

  19. Sch9 regulates intracellular protein ubiquitination by controlling stress responses

    PubMed Central

    Qie, Beibei; Lyu, Zhou; Lyu, Lei; Liu, Jun; Gao, Xuejie; Liu, Yanyan; Duan, Wei; Zhang, Nianhui; Du, Linfang; Liu, Ke

    2015-01-01

    Protein ubiquitination and the subsequent degradation are important means by which aberrant proteins are removed from cells, a key requirement for long-term survival. In this study, we found that the overall level of ubiquitinated proteins dramatically decreased as yeast cell grew from log to stationary phase. Deletion of SCH9, a gene encoding a key protein kinase for longevity control, decreased the level of ubiquitinated proteins in log phase and this effect could be reversed by restoring Sch9 function. We demonstrate here that the decrease of ubiquitinated proteins in sch9? cells in log phase is not caused by changes in ubiquitin expression, proteasome activity, or autophagy, but by enhanced expression of stress response factors and a decreased level of oxidative stress. Our results revealed for the first time how Sch9 regulates the level of ubiquitinated proteins and provides new insight into how Sch9 controls longevity. PMID:26087116

  20. Magnetospheric Image Unfolding

    NASA Technical Reports Server (NTRS)

    1998-01-01

    The Grant was a three year grant funded under the Space Physics Supporting Research and Technology and Suborbital Program. Our objective was to develop automated techniques needed to unfold or "invert" global images of the magnetospheric ion populations obtained by the new magnetospheric imaging techniques (ENA, EUV) in anticipation of future missions such as the Magnetospheric Imager and, now, IMAGE. Our focus on the present three year grant is to determine the degree to which such images can quantitatively constrain the global electromagnetic properties of the magnetosphere. In a previous three year grant period we successfully automated a forward modeling inversion algorithm, demonstrated that these inversions are robust in the face of realistic instrumental considerations such as counting statistics and backgrounds, applied error analysis techniques to the extracted parameters using variational procedures, implemented very realistic magnetospheric test images to test the inversion algorithms using the Rice University Magnetospheric Specification Model, and began the process of generating parametric models with the flexibility to handle the realistic magnetospheric images (e.g. Roelof et al, 1992; 1993). Our plan for the present 3 year grant period was to complete the development of the inversion tools needed to handle realistic magnetospheric images, assess the degree to which global electrodynamics is quantitatively constrained by ENA images of the magnetosphere, and bring the inversion of EUV images up to the maturity that we will have achieved for the ENA imaging. Below the accomplishments of our three year effort are present followed by a list of our presentations and publications. The accomplishments of all three years are presented here, and thus some of these items appeared on interim progress reports.

  1. Thermolability of mutant MMACHC protein in the vitamin B12-responsive cblC disorder

    PubMed Central

    Froese, D.S.; Healy, S.; McDonald, M.; Kochan, G.; Oppermann, U.; Niesen, F.H.; Gravel, R.A.

    2010-01-01

    Methylmalonic aciduria and homocystinuria, cblC type, is the most common inborn error of cellular vitamin B12 metabolism. We previously showed that the protein carrying the mutation responsible for late-onset cblC (MMACHC-R161Q), treatable with high dose OHCbl, is able to bind OHCbl with wild-type affinity, leaving undetermined the disease mechanism involved [Froese et al., Mechanism of responsiveness, Mol. Genet. Metab. (2009).]. To assess whether the mutation renders the protein unstable, we investigated the thermostability of the wild-type and mutant MMACHC proteins, either unbound or bound to different cobalamins (Cbl), using differential scanning fluorimetry. We found that MMACHC-wt and MMACHC-R161Q are both very thermolabile proteins in their apo forms, with melting temperatures (Tm) of 39.3 ± 1.0 and 37.1 ± 0.7 °C, respectively; a difference confirmed by unfolding of MMACHC-R161Q but not MMACHC-wt by isothermal denaturation at 35 °C over 120 min. However, with the addition of OHCbl, MMACHC-wt becomes significantly stabilized (?Tm max = 8 °C, half-maximal effective ligand concentration, AC50 = 3 ?M). We surveyed the effect of different cobalamins on the stabilization of the wild-type protein and found that AdoCbl was the most stabilizing, exerting a maximum increase in Tm of ?16 °C, followed by MeCbl at ?13 °C, each evaluated at 50 ?M cofactor. The other cobalamins stabilized in the order (CN)2Cbi > OHCbl > CNCbl. Interestingly, the AC50’s for AdoCbl, MeCbl, (CN)2Cbi and OHCbl were similar and ranged from 1–3 ?M, which compares well with the Kd of 6 ?M for OHCbl [Froese et al., Mechanism of responsiveness, Mol. Genet. Metab. (2009).]. Unlike MMACHC-wt, the mutant protein MMACHC-R161Q is only moderately stabilized by OHCbl (?Tm max = 4 °C). The dose–response curve also shows a lower effectivity of OHCbl with respect to stabilization, with an AC50 of 7 ?M. MMACHC-R161Q showed the same order of stabilization as MMACHC-wt, but each cobalamin stabilized this mutant protein less than its wild-type counterpart. Additionally, MMACHC-R161Q had a higher AC50 for each cobalamin form compared to MMACHC-wt. Finally, we show that MMACHC-R161Q is able to support the base-off transition for AdoCbl and CNCbl, indicating this mutant is not blocked in that respect. Taken together, our results suggest that protein stability, as well as propensity for ligand-induced stabilization, contributes to the disease mechanism in late-onset cblC disorder. Our results underscore the importance of cofactor stabilization of MMACHC and suggest that even small increases in the concentration of cobalamin complexed with MMACHC may have therapeutic benefit in children with the late-onset, vitamin responsive cblC disease. PMID:20219402

  2. Kinetic response of a photoperturbed allosteric protein

    PubMed Central

    Buchli, Brigitte; Waldauer, Steven A.; Walser, Reto; Donten, Mateusz L.; Pfister, Rolf; Blöchliger, Nicolas; Steiner, Sandra; Caflisch, Amedeo; Zerbe, Oliver; Hamm, Peter

    2013-01-01

    By covalently linking an azobenzene photoswitch across the binding groove of a PDZ domain, a conformational transition, similar to the one occurring upon ligand binding to the unmodified domain, can be initiated on a picosecond timescale by a laser pulse. The protein structures have been characterized in the two photoswitch states through NMR spectroscopy and the transition between them through ultrafast IR spectroscopy and molecular dynamics simulations. The binding groove opens on a 100-ns timescale in a highly nonexponential manner, and the molecular dynamics simulations suggest that the process is governed by the rearrangement of the water network on the protein surface. We propose this rearrangement of the water network to be another possible mechanism of allostery. PMID:23818626

  3. Dynamic and static components power unfolding in topologically closed rings of a AAA+ proteolytic machine

    E-print Network

    Glynn, Steven E.

    In the Escherichia coli ClpXP protease, a hexameric ClpX ring couples ATP binding and hydrolysis to mechanical protein unfolding and translocation into the ClpP degradation chamber. Rigid-body packing between the small ...

  4. 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

  5. Pea lectin unfolding reveals a unique molten globule fragment chain.

    PubMed

    Sen, Debasish; Mandal, Dipak K

    2011-03-01

    Pea lectin (PSL) is a dimeric protein in which each subunit comprises two intertwined, post-translationally processed polypeptide chains--a long ?-fragment and a short ?-fragment. Using guanidine hydrochloride-induced denaturation, we have investigated and characterized the species obtained in the unfolding equilibrium of PSL by steady-state and time-resolved fluorescence, phosphorescence, and selective chemical modification. During unfolding, the fragment chains become separated, and the unfolding pattern reveals a ?-fragment as intermediate that has the molten globule characteristics. As examined by 8-anilino-1-naphthalenesulfonate (ANS) binding, the fragment intermediate shows ~20 fold increase in ANS fluorescence, and a large increase in ANS lifetime (12.8 ns). The tryptophan environment of the molten globule ?-fragment has been probed by selective modification with N-bromosuccinimide (NBS), which shows that two tryptophans, possibly Trp 53 and Trp 152 are oxidized while the other Trp 128 remains resistant to oxidation. The different types of tryptophan environment for the intermediate are supported by phosphorescence studies at 77 K, which gives a (0,0) band at 410 nm. These results seem to indicate that the larger fragment chain of PSL can independently behave as a monomeric or single domain protein that undergoes unfolding through intermediate state(s), and may provide important insight into the folding problem of oligomeric proteins in general and lectins in particular. PMID:21078359

  6. A disease state mutation unfolds the parkin ubiquitin-like domain.

    PubMed

    Safadi, Susan S; Shaw, Gary S

    2007-12-11

    E3 ubiquitin ligases are essential enzymes in the ubiquitination pathway responsible for the recognition of specific E2 conjugating enzymes and for transferring ubiquitin to a substrate targeted for degradation. In autosomal recessive juvenile Parkinson's disease, an early onset form of Parkinson's disease, point mutations in the E3 ligase parkin are one of the most commonly observed traits. Parkin is a multidomain E3 ligase that contains an N-terminal ubiquitin-like domain that interacts with, and effects the ubiquitination of, substrates such as cyclin E, p38 and synphilin. In this work we have examined the folding and structure of the parkin ubiquitin-like domain (Ubld) and of the protein with two causative disease mutations (K48A and R42P). Parallel experiments with the protein ubiquitin were done in order to determine if the same mutations were detrimental to the ubiquitin structure and stability. Despite similar folds between the parkin Ubld and ubiquitin, urea unfolding experiments show that the parkin Ubld is surprisingly approximately 10.6 kJ/mol less stable than ubiquitin. The K48A mutation had little effect on the stability of the parkin Ubld or ubiquitin indicating that this mutation contributes to defective protein-protein interactions. In contrast, the single point mutation R42P in parkin's Ubld caused poor expression and degradation of the protein. To avoid these problems, a GB1-Ubld fusion protein was characterized by NMR spectroscopy to show that the R42P mutation causes the complete unfolding of the parkin Ubld. This observation provides a rationale for the more rapid degradation of parkin carrying the R42P mutation in vivo, and its inability to interact with some substrate proteins. Our work provides the first structural and folding insight into the effects of causative mutations within the ubiquitin-like domain in autosomal recessive juvenile Parkinson's disease. PMID:18004887

  7. 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.

  8. Safe Folding/Unfolding with Conditional Narrowing?

    E-print Network

    Alpuente, María

    Safe Folding/Unfolding with Conditional Narrowing? M study the combination of this technique with a folding transformation rule in the case of innermost conditional narrowing. We also discuss a relationship between unfold/fold transformations

  9. Proteins containing non-native disulfide bonds generated by oxidative stress can act as signals for the induction of the heat shock response.

    PubMed

    McDuffee, A T; Senisterra, G; Huntley, S; Lepock, J R; Sekhar, K R; Meredith, M J; Borrelli, M J; Morrow, J D; Freeman, M L

    1997-05-01

    While oxidative stress can induce a heat shock response, the primary signals that initiate activation have not been identified. To identify such signals, HepG2 and V 79 cells were exposed to menadione, a compound that redox-cycles to generate superoxide. The oxidative stress generated by menadione resulted in oxidation of protein thiols in a dose-dependent manner. This was followed by protein destabilization and denaturation, as determined by differential scanning calorimetry of whole cells. To directly evaluate the effect of non-native disulfides on protein conformation, Ca2(+)-ATPase, isolated from rabbit sarcoplasmic reticulum, was chemically modified to contain non-native intermolecular or glutathione (GHS)-mixed disulfides. Differential scanning calorimetry profiles and 1-anilinonaphthalene-8-sulfonic acid fluorescence indicated that formation of non-native disulfides produced protein destabilization, denaturation, and exposure of hydrophobic domains. Cellular proteins shown to contain oxidized thiols formed detergent-insoluble aggregates. Cells treated with menadione exhibited activation of HSF-1, accumulated Hsp 70 mRNA, and increased synthesis of Hsp 70. This work demonstrates that formation of physiologically relevant, non-native intermolecular and GSH-mixed disulfides causes proteins to destabilize, unfold such that hydrophobic domains are exposed, and initiate a signal for induction of the heat shock response. PMID:9130461

  10. Molecular basis for AUXIN RESPONSE FACTOR protein interaction and the control of auxin response repression

    PubMed Central

    Korasick, David A.; Westfall, Corey S.; Lee, Soon Goo; Nanao, Max H.; Dumas, Renaud; Hagen, Gretchen; Guilfoyle, Thomas J.; Jez, Joseph M.; Strader, Lucia C.

    2014-01-01

    In plants, the AUXIN RESPONSE FACTOR (ARF) transcription factor family regulates gene expression in response to auxin. In the absence of auxin, ARF transcription factors are repressed by interaction with AUXIN/INDOLE 3-ACETIC ACID (Aux/IAA) proteins. Although the C termini of ARF and Aux/IAA proteins facilitate their homo- and heterooligomerization, the molecular basis for this interaction remained undefined. The crystal structure of the C-terminal interaction domain of Arabidopsis ARF7 reveals a Phox and Bem1p (PB1) domain that provides both positive and negative electrostatic interfaces for directional protein interaction. Mutation of interface residues in the ARF7 PB1 domain yields monomeric protein and abolishes interaction with both itself and IAA17. Expression of a stabilized Aux/IAA protein (i.e., IAA16) bearing PB1 mutations in Arabidopsis suggests a multimerization requirement for ARF protein repression, leading to a refined auxin-signaling model. PMID:24706860

  11. Highly Perturbed pKa Values in the Unfolded State of Hen Egg White Lysozyme

    PubMed Central

    Bradley, John; O'Meara, Fergal; Farrell, Damien; Nielsen, Jens Erik

    2012-01-01

    The majority of pKa values in protein unfolded states are close to the amino acid model pKa values, thus reflecting the weak intramolecular interactions present in the unfolded ensemble of most proteins. We have carried out thermal denaturation measurements on the WT and eight mutants of HEWL from pH 1.5 to pH 11.0 to examine the unfolded state pKa values and the pH dependence of protein stability for this enzyme. The availability of accurate pKa values for the folded state of HEWL and separate measurements of mutant-induced effects on the folded state pKa values, allows us to estimate the pKa values of seven acidic residues in the unfolded state of HEWL. Asp-48 and Asp-66 display pKa values of 2.9 and 3.1 in our analysis, thus representing the most depressed unfolded state pKa values observed to date. We observe a strong correlation between the folded state pKa values and the unfolded state pKa values of HEWL, thus suggesting that the unfolded state of HEWL possesses a large degree of native state characteristics. PMID:22500764

  12. Band Unfoldings and Prismatoids: A Counterexample

    E-print Network

    O'Rourke, Joseph

    Band Unfoldings and Prismatoids: A Counterexample Joseph O'Rourke October 4, 2007 Abstract polyhedral band without overlap might lead to an algorithm for unfolding any prismatoid without overlap that every placement of the prisma- toid top face overlaps with the band unfolding. 1 Introduction An edge

  13. Unfolding CR Singularities Adam Co#man

    E-print Network

    Coffman, Adam

    Unfolding CR Singularities Adam Co#man Author address: Department of Mathematical Sciences, Indiana: CoffmanA@ipfw.edu #12; Contents List of Figures vii Unfolding CR singularities 1 1. Introduction 1 2 construction 14 5. Real surfaces in C 2 17 5.1. Normal forms 17 5.2. Unfolding CR singularities of surfaces 23

  14. Kinesin: the tail unfolds

    Microsoft Academic Search

    Robert Cross; Jonathan Scholey

    1999-01-01

    The cargo-binding tail of the motor protein kinesin acts as a regulator of kinesin-driven vesicle transport. In the absence of bound cargo, the kinesin tail interacts with the motor domains and inhibits their activity. Cargo binding blocks this interaction and relieves the inhibition.

  15. 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.

  16. 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

  17. 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 ...

  18. 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.

  19. 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.

  20. Antibody responses to vaccinia membrane proteins following smallpox vaccination

    PubMed Central

    Lawrence, Steven J.; Lottenbach, Kathleen R.; Newman, Frances K.; Buller, R. Mark L.; Bellone, Clifford J.; Chen, John J.; Cohen, Gary H.; Eisenberg, Roselyn J.; Belshe, Robert B.; Stanley, Samuel L.; Frey, Sharon E.

    2008-01-01

    Background Vaccinia virus (VV) membrane proteins are candidates for orthopoxvirus subunit vaccines and potential targets for therapeutic antibodies. Human antibody responses to these proteins following VV vaccination have not been well characterized. Methods Pre- and day 26?30 post-vaccination sera from 80 VV vaccine recipients were examined for anti-B5, -A33, -A27, and -L1 specific IgG antibodies by enzyme-linked immunoassay (ELISA). Responses were compared between vaccinia-naïve and previously vaccinated (non-naïve) recipients, and between non-naïve recipients of undiluted and 1:10 diluted vaccine. Results VV vaccination elicited anti-A33 (100%) and -A27 (93%) antibodies in nearly all vaccinia-naïve subjects. Pre-existing antibodies were commonly detected in non-naïve subjects (anti-B5, 68%; -A33, 59%; -A27, 38%; -L1, 10%). Anti-B5 antibodies were strongly boosted by undiluted vaccine (geometric mean titer [GMT], 151 vs. 1010, p<0.001, pre- vs. post-vaccination, respectively), while anti-L1 antibody responses were less robust (31% detected, GMT, 75) in non-naïve subjects. Diluted vaccine elicited antibody responses that were similar to undiluted vaccine responses. Conclusions Vaccination with VV elicits long-lived specific antibody responses directed against VV membrane proteins that vary by previous vaccination status, but not by 10-fold dilution of vaccine. B5, A33 and A27 should be considered for inclusion in future human orthopoxvirus subunit vaccines. PMID:17570109

  1. Oxidatively Responsive Chain Extension to Topologically Entangle Engineered Protein Hydrogels

    NASA Astrophysics Data System (ADS)

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

    2014-03-01

    Hydrogels with increased toughness and extensibility have attracted a great deal of interest as mimics for natural tissues in biomedical applications. Artificial protein polymers provide particularly attractive systems for these applications due to their similarity to the chemistry of the natural extracellular matrix. Here, we show that entanglements can be incorporated into physically associating protein gels using simple oxidative chain extension chemistries, producing hydrogels with multiple time and length scales of relaxation. These oxidative chemistries follow the Jacobson-Stockmayer theory and are fully reversible, enabling responsive formation of entanglements within a material. The entangled protein gels demonstrate extensibility up to engineering strains of greater than 3,000%, a toughness of 65,000 J/m?3, and significant reductions in creep compliance and increases in elastic recovery. The rheology of the materials is compared to sticky reptation theory as a function of gel concentration, providing insights into the effect of network structure on different modes of molecular relaxation.

  2. 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

  3. Structure of the periplasmic stress response protein CpxP.

    PubMed

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

    2011-05-01

    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

  4. A Euclidean Perspective on the Unfolding of Azurin: Chain Motion

    PubMed Central

    Gray, Harry B.; Warren, Jeffery J.; Winkler, Jay R.

    2014-01-01

    We present a new approach to visualizing and quantifying the displacement of segments of P. aeruginosa azurin in the early stages of denaturation. Our method is based on a geometrical method developed previously by the authors, and elaborated extensively for azurin. In this study, we quantify directional changes in three ?-helical regions, two regions having ?-strand residues, and three unstructured regions of azurin. Snapshots of these changes as the protein unfolds are displayed and described quantitatively by introducing a scaling diagnostic. In accord with MD simulations, we show that the long ?-helix in azurin (residues 54–67) is displaced from the polypeptide scaffolding and then pivots first in one direction, and then in the opposite direction as the protein continues to unfold. The two ?-strand chains remain essentially intact and, except in the earliest stages, move in tandem. We show that unstructured regions 72–81 and 84–91, hinged by ?-strand residues 82–83, pivot oppositely. The region comprised of residues 72–91 (40% hydrophobic and 16% of the 128 total residues), forms an effectively stationary region that persists as the protein unfolds. This static behavior is a consequence of a dynamic balance between the competing motion of two segments, residues 72–81 and 84–91. PMID:24378983

  5. 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

  6. A Generalization of the Source Unfolding of Convex Polyhedra

    E-print Network

    Demaine, Erik

    "sun unfoldings" encompass source unfolding from a point, source unfolding from an open geodesic curve Figure 3. Our result generalizes source unfolding from a point or an open geodesic, by taking CA Generalization of the Source Unfolding of Convex Polyhedra Erik D. Demaine1 and Anna Lubiw2 1 MIT

  7. Grid Vertex-Unfolding Orthogonal Polyhedra

    Microsoft Academic Search

    Mirela Damian; Robin Y. Flatland; Joseph O’Rourke

    2008-01-01

    An edge-unfolding of a polyhedron is produced by cutting along edges and flattening the faces to a net, a connected planar piece with no overlaps. A grid unfolding allows additional cuts along grid edges induced by coordinate planes passing through every vertex. A vertex-unfolding allows faces in the net to be connected at single vertices, not necessarily along edges. We show that

  8. Band Unfoldings and Prismatoids: A Counterexample

    E-print Network

    O'Rourke, Joseph

    2007-01-01

    This note shows that the hope expressed in [ADL+07]--that the new algorithm for edge-unfolding any polyhedral band without overlap might lead to an algorithm for unfolding any prismatoid without overlap--cannot be realized. A prismatoid is constructed whose sides constitute a nested polyhedral band, with the property that every placement of the prismatoid top face overlaps with the band unfolding.

  9. Capillary electrophoresis with lamp-based wavelength-resolved fluorescence detection for the probing of protein conformational changes.

    PubMed

    de Kort, Bregje J; ten Kate, Geert A; de Jong, Gerhardus J; Somsen, Govert W

    2011-08-01

    Native protein fluorescence spectra encompass information on protein conformation. In this study, capillary electrophoresis (CE) combined with lamp-based wavelength-resolved fluorescence detection (wrFlu) is presented as a novel tool for the analysis of protein mixtures and the monitoring of protein unfolding. The CE-wrFlu system provides three-dimensional data (time, emission wavelength, intensity) from which electropherograms and accurate emission spectra of separated proteins can be extracted. For model proteins, linear detector responses (peak height vs concentration) were obtained (R(2) > 0.96) with detection limits (LODs) in the 6-32 nM range. The minimum protein concentration required for precise determination of the maximum emission wavelength by CE-wrFlu was about 15 times the LOD. Unfolding of various model proteins was induced by protein incubation and analysis in background electrolyte (BGE) containing 7.0 M urea. CE-wrFlu of the unfolded species revealed peaks with clear red-shifted spectra, which adequately corresponded to reference spectra obtained on a standard spectrophotometer. Moreover, unfolded proteins showed a significant decrease in effective electrophoretic mobility (after correction for BGE viscosity) due to the increase of their molecular hydrodynamic radii. It is concluded that the CE-wrFlu system provides two independent indicators for changes in protein folding and will allow the simultaneous assessment of protein purity and conformation. PMID:21699162

  10. 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.

  11. Human insulinotropic response to oral ingestion of native and hydrolysed whey protein

    Microsoft Academic Search

    O. Power; A. Hallihan; P. Jakeman

    2009-01-01

    The insulinotropic response to the ingestion of whey protein and whey protein hydrolysate, independent of carbohydrate, is\\u000a not known. This study examined the effect of protein hydrolysis on the insulinotropic response to the ingestion of whey protein.\\u000a Sixteen healthy males ingested a 500 mL solution containing either 45 g of whey protein (WPI) or whey protein hydrolysate\\u000a (WPH). The estimated

  12. Renal, metabolic and hormonal responses to ingestion of animal and vegetable proteins

    Microsoft Academic Search

    Panayotis Kontessis; Sharon Jones; Rosemary Dodds; Roberto Trevisan; Romano Nosadini; Paola Fioretto; Mauro Borsato; Davide Sacerdoti; GianCarlo Viberti; G C Viberti

    1990-01-01

    Renal, metabolic and hormonal responses to ingestion of animal and vegetable proteins. Renal and hormonal responses were studied in a group of healthy individuals fed, in random order, for three weeks, a vegetable protein diet (N = 10), an animal protein diet (N = 10), or an animal protein diet supplemented with fiber (N = 7), all containing the same

  13. Primary photoinduced protein response in bacteriorhodopsin and sensory rhodopsin II.

    PubMed

    Gross, Ruth; Wolf, Matthias M N; Schumann, Christian; Friedman, Noga; Sheves, Mordechai; Li, Lin; Engelhard, Martin; Trentmann, Oliver; Neuhaus, H Ekkehard; Diller, Rolf

    2009-10-21

    Essential for the biological function of the light-driven proton pump, bacteriorhodopsin (BR), and the light sensor, sensory rhodopsin II (SRII), is the coupling of the activated retinal chromophore to the hosting protein moiety. In order to explore the dynamics of this process we have performed ultrafast transient mid-infrared spectroscopy on isotopically labeled BR and SRII samples. These include SRII in D(2)O buffer, BR in H(2)(18)O medium, SRII with (15)N-labeled protein, and BR with (13)C(14)(13)C(15)-labeled retinal chromophore. Via observed shifts of infrared difference bands after photoexcitation and their kinetics we provide evidence for nonchromophore bands in the amide I and the amide II region of BR and SRII. A band around 1550 cm(-1) is very likely due to an amide II vibration. In the amide I region, contributions of modes involving exchangeable protons and modes not involving exchangeable protons can be discerned. Observed bands in the amide I region of BR are not due to bending vibrations of protein-bound water molecules. The observed protein bands appear in the amide I region within the system response of ca. 0.3 ps and in the amide II region within 3 ps, and decay partially in both regions on a slower time scale of 9-18 ps. Similar observations have been presented earlier for BR5.12, containing a nonisomerizable chromophore (R. Gross et al. J. Phys. Chem. B 2009, 113, 7851-7860). Thus, the results suggest a common mechanism for ultrafast protein response in the artificial and the native system besides isomerization, which could be induced by initial chromophore polarization. PMID:19778046

  14. An improved physics-based approach for unfolding megavoltage bremsstrahlung spectra using transmission analysis

    SciTech Connect

    Ali, E. S. M.; Rogers, D. W. O. [Carleton Laboratory for Radiotherapy Physics, Department of Physics, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario K1S 5B6 (Canada)

    2012-03-15

    Purpose: To develop a physics-based approach to improve the accuracy and robustness of the ill-conditioned problem of unfolding megavoltage bremsstrahlung spectra from transmission data. Methods: Spectra are specified using a rigorously-benchmarked functional form. Since ion chambers are the typical detector used in transmission measurements, the energy response of a Farmer chamber is calculated using the EGSnrc Monte Carlo code, and the effect of approximating the energy response on the accuracy of the unfolded spectra is studied. A proposal is introduced to enhance spectral sensitivity by combining transmission data measured with multiple detectors of different energy response and by combining data from multiple attenuating materials. Monte Carlo methods are developed to correct for nonideal exponential attenuation (e.g., scatter effects and secondary attenuation). The performance of the proposed methods is evaluated for a diverse set of validated clinical spectra (3.5-25 MV) using analytical transmission data with simulated experimental noise. Results: The approximations commonly used in previous studies for the ion-chamber energy response lead to significant errors in the unfolded spectra. Of the configurations studied, the one with best spectral sensitivity is to measure four full transmission curves using separate low-Z and high-Z attenuators in conjunction with two detectors of different energy response (the authors propose a Farmer-type ion chamber, once with a low-Z, and once with a high-Z buildup cap material), then to feed the data simultaneously to the unfolding algorithm. Deviations from ideal exponential attenuation are as much as 1.5% for the smallest transmission signals, and the proposed methods properly correct for those deviations. The transmission data with enhanced spectral sensitivity, combined with the accurate and flexible spectral functional form, lead to robust unfolding without requiring a priori knowledge of the spectrum. Compared with the commonly-used methods, the accuracy is improved for the unfolded spectra and for the unfolded mean incident electron kinetic energy by at least factors of three and four, respectively. With simulated experimental noise and a lowest transmission of 1%, the unfolded energy fluence spectra agree with the original spectra with a normalized root-mean-square deviation, %{Delta}({psi}), of 2.3%. The unfolded mean incident electron kinetic energies agree, on average, with the original values within 1.4%. A lowest transmission of only 10% still allows unfolding with %{Delta}({psi}) of 3.3%. Conclusions: In the presence of realistic experimental noise, the proposed approach significantly improves the accuracy and robustness of the spectral unfolding problem for all therapy and MV imaging beams of clinical interest.

  15. Vertex-Unfoldings of Simplicial Manifolds Erik D. Demaine

    E-print Network

    Erickson, Jeff

    has been termed an edge- unfolding; the unfolding consists of the facets of the polyhe- dron joined] for a history of the edge-unfolding problem and its applications to manufactur- ing. Recently it was established

  16. 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

  17. Model process for separation based on unfolding and refolding of chymotrypsin inhibitor 2 in thermoseparating polymer two-phase systems

    Microsoft Academic Search

    Hiroshi Umakoshi; Josefine Persson; Maj Kroon; Hans-Olof Johansson; Daniel E Otzen; Ryoichi Kuboi; Folke Tjerneld

    2000-01-01

    For the design of a new separation process based on unfolding and refolding of protein, the partitioning behaviour of proteins was studied in thermoseparating polymer two-phase systems with varying pH and temperature. Chymotrypsin inhibitor 2 (CI2), which unfolds reversibly in a simple two-state manner, was partitioned in an aqueous two-phase system (ATPS) composed of a random copolymer of ethylene oxide

  18. Dimer Dissociation and Unfolding Mechanism of Coagulation Factor XI Apple 4 Domain: Spectroscopic

    E-print Network

    Roder, Heinrich

    Dimer Dissociation and Unfolding Mechanism of Coagulation Factor XI Apple 4 Domain: Spectroscopic of Medicine, Philadelphia, PA 19140, USA The blood coagulation protein factor XI (FXI) consists of a pair Ltd. All rights reserved. *Corresponding authors Keywords: FXI; blood coagulation; protein folding

  19. Glycine–alanine repeats impair proper substrate unfolding by the proteasome

    Microsoft Academic Search

    Martin A Hoyt; Judith Zich; Junko Takeuchi; Mingsheng Zhang; Cedric Govaerts; Philip Coffino

    2006-01-01

    Proteasome ATPases unravel folded proteins. Introducing a sequence containing only glycine and alanine residues (GAr) into substrates can impair their digestion. We pre- viously proposed that a GAr interferes with the unfolding capacity of the proteasome, leading to partial degradation of products. Here we tested that idea in several ways. Stabilizing or destabilizing a folded domain within sub- strate proteins

  20. Grid Vertex-Unfolding Orthogonal Polyhedra

    Microsoft Academic Search

    Mirela Damian; Robin Y. Flatland; Joseph O’Rourke

    2005-01-01

    Abstract An edge-unfolding of a polyhedron is produced by cutting along edges and ?attening the faces to a net, a connected planar piece with no overlaps. A grid unfolding allows additional cuts along grid edges induced by coordinate planes passing through every vertex. A vertexunfolding permits faces in the net to be connected at single vertices, not necessarily along edges.

  1. Unfolding First-Principles Band Structures

    Microsoft Academic Search

    Wei Ku; Tom Berlijn; Chi-Cheng Lee

    2010-01-01

    A general method is presented to unfold band structures of first-principles supercell calculations with proper spectral weight, allowing easier visualization of the electronic structure and the degree of broken translational symmetry. The resulting unfolded band structures contain additional rich information from the Kohn-Sham orbitals, and absorb the structure factor that makes them ideal for a direct comparison with angle resolved

  2. Unfolding first-principles band structures

    Microsoft Academic Search

    Wei Ku; T. Berlijn; C.-C. Lee

    2011-01-01

    A general method [1] is presented to unfold band structures of first-principles supercell calculations with proper spectral weight, allowing easier visualization of the electronic structure and the degree of broken translational symmetry. The resulting unfolded band structures contain additional rich information from the Kohn-Sham orbitals, and absorb the structure factor that makes them ideal for a direct comparison with angle

  3. Unfolding Polyhedra Joseph O'Rourke

    E-print Network

    O'Rourke, Joseph

    Unfolding Polyhedra Joseph O'Rourke July 10, 2008 Contents 1 Introduction 1 2 Edge Unfolding 1 2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 3.2.1 Orthogonal Polyhedra . . . . . . . . . . . . . . . . . . . . 8 4 Summary & Prospects 8 Dept of rectan- gular towers rising from a rectangular base. Polyhedra in this class are known as orthogonal

  4. Solid-state synthesis and mechanical unfolding of polymers of T4 lysozyme

    PubMed Central

    Yang, Guoliang; Cecconi, Ciro; Baase, Walter A.; Vetter, Ingrid R.; Breyer, Wendy A.; Haack, Julie A.; Matthews, Brian W.; Dahlquist, Frederick W.; Bustamante, Carlos

    2000-01-01

    Recent advances in single molecule manipulation methods offer a novel approach to investigating the protein folding problem. These studies usually are done on molecules that are naturally organized as linear arrays of globular domains. To extend these techniques to study proteins that normally exist as monomers, we have developed a method of synthesizing polymers of protein molecules in the solid state. By introducing cysteines at locations where bacteriophage T4 lysozyme molecules contact each other in a crystal and taking advantage of the alignment provided by the lattice, we have obtained polymers of defined polarity up to 25 molecules long that retain enzymatic activity. These polymers then were manipulated mechanically by using a modified scanning force microscope to characterize the force-induced reversible unfolding of the individual lysozyme molecules. This approach should be general and adaptable to many other proteins with known crystal structures. For T4 lysozyme, the force required to unfold the monomers was 64 ± 16 pN at the pulling speed used. Refolding occurred within 1 sec of relaxation with an efficiency close to 100%. Analysis of the force versus extension curves suggests that the mechanical unfolding transition follows a two-state model. The unfolding forces determined in 1 M guanidine hydrochloride indicate that in these conditions the activation barrier for unfolding is reduced by 2 kcal/mol. PMID:10618384

  5. Receptor-like protein kinases: the keys to response.

    PubMed

    Morris, Erin R; Walker, John C

    2003-08-01

    Plants are constantly challenged by changes in temperature, light, nutrient conditions, and exposure to pathogens and by other fluctuations in their environment. The molecular basis of how plants respond to these external factors is an active area of investigation. Plant cells often use receptors at the cell surface to sense environmental changes, and then transduce this information via activated signaling pathways to trigger adaptive responses. In Arabidopsis, the receptor-like protein kinase (RLK) gene family contains more than 600 members, many of which are likely to respond to the external challenges presented by an ever-changing environment. RLKs are involved in hormonal response pathways, cell differentiation, plant growth and development, self-incompatibility, and symbiont and pathogen recognition. PMID:12873528

  6. 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

  7. Molecular cloning and expression of chicken carbohydrate response element binding protein and Max-like protein X gene homologues

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Carbohydrate response element binding protein (ChREBP) and sterol regulatory element binding protein-1c (SREBP-1c) are transcription factors that are known to be key regulators of glucose metabolism and lipid synthesis in mammals. Since ChREBP and its co-activator Max-like protein X (Mlx) have not ...

  8. Effects of C-reactive protein on human lymphocyte responsiveness.

    PubMed

    Vetter, M L; Gewurz, H; Hansen, B; James, K; Baum, L L

    1983-05-01

    C-reactive protein (CRP), a trace serum protein that increases markedly in concentration during inflammatory reactions, was recently shown to bind to a subset of human IgG-FcR-bearing peripheral blood lymphocytes in the presence of a ligand such as pneumococcal C-polysaccharide (CPS). CRP has also been detected on a small percentage of PBL that are associated with NK activity. In the present study, we assessed the effects of CRP and CRP-CPS complexes on a variety of human lymphocyte functions in vitro. CRP and CRP complexes significantly enhanced (generally two to threefold) cell-mediated cytotoxicity, minimally enhanced the MLC reaction, and induced a small but regularly detectable blastogenic response in resting PBL. CRP or CRP-CPS complexes had no effect on mitogen-induced blastogenesis, PWM-induced generation of IgM plaque-forming cells, E-rosette formation, antibody-dependent cell-mediated cytotoxicity, or NK activity. The basis for the preferential ability of CRP to enhance cytotoxicity responses in vitro is under further investigation. PMID:6220082

  9. 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

  10. Testosterone Regulates Tight Junction Proteins and Influences Prostatic Autoimmune Responses

    PubMed Central

    Meng, Jing; Mostaghel, Elahe A.; Vakar-Lopez, Funda; Montgomery, Bruce; True, Larry; Nelson, Peter S.

    2015-01-01

    Testosterone and inflammation have been linked to the development of common age-associated diseases affecting the prostate gland including prostate cancer, prostatitis, and benign prostatic hypertrophy. We hypothesized that testosterone regulates components of prostate tight junctions which serve as a barrier to inflammation, thus providing a connection between age- and treatment-associated testosterone declines and prostatic pathology. We examined the expression and distribution of tight junction proteins in prostate biospecimens from mouse models and a clinical study of chemical castration, using transcript profiling, immunohistochemistry and electron microscopy. We determined that low serum testosterone is associated with reduced transcript and protein levels of Claudin 4 and Claudin 8, resulting in defective tight junction ultrastructure in benign prostate glands. Expression of Claudin 4 and Claudin 8 was negatively correlated with the mononuclear inflammatory infiltrate caused by testosterone deprivation. Testosterone suppression also induced an auto-immune humoral response directed toward prostatic proteins. Testosterone supplementation in castrate mice resulted in re-expression of tight junction components in prostate epithelium and significantly reduced prostate inflammatory cell numbers. These data demonstrate that tight junction architecture in the prostate is related to changes in serum testosterone levels, and identify an androgen-regulated mechanism that potentially contributes to the development of prostate inflammation and consequent pathology. PMID:21761342

  11. 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

  12. 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

  13. Grid Vertex-Unfolding Orthogonal Polyhedra Mirela Damian

    E-print Network

    O'Rourke, Joseph

    Grid Vertex-Unfolding Orthogonal Polyhedra Mirela Damian Robin Flatland Joseph O'Rourke§ September polyhedra of genus zero has a grid vertex-unfolding. (There are orthogonal polyhedra that cannot be vertex grid. Keywords: vertex-unfolding, grid unfolding, orthogonal polyhedra, genus-zero. 1 Introduction Two

  14. The accumulation of misfolded proteins in the mitochondrial matrix is sensed by PINK1 to induce PARK2/Parkin-mediated mitophagy of polarized mitochondria.

    PubMed

    Jin, Seok Min; Youle, Richard J

    2013-11-01

    Defective mitochondria exert deleterious effects on host cells. To manage this risk, mitochondria display several lines of quality control mechanisms: mitochondria-specific chaperones and proteases protect against misfolded proteins at the molecular level, and fission/fusion and mitophagy segregate and eliminate damage at the organelle level. An increase in unfolded proteins in mitochondria activates a mitochondrial unfolded protein response (UPR(mt)) to increase chaperone production, while the mitochondrial kinase PINK1 and the E3 ubiquitin ligase PARK2/Parkin, whose mutations cause familial Parkinson disease, remove depolarized mitochondria through mitophagy. It is unclear, however, if there is a connection between those different levels of quality control (QC). Here, we show that the expression of unfolded proteins in the matrix causes the accumulation of PINK1 on energetically healthy mitochondria, resulting in mitochondrial translocation of PARK2, mitophagy and subsequent reduction of unfolded protein load. Also, PINK1 accumulation is greatly enhanced by the knockdown of the LONP1 protease. We suggest that the accumulation of unfolded proteins in mitochondria is a physiological trigger of mitophagy. PMID:24149988

  15. The accumulation of misfolded proteins in the mitochondrial matrix is sensed by PINK1 to induce PARK2/Parkin-mediated mitophagy of polarized mitochondria

    PubMed Central

    Jin, Seok Min; Youle, Richard J

    2013-01-01

    Defective mitochondria exert deleterious effects on host cells. To manage this risk, mitochondria display several lines of quality control mechanisms: mitochondria-specific chaperones and proteases protect against misfolded proteins at the molecular level, and fission/fusion and mitophagy segregate and eliminate damage at the organelle level. An increase in unfolded proteins in mitochondria activates a mitochondrial unfolded protein response (UPRmt) to increase chaperone production, while the mitochondrial kinase PINK1 and the E3 ubiquitin ligase PARK2/Parkin, whose mutations cause familial Parkinson disease, remove depolarized mitochondria through mitophagy. It is unclear, however, if there is a connection between those different levels of quality control (QC). Here, we show that the expression of unfolded proteins in the matrix causes the accumulation of PINK1 on energetically healthy mitochondria, resulting in mitochondrial translocation of PARK2, mitophagy and subsequent reduction of unfolded protein load. Also, PINK1 accumulation is greatly enhanced by the knockdown of the LONP1 protease. We suggest that the accumulation of unfolded proteins in mitochondria is a physiological trigger of mitophagy. PMID:24149988

  16. Quantum Unfolding: A program for unfolding electronic energy bands of materials

    NASA Astrophysics Data System (ADS)

    Zheng, Fawei; Zhang, Ping; Duan, Wenhui

    2015-04-01

    We present Quantum Unfolding, a Fortran90 program for unfolding first-principles electronic energy bands. It unfolds energy bands accurately by handling the Fourier components of Bloch wavefunctions, which are reconstructed from Wannier functions from Wannier90. Due to the wide application of Wannier90 package and the possibility of focusing only on the most important energy bands, the present code works very conveniently.

  17. 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

  18. Is annexin 1 a multifunctional protein during stress responses?

    PubMed Central

    Clark, Greg; Hennig, Jacek; Roux, Stanley

    2010-01-01

    Accumulating evidence suggest that certain annexins can play a role in abiotic stress responses in plants. We found that for one member of the Arabidopsis thaliana annexin gene family, annexin 1 (AnnAt1), loss-of-function mutants are more sensitive to drought stress and gain-of-function mutants are more tolerant.1 We also found that AnnAt1 is able to regulate accumulation of H2O2 in vivo in Arabidopsis cells based on the observation that the level of ROS accumulation following induction by ABA correlates with the level of AnnAt1 protein in transgenic Arabidopsis plants. Here we provide more commentary on the antioxidant activity of AnnAt1, critically assess the evidence that AnnAt1 and other annexins possess peroxidase activity, emphasize a redox-induced posttranslational modification which occurs to AnnAt1 during ABA signaling, and discuss ways this annexin’s membrane associations could mediate stress signaling while addressing the potential that AnnAt1 is a multifunctional protein in plants. PMID:20215861

  19. Investigations of the Low Frequency Spectral Density of Cytochrome c upon Equilibrium Unfolding

    PubMed Central

    Sun, Yuhan; Karunakaran, Venugopal; Champion, Paul M.

    2013-01-01

    The equilibrium unfolding process of ferric horse heart cytochrome c (cyt c), induced by guanidinium hydrochloride (GdHCl), was studied using UV-vis absorption spectroscopy, resonance Raman spectroscopy and vibrational coherence spectroscopy (VCS). The unfolding process was successfully fit using a three-state model35 which included the fully folded (N) and unfolded (U) states, along with an intermediate (I) assigned to a Lys bound heme. The VCS spectra revealed for the first time several low frequency heme modes that are sensitive to cytochrome c unfolding: ?a (~50 cm?1), ?b (~80cm?1), ?c (~100cm?1), and vs(His-Fe-His) at 205 cm?1. These out-of-plane modes have potential functional relevance and are activated by protein-induced heme distortions. The free energies for the N-I and the I-U transitions at pH 7.0 and 20°C were found to be 4.6 kcal/M and 11.6 kcal/M, respectively. Imidazole was also introduced to replace the methionine ligand so the unfolding can be modeled as a two-state system. The intensity of the mode ?b~80 cm?1 remains nearly constant during the unfolding process, while the amplitudes of the other low frequency modes track with spectral changes observed at higher frequency. This confirms that the heme deformation changes are coupled to the protein tertiary structural changes that take place upon unfolding. These studies also reveal that damping of the coherent oscillations depends sensitively on the coupling between heme and the surrounding water solvent. PMID:23863217

  20. Vertex-Unfoldings of Simplicial Manifolds

    E-print Network

    Demaine, Erik

    Introduction It is a long-standing open problem to determine whether every convex polyhe- dron can be cut along] gives a history of the edge-unfolding problem and its applications to manufacturing. Recently

  1. The Placental Protein Syncytin-1 Impairs Antiviral Responses and Exaggerates Inflammatory Responses to Influenza

    PubMed Central

    Tolosa, Jorge M.; Parsons, Kristy S.; Hansbro, Philip M.; Smith, Roger; Wark, Peter A. B.

    2015-01-01

    Background Pregnancy increases susceptibility to influenza. The placenta releases an immunosuppressive endogenous retroviral protein syncytin-1. We hypothesised that exposure of peripheral monocytes (PBMCs) to syncytin-1 would impair responses to H1N1pdm09 influenza. Methods and Findings Recombinant syncytin-1 was produced. PBMCs from non-pregnant women (n=10) were exposed to H1N1pdm09 in the presence and absence of syncytin-1 and compared to responses of PBMCs from pregnant women (n=12). PBMCs were characterised using flow cytometry, release of interferon (IFN)-?, IFN-?, IFN-?, IL-10, IL-2, IL-6 and IL-1? were measured by cytometric bead array or ELISA. Exposure of PBMCs to H1N1pdm09 resulted in the release of IFN-?, (14,787 pg/mL, 95% CI 7311-22,264 pg/mL) IFN-? (1486 pg/mL, 95% CI 756-2216 pg/mL) and IFN-? (852 pg/mL, 95% CI 193-1511 pg/mL) after 48 hours. This was significantly impaired in pregnant women (IFN-?; p<0.0001 and IFN-?; p<0.001). Furthermore, in the presence of syncytin-1, PBMCs demonstrated marked reductions in IFN-? and IFN-?, while enhanced release of IL-10 as well as IL-6 and IL-1?. Conclusions Our data indicates that a placental derived protein, syncytin-1 may be responsible for the heightened vulnerability of pregnant women to influenza. PMID:25831059

  2. Web-based unfolding cases: a strategy to enhance and evaluate clinical reasoning skills.

    PubMed

    Johnson, Gail; Flagler, Susan

    2013-10-01

    Clinical reasoning involves the use of both analytical and nonanalytical intuitive cognitive processes. Fostering student development of clinical reasoning skills and evaluating student performance in this cognitive arena can challenge educators. The use of Web-based unfolding cases is proposed as a strategy to address these challenges. Unfolding cases mimic real-life clinical situations by presenting only partial clinical information in sequential segments. Students receive immediate feedback after submitting a response to a given segment. The student's comparison of the desired and submitted responses provides information to enhance the development of clinical reasoning skills. Each student's set of case responses are saved for the instructor in an individual-student electronic file, providing a record of the student's knowledge and thinking processes for faculty evaluation. For the example case given, the approaches used to evaluate individual components of clinical reasoning are provided. Possible future uses of Web-based unfolding cases are described. PMID:24044385

  3. Band Unfoldings and Prismatoids: A Counterexample

    Microsoft Academic Search

    Joseph O'rourke

    2007-01-01

    Abstract This note shows that the hope expressed in [ADL,07]|that the new algorithm for edge-unfolding any polyhedral band without overlap might lead to an algorithm for unfolding any prismatoid without overlap|cannot be realized. A prismatoid is constructed whose sides constitute a nested polyhedral band, with the property that every placement of the prisma- toid top face overlaps with the band

  4. Hierarchical cascades of instability govern the mechanics of coiled coils: helix unfolding precedes coil unzipping.

    PubMed

    Hamed, Elham; Keten, Sinan

    2014-07-15

    Coiled coils are a fundamental emergent motif in proteins found in structural biomaterials, consisting of ?-helical secondary structures wrapped in a supercoil. A fundamental question regarding the thermal and mechanical stability of coiled coils in extreme environments is the sequence of events leading to the disassembly of individual oligomers from the universal coiled-coil motifs. To shed light on this phenomenon, here we report atomistic simulations of a trimeric coiled coil in an explicit water solvent and investigate the mechanisms underlying helix unfolding and coil unzipping in the assembly. We employ advanced sampling techniques involving steered molecular dynamics and metadynamics simulations to obtain the free-energy landscapes of single-strand unfolding and unzipping in a three-stranded assembly. Our comparative analysis of the free-energy landscapes of instability pathways shows that coil unzipping is a sequential process involving multiple intermediates. At each intermediate state, one heptad repeat of the coiled coil first unfolds and then unzips due to the loss of contacts with the hydrophobic core. This observation suggests that helix unfolding facilitates the initiation of coiled-coil disassembly, which is confirmed by our 2D metadynamics simulations showing that unzipping of one strand requires less energy in the unfolded state compared with the folded state. Our results explain recent experimental findings and lay the groundwork for studying the hierarchical molecular mechanisms that underpin the thermomechanical stability/instability of coiled coils and similar protein assemblies. PMID:25028889

  5. Unfold dynamics of generalized Gaussian structures

    NASA Astrophysics Data System (ADS)

    Schiessel, H.

    1998-05-01

    We consider the unfold dynamics of generalized Gaussian structures (GGSs) exposed to different kinds of external forces. A GGS consists of N monomers connected by harmonic springs into a network; when its spectral dimension ds exceeds the critical value of 2 the GGS is in a collapsed state. Sommer and Blumen [J. Phys. A 28, 6669 (1995)] showed that collapsed structures can be unfolded under external forces; they demonstrate this for the case where each monomer is exposed to a force with a random direction: Then networks with a spectral dimension up to 4 become unfolded. In the present paper we focus on the dynamics of such unfold processes. We investigate GGSs exposed to different kinds of external forces (pulling one monomer, uncorrelated forces, long-range correlated forces, and diblocklike forces). We show that external perturbations that act only on a few monomers are not able to unfold a collapsed structure; on the other hand, more general kinds of forces lead to a stretching of GGSs even for ds>2 as long as dsunfold process the size R of the GGS grows via a power law R~t? (0<=?<=1), where ? depends on ds as well as on the kind of force field that is applied.

  6. Photoinduced partial unfolding of tubulin bound to meso-tetrakis(sulfonatophenyl) porphyrin leads to inhibition of microtubule formation in vitro.

    PubMed

    McMicken, Brady; Thomas, Robert J; Brancaleon, Lorenzo

    2014-11-01

    The irradiation of the complex formed by meso-tetrakis (sulfonatophenyl) porphyrin (TSPP) and tubulin was investigated as well as its effects on the structure and function of the protein. We have used tubulin as a model target to investigate whether photoactive ligands docked to the protein can affect the structure and function of the protein upon exposure to visible light. We observed that laser irradiation prompts bleaching of the porphyrin which is accompanied by a sharp decrease (?2 ns) in the average fluorescence lifetime of the protein and a change in the dichroic spectrum consistent with a decrease of helical structure. The result indicated the photoinduced partial unfolding of tubulin. We also observed that such partial conformational change inhibits the formation of microtubules in vitro. We investigated whether photosensitization of reactive oxygen species was responsible for these effects. Even upon removal of O2 the protein still undergoes conformational changes indicating that irradiation of the bound porphyrin does not require the presence of O2 to prompt conformational and functional effects opening the possibility that other mechanisms (e.g., charge transfer) are responsible for the photoinduced mechanism. PMID:23893937

  7. 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

  8. 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...

  9. DELLA proteins and their interacting RING Finger proteins repress gibberellin responses by binding to the promoters of a subset of gibberellin-responsive genes in Arabidopsis.

    PubMed

    Park, Jeongmoo; Nguyen, Khoa Thi; Park, Eunae; Jeon, Jong-Seong; Choi, Giltsu

    2013-03-01

    DELLA proteins, consisting of GA INSENSITIVE, REPRESSOR OF GA1-3, RGA-LIKE1 (RGL1), RGL2, and RGL3, are central repressors of gibberellin (GA) responses, but their molecular functions are not fully understood. We isolated four DELLA-interacting RING domain proteins, previously designated as BOTRYTIS SUSCEPTIBLE1 INTERACTOR (BOI), BOI-RELATED GENE1 (BRG1), BRG2, and BRG3 (collectively referred to as BOIs). Single mutants of each BOI gene failed to significantly alter GA responses, but the boi quadruple mutant (boiQ) showed a higher seed germination frequency in the presence of paclobutrazol, precocious juvenile-to-adult phase transition, and early flowering, all of which are consistent with enhanced GA signaling. By contrast, BOI overexpression lines displayed phenotypes consistent with reduced GA signaling. Analysis of a gai-1 boiQ pentuple mutant further indicated that the GAI protein requires BOIs to inhibit a subset of GA responses. At the molecular level, BOIs did not significantly alter the stability of a DELLA protein. Instead, BOI and DELLA proteins are targeted to the promoters of a subset of GA-responsive genes and repress their expression. Taken together, our results indicate that the DELLA and BOI proteins inhibit GA responses by interacting with each other, binding to the same promoters of GA-responsive genes, and repressing these genes. PMID:23482857

  10. 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...

  11. The Utilization of Competing Unfolding Pathways of Monellin Is Dictated by Enthalpic Barriers

    E-print Network

    a change in unfolding conditions. The m-N and Cp values indicate that TSL is more compact than TSH and not entropic in origin. The availability of multiple pathways by which a protein can fold adds robustness analysis39-44 in terms of activation parameters H , S , and Cp provides a general picture of the extent

  12. The Trp Cage: Folding Kinetics and Unfolded State Topology via Molecular Dynamics Simulations

    E-print Network

    Snow, Christopher

    The Trp Cage: Folding Kinetics and Unfolded State Topology via Molecular Dynamics Simulations dynamics simulations in a generalized-Born implicit solvent (in atomic detail) to simulate the folding dynamics of the Trp cage mini-protein under experi- mental conditions (27 °C with full solvent viscosity

  13. 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

  14. 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…

  15. Development, characterization, and optimization of protein level in date bars using response surface methodology.

    PubMed

    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. 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

  17. 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.

  18. Effects of previous nutrition on the response to protein by weaner steers

    E-print Network

    Paris-Sud XI, Université de

    by increasing protein intake during the summer. An experiment was conducted in which 52 weaner Bos indicusEffects of previous nutrition on the response to protein by weaner steers SR McLennan DP Poppi the subsequent wet summer period often erodes much of this response. Using intra-gastric infused animals, Hovell

  19. Phagocyte-specific S100 proteins in the local response to the Echinococcus granulosus larva

    E-print Network

    Paris-Sud XI, Université de

    Phagocyte-specific S100 proteins in the local response to the Echinococcus granulosus larva TATIANA Infection by larval Echinococcus granulosus is usually characterized by tight inflammatory control. However be involved in this granulomatous response. Key words: Echinococcus granulosus, S100 proteins, granuloma

  20. Temperature-Responsive Protein Pores Yuni Jung,, Hagan Bayley, and Liviu Movileanu*,|

    E-print Network

    Movileanu, Liviu

    Temperature-Responsive Protein Pores Yuni Jung,, Hagan Bayley,§ and Liviu Movileanu*,| Contribution, 2006; E-mail: lmovilea@physics.syr.edu Abstract: We describe temperature-responsive protein pores spherical with a molecular surface volume of about 39 500 Å3 . In an applied potential, the wild-type RHL

  1. 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...

  2. 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

  3. Transcriptome response of Lactobacillus sakei to meat protein environment.

    PubMed

    Xu, Hui-qing; Gao, Lu; Jiang, Yun-shen; Tian, Ying; Peng, Jin; Xa, Qi-quan; Chen, Yu

    2015-04-01

    Lactobacillus sakei is a heterofermentative species of lactic acid bacteria that is used in industrial meat fermentation. To investigate adaptation in a meat environment, whole-genome DNA microarrays were used to analyze the gene expression related to growth and survival of L. sakei strain La22 when grown in sarcoplasmic (S-) or myofibrillar (M-) protein-supplemented chemically defined medium (CDM). Differential expression was detected in 551 genes. Genes encoding enzymes involved in peptide hydrolysis were differentially upregulated in M-CDM or/and S-CDM, and only oppB and oppC, involved in the amino acid and peptide transport system, were upregulated. Most genes related to metabolism of peptides, amino acids and related molecules were over-expressed in M-CDM and S-CDM, except for glnA and metK. Expression of certain genes was according to the differential substrate environment. The expression of genes involved in the stress response was not induced by growth in M-CDM. PMID:25384669

  4. Study of global transcriptional changes of N-GlcNAc2 proteins-producing T24 bladder carcinoma cells under glucose deprivation.

    PubMed

    Isono, Takahiro; Chano, Tokuhiro; Okabe, Hidetoshi; Suzaki, Masafumi

    2013-01-01

    Increased levels of N-linked (?-N- acetylglucosamine)2 [N-GlcNAc2]-modified proteins have been recognized to be an effective response to glucose deprivation. In the first step of this study, using a next generation sequencer, we investigated the global transcriptional changes induced by glucose deprivation in a T24 bladder carcinoma cell line, producing N-GlcNAc2-modified proteins under glucose deprivation. Our transcriptome analysis revealed significant up-regulation of the UDP-GlcNAc biosynthesis pathway and unfolded protein response genes, and down-regulation of G2/M transition-related genes containing mitotic kinases. Our biological analysis confirmed that N-GlcNAc2-modified proteins were localized with BiP proteins in the ER. G2/M arrest was caused by glucose deprivation in T24 cells. Moreover, the knockdown of unfolded protein response genes induced the expressional recovery of mitotic kinases under glucose deprivation. Taken together, our results suggest N-GlcNAc2-modified proteins produced under glucose deprivation caused unfolded protein response in the ER, and that this response induced G2/M arrest. PMID:23560094

  5. Study of Global Transcriptional Changes of N-GlcNAc2 Proteins-Producing T24 Bladder Carcinoma Cells under Glucose Deprivation

    PubMed Central

    Isono, Takahiro; Chano, Tokuhiro; Okabe, Hidetoshi; Suzaki, Masafumi

    2013-01-01

    Increased levels of N-linked (?-N- acetylglucosamine)2 [N-GlcNAc2]-modified proteins have been recognized to be an effective response to glucose deprivation. In the first step of this study, using a next generation sequencer, we investigated the global transcriptional changes induced by glucose deprivation in a T24 bladder carcinoma cell line, producing N-GlcNAc2-modified proteins under glucose deprivation. Our transcriptome analysis revealed significant up-regulation of the UDP-GlcNAc biosynthesis pathway and unfolded protein response genes, and down-regulation of G2/M transition-related genes containing mitotic kinases. Our biological analysis confirmed that N-GlcNAc2-modified proteins were localized with BiP proteins in the ER. G2/M arrest was caused by glucose deprivation in T24 cells. Moreover, the knockdown of unfolded protein response genes induced the expressional recovery of mitotic kinases under glucose deprivation. Taken together, our results suggest N-GlcNAc2-modified proteins produced under glucose deprivation caused unfolded protein response in the ER, and that this response induced G2/M arrest. PMID:23560094

  6. Dynamics of the coiled-coil unfolding transition of myosin rod probed by dissipation force spectrum.

    PubMed

    Taniguchi, Yukinori; Khatri, Bhavin S; Brockwell, David J; Paci, Emanuele; Kawakami, Masaru

    2010-07-01

    The motor protein myosin II plays a crucial role in muscle contraction. The mechanical properties of its coiled-coil region, the myosin rod, are important for effective force transduction during muscle function. Previous studies have investigated the static elastic response of the myosin rod. However, analogous to the study of macroscopic complex fluids, how myosin will respond to physiological time-dependent loads can only be understood from its viscoelastic response. Here, we apply atomic force microscopy using a magnetically driven oscillating cantilever to measure the dissipative properties of single myosin rods that provide unique dynamical information about the coiled-coil structure as a function of force. We find that the friction constant of the single myosin rod has a highly nontrivial variation with force; in particular, the single-molecule friction constant is reduced dramatically and increases again as it passes through the coiled-uncoiled transition. This is a direct indication of a large free-energy barrier to uncoiling, which may be related to a fine-tuned dynamic mechanosignaling response to large and unexpected physiological loads. Further, from the critical force at which the minimum in friction occurs we determine the asymmetry of the bistable landscape that controls uncoiling of the coiled coil. This work highlights the sensitivity of the dissipative signal in force unfolding to dynamic molecular structure that is hidden to the elastic signal. PMID:20655854

  7. Proteomics of Arabidopsis redox proteins in response to methyl jasmonate

    Microsoft Academic Search

    Sophie Alvarez; Mengmeng Zhu; Sixue Chen

    2009-01-01

    Protein redox regulation is increasingly recognized as an important switch of protein activity in yeast, bacteria, mammals and plants. In this study, we identified proteins with potential thiol switches involved in jasmonate signaling, which is essential for plant defense. Methyl jasmonate (MeJA) treatment led to enhanced production of hydrogen peroxide in Arabidopsis leaves and roots, indicating in vivo oxidative stress.

  8. Immune Response to Chlamydial 60-Kilodalton Heat Shock Protein in Tears from Nepali Trachoma Patients

    Microsoft Academic Search

    TRACEY HESSEL; S. P. Dhital; REBECA PLANK; DEBORAH DEAN

    2001-01-01

    Although the host immune response to the 60-kDa chlamydial heat shock protein (hsp60) has been impli- cated in trachoma pathogenesis, no studies have examined mucosal immune responses to hsp60 in populations for which chlamydia is endemic. Tears and sera from Nepali villagers were reacted against hsp60 fusion proteins, whole hsp60, and the major outer membrane protein (MOMP). Tears from villagers

  9. Picosecond spectroscopic studies of equilibrium structural fluctuations of native and partially unfolded states of Zinc II-substituted and metal-free cytochromes C

    NASA Astrophysics Data System (ADS)

    Tripathy, Jagnyaseni

    Picosecond time-resolved fluorescence spectroscopy was employed to characterize the equilibrium and non-equilibrium protein structural fluctuations in Zn II-substituted (ZnCytc) and metal-free (fbCytc) cytochromes c using dynamic fluorescence Stokes shift (FSS) and fluorescence anisotropy (FA) measurements. The intrinsic porphyrin chromophore is used as the probe for the structural fluctuations of the surrounding protein and solvent. The FSS experiments examine how the time scales detected from the dynamic solvation of a chromoprotein report changes in the character of motion. ZnCytc and fbCytc serve as limited, single-chromophore models for photosynthetic reaction center and light-harvesting proteins. The dynamic solvation of redox and light-harvesting chromophores in photosynthesis plays an important role in the quantum efficiency of electron transfer and energy transfer performed by these systems, respectively. The FSS response function of fbCytc in water is biexponential over the 100-ps--50-ns regime and the two time constants are 1.4 ns and 9.1 ns. ZnCytc under similar solution conditions shows a biexponential FSS response but with time constants of 0.2 ns and 1.5 ns. The two correlation times from the FSS response function correspond to motions of the hydrophobic core and the solvent-contact layer, respectively. Both FSS correlation times were lengthened and the solvation reorganization energy was reduced from 43 cm-1 to 33 cm-1 in the presence of 50% (v/v) glycerol. A Brownian diffusion model with thermally activated barrier crossings on the protein-folding energy landscape is used to interpret these results. The conclusion is that the mean-squared deviations of the fluctuations exhibited by fbCytc are perhaps a factor of ten larger than those in ZnCytc, which is consistent with the suggestion that fbCytc assumes a dynamic, partially unfolded structure with some of the characteristics of a molten globule. The nature of the motion associated with the unfolding reaction coordinate was then studied in ZnCytc using the FA response of the Zn II-porphyrin probe as guanidinium ion (Gdm+) is added to the solution. An unfolding transition-state-like intermediate is formed at ˜1.0 M Gdm +, well below the unfolding transition concentration at ˜2.0 M. The intermediate is characterized by an enhanced angular fluctuation of the porphyrin compared to the native and the denatured structures. The correlation time for internal angular motion returned by the FA response for the 1.0 M intermediate is similar to those observed for the FSS response of fbCytc, which suggests that the axial ligands for the ZnII porphyrin have been lost. The FSS response was then obtained as ZnCytc is titrated with Gdm + using the same data sets as the FA studies. The FSS results show that even at the lowest concentrations of Gdm+, the native fold is destabilized so that the optical excitation of the ZnII porphyrin chromophore perturbs the motions of the surrounding protein and solvent so that they depart from the linear-response regime. This conclusion is supported by the observation of an unusual bidirectional FSS response. This action of Gdm+ appears to saturate well prior to the formation of the 1-M intermediate structure noted in the FA studies. The main implication of these results is that structural changes in the hydration layer surrounding the folded protein are the origin of the dynamical changes reported in the FSS response in the presence of Gdm+.

  10. 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.

  11. The unfolding of alpha-momorcharin proceeds through the compact folded intermediate.

    PubMed

    Fukunaga, Yukihiro; Nishimoto, Etsuko; Otosu, Takuhiro; Murakami, Yasutaka; Yamashita, Shoji

    2008-10-01

    The unfolding of alpha-momorcharin was systematically investigated using steady-state and time-resolved tryptophan fluorescence, circular dichroism and 8-anilino-1-naphthalenesulfonic acid (ANS) binding. These spectroscopic studies demonstrated that alpha-momorcharin unfolded through a compact folded intermediate state. The content of alpha-helix was increased, Trp192 approached closer to the side of active site and its rotational motion was restricted by being equilibrated with 2-3 M of guanidine hydrochloride. Furthermore, the binding of ANS with alpha-momorcharin was more suppressed to show that the hydrophobic parts would not be accessed to the protein surface but rather be sealed off in this specific conformation state. These results suggest that the structure of alpha-momorcharin holds the more compact conformation as an incipient state for unfolding, which is the sharp contrast to beta-momorcharin that gives the characteristics of the generally known molten globule state. PMID:18603588

  12. Collagen Unfolding Accelerates Water Influx, Determining Hydration in the Interstitial Matrix

    PubMed Central

    McGee, Maria P.; Morykwas, Michael; Shelton, Julie; Argenta, Louis

    2012-01-01

    In the interstitial matrix, collagen unfolding at physiologic temperatures is thought to facilitate interactions with enzymes and scaffold molecules during inflammation, tissue remodeling, and wound healing. We tested the hypothesis that it also plays a role in modulating flows and matrix hydration potential. After progressively unfolding dermal collagen in situ, we measured the hydration parameters by osmotic stress techniques and modeled them as linear functions of unfolded collagen, quantified by differential scanning calorimetry after timed heat treatment. Consistent with the hypothetical model, the thermodynamic and flow parameters obtained experimentally were related linearly to the unfolded collagen fraction. The increases in relative humidity and intensity of T2 maps were also consistent with interfacial energy contributions to the hydration potential and the hydrophobic character of the newly formed protein/water interfaces. As a plausible explanation, we propose that increased tension at interfaces formed during collagen unfolding generate local gradients in the matrix that accelerate water transfer in the dermis. This mechanism adds a convective component to interstitial transfer of biological fluids that, unlike diffusion, can speed the dispersion of water and large solutes within the matrix. PMID:23200049

  13. Hexosamine pathway and (ER) protein quality control.

    PubMed

    Denzel, Martin S; Antebi, Adam

    2015-04-01

    Aminosugars produced in the hexosamine pathway (HP) are utilized in protein glycosylation reactions involved in protein maturation and cellular signaling. Recent evidence revealed a role of the HP in protein quality control and ageing. Elevation of the HP product UDP-N-acetylglucosamine in the nematode Caenorhabditis elegans results in resistance towards toxic aggregation-prone proteins, and extended lifespan. Glutamine-fructose 6 phosphate aminotransferase (GFAT-1), the HP's key enzyme, is a target of the unfolded protein response (UPR). Thus, cardiac stress in mice results in GFAT-1 activation that triggers a cytoprotective response. Feeding of glucosamine to aged mice increases their life expectancy. Here we discuss HP activation and cellular protein quality control mechanisms that result in stress resistance and suppression of age-related proteotoxicity. PMID:25463841

  14. Pressure-Jump-Induced Kinetics Reveals a Hydration Dependent Folding/Unfolding Mechanism of Ribonuclease A

    PubMed Central

    Font, J.; Torrent, J.; Ribó, M.; Laurents, D. V.; Balny, C.; Vilanova, M.; Lange, R.

    2006-01-01

    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°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°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

  15. Microsecond Molecular Dynamics Simulations of Intrinsically Disordered Proteins Involved in the Oxidative Stress Response

    Microsoft Academic Search

    Elio A. Cino; Jirasak Wong-ekkabut; Mikko Karttunen; Wing-Yiu Choy

    2011-01-01

    Intrinsically disordered proteins (IDPs) are abundant in cells and have central roles in protein-protein interaction networks. Interactions between the IDP Prothymosin alpha (ProT?) and the Neh2 domain of Nuclear factor erythroid 2-related factor 2 (Nrf2), with a common binding partner, Kelch-like ECH-associated protein 1(Keap1), are essential for regulating cellular response to oxidative stress. Misregulation of this pathway can lead to

  16. Probing the role of hydration in the unfolding transitions of carbonmonoxy myoglobin and apomyoglobin.

    PubMed

    Guo, Lin; Park, Jaeheung; Lee, Taegon; Chowdhury, Pramit; Lim, Manho; Gai, Feng

    2009-04-30

    We show that the equilibrium unfolding transition of horse carbonmonoxy myoglobin monitored by the stretching vibration of the CO ligand, a local environmental probe, is very sharp and, thus, quite different from those measured by global conformational reporters. In addition, the denatured protein exhibits an A(0)-like CO band. We hypothesize that this sharp transition reports penetration of water into the heme pocket of the protein. Parallel experiments on horse apomyoglobin, wherein an environment-sensitive fluorescent probe, nile red, was used, also reveals a similar putative hydration event. Given the importance of dehydration in protein folding and also the recent debate over the interpretation of probe-dependent unfolding transitions, these results have strong implications on the mechanism of protein folding. PMID:19348439

  17. 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

  18. 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

  19. The muscle protein synthetic response to the combined ingestion of protein and carbohydrate is not impaired in healthy older men.

    PubMed

    Kiskini, Alexandra; Hamer, Henrike M; Wall, Benjamin T; Groen, Bart B L; de Lange, Anneke; Bakker, Jaap A; Senden, Joan M G; Verdijk, Lex B; van Loon, Luc J C

    2013-12-01

    Aging is associated with a progressive decline in skeletal muscle mass. It has been hypothesized that an attenuated muscle protein synthetic response to the main anabolic stimuli may contribute to the age-related loss of muscle tissue. The aim of the present study was to compare the muscle protein synthetic response following ingestion of a meal-like amount of dietary protein plus carbohydrate between healthy young and older men. Twelve young (21 ± 1 years) and 12 older (75 ± 1 years) men consumed 20 g of intrinsically L-[1-(13)C]phenylalanine-labeled protein with 40 g of carbohydrate. Ingestion of specifically produced intrinsically L-[1-(13)C]phenylalanine-labeled protein allowed us to assess the subsequent incorporation of casein-derived amino acids into muscle protein. Blood samples were collected at regular intervals, with muscle biopsies obtained prior to and 2 and 6 h after protein plus carbohydrate ingestion. The acute post-prandial rise in plasma glucose and insulin concentrations was significantly greater in the older compared with the younger males. Plasma amino acid concentrations increased rapidly following drink ingestion in both groups. However, plasma leucine concentrations were significantly lower at t = 90 min in the older when compared with the young group (P < 0.05). Muscle protein-bound L-[1-(13)C]phenylalanine enrichments increased to 0.0071 ± 0.0016 and 0.0072 ± 0.0013 mole percent excess (MPE) at 2 h and 0.0229 ± 0.0016 and 0.0213 ± 0.0024 MPE at 6 h following ingestion of the intrinsically labeled protein in the young and older males, respectively, with no differences between groups (P > 0.05). We conclude that the use of dietary protein-derived amino acids for muscle protein synthesis is not impaired in healthy older men following intake of protein plus carbohydrate. PMID:23529503

  20. Sequence Motifs in MADS Transcription Factors Responsible for Specificity and Diversification of Protein-Protein Interaction

    Microsoft Academic Search

    Aalt D. J. van Dijk; Giuseppa Morabito; Martijn Fiers; Roeland C. H. J. van Ham; Gerco C. Angenent; Richard G. H. Immink

    2010-01-01

    Protein sequences encompass tertiary structures and contain information about specific molecular interactions, which in turn determine biological functions of proteins. Knowledge about how protein sequences define interaction specificity is largely missing, in particular for paralogous protein families with high sequence similarity, such as the plant MADS domain transcription factor family. In comparison to the situation in mammalian species, this important

  1. Unfolding first-principles band structures.

    PubMed

    Ku, Wei; Berlijn, Tom; Lee, Chi-Cheng

    2010-05-28

    A general method is presented to unfold band structures of first-principles supercell calculations with proper spectral weight, allowing easier visualization of the electronic structure and the degree of broken translational symmetry. The resulting unfolded band structures contain additional rich information from the Kohn-Sham orbitals, and absorb the structure factor that makes them ideal for a direct comparison with angle resolved photoemission spectroscopy experiments. With negligible computational expense via the use of Wannier functions, this simple method has great practical value in the studies of a wide range of materials containing impurities, vacancies, lattice distortions, or spontaneous long-range orders. PMID:20867120

  2. Fitting and Testing Carroll's Weighted