Enthalpy-entropy compensation in protein unfolding
Institute of Scientific and Technical Information of China (English)
无
2000-01-01
Enthalpy-entropy compensation was found to be a universal law in protein unfolding based on over 3 000 experimental data. Water molecular reorganization accompanying the protein unfolding was suggested as the origin of the enthalpy-entropy compensation in protein unfolding. It is indicated that the enthalpy-entropy compensation constitutes the physical foundation that satisfies the biological need of the small free energy changes in protein unfolding, without the sacrifice of the bio-diversity of proteins. The enthalpy-entropy compensation theory proposed herein also provides valuable insights into the Privalov's puzzle of enthalpy and entropy convergence in protein unfolding.
Protein unfolding with a steric trap.
Blois, Tracy M; Hong, Heedeok; Kim, Tae H; Bowie, James U
2009-10-07
The study of protein folding requires a method to drive unfolding, which is typically accomplished by altering solution conditions to favor the denatured state. This has the undesirable consequence that the molecular forces responsible for configuring the polypeptide chain are also changed. It would therefore be useful to develop methods that can drive unfolding without the need for destabilizing solvent conditions. Here we introduce a new method to accomplish this goal, which we call steric trapping. In the steric trap method, the target protein is labeled with two biotin tags placed close in space so that both biotin tags can only be bound by streptavidin when the protein unfolds. Thus, binding of the second streptavidin is energetically coupled to unfolding of the target protein. Testing the method on a model protein, dihydrofolate reductase (DHFR), we find that streptavidin binding can drive unfolding and that the apparent binding affinity reports on changes in DHFR stability. Finally, by employing the slow off-rate of wild-type streptavidin, we find that DHFR can be locked in the unfolded state. The steric trap method provides a simple method for studying aspects of protein folding and stability in native solvent conditions, could be used to specifically unfold selected domains, and could be applicable to membrane proteins.
Peripheral Protein Unfolding Drives Membrane Bending.
Siaw, Hew Ming Helen; Raghunath, Gokul; Dyer, R Brian
2018-06-20
Dynamic modulation of lipid membrane curvature can be achieved by a number of peripheral protein binding mechanisms such as hy-drophobic insertion of amphipathic helices and membrane scaffolding. Recently, an alternative mechanism was proposed in which crowding of peripherally bound proteins induces membrane curvature through steric pressure generated by lateral collisions. This effect was enhanced using intrinsically disordered proteins that possess high hydrodynamic radii, prompting us to explore whether membrane bending can be triggered by the folding-unfolding transition of surface-bound proteins. We utilized histidine-tagged human serum albumin bound to Ni-NTA-DGS containing liposomes as our model system to test this hypothesis. We found that reduction of the disulfide bonds in the protein resulted in unfolding of HSA, which subsequently led to membrane tubule formation. The frequency of tubule formation was found to be significantly higher when the proteins were unfolded while being localized to a phase-separated domain as opposed to randomly distributed in fluid phase liposomes, indicating that the steric pressure generated from protein unfolding is directly responsible for membrane deformation. Our results are critical for the design of peripheral membrane protein-immobilization strategies and open new avenues for exploring mechanisms of membrane bending driven by conformational changes of peripheral membrane proteins.
Indian Academy of Sciences (India)
Unknown
2005-01-03
Jan 3, 2005 ... covering all the systems, so far discovered.5,7,8,12. With the increasing ... Structural investigations on proteins by NMR are, currently ... rapid analysis of unfolded proteins. ...... and hence help in design of drugs against them.
International Nuclear Information System (INIS)
Beddard, G S; Brockwell, D J
2010-01-01
A statistical calculation is described with which the saw-tooth-like unfolding patterns of concatenated heteropolymeric proteins can be used to estimate the forced unfolding parameters of a previously uncharacterized protein. The chance of observing the various sequences of unfolding events, such as ABAABBB or BBAAABB etc, for two proteins of types A and B is calculated using proteins with various ratios of A and B and at different values of effective unfolding rate constants. If the experimental rate constant for forced unfolding, k 0 , and distance to the transition state x u are known for one protein, then the calculation allows an estimation of values for the other. The predictions are compared with Monte Carlo simulations and experimental data. (communication)
Bian, Liujiao; Ji, Xu
2014-01-01
Extensive and intensive studies on the unfolding of proteins require appropriate theoretical model and parameter to clearly illustrate the feature and characteristic of the unfolding system. Over the past several decades, four approaches have been proposed to describe the interaction between proteins and denaturants, but some ambiguity and deviations usually occur in the explanation of the experimental data. In this work, a theoretical model was presented to show the dependency of the residual activity ratio of the proteins on the molar denaturant concentration. Through the characteristic unfolding parameters ki and Δmi in this model, the distribution, transition and thermodynamic stability of protein conformations during the unfolding process can be quantitatively described. This model was tested with the two-state unfolding of bovine heart cytochrome c and the three-state unfolding of hen egg white lysozyme induced by both guanidine hydrochloride and urea, the four-state unfolding of bovine carbonic anhydrase b induced by guanidine hydrochloride and the unfolding of some other proteins induced by denaturants. The results illustrated that this model could be used accurately to reveal the distribution and transition of protein conformations in the presence of different concentrations of denaturants and to evaluate the unfolding tendency and thermodynamic stability of different conformations. In most denaturant-induced unfolding of proteins, the unfolding became increasingly hard in next transition step and the proteins became more unstable as they attained next successive stable conformation. This work presents a useful method for people to study the unfolding of proteins and may be used to describe the unfolding and refolding of other biopolymers induced by denaturants, inducers, etc.
Unfolding study of a trimeric membrane protein AcrB.
Ye, Cui; Wang, Zhaoshuai; Lu, Wei; Wei, Yinan
2014-07-01
The folding of a multi-domain trimeric α-helical membrane protein, Escherichia coli inner membrane protein AcrB, was investigated. AcrB contains both a transmembrane domain and a large periplasmic domain. Protein unfolding in sodium dodecyl sulfate (SDS) and urea was monitored using the intrinsic fluorescence and circular dichroism spectroscopy. The SDS denaturation curve displayed a sigmoidal profile, which could be fitted with a two-state unfolding model. To investigate the unfolding of separate domains, a triple mutant was created, in which all three Trp residues in the transmembrane domain were replaced with Phe. The SDS unfolding profile of the mutant was comparable to that of the wild type AcrB, suggesting that the observed signal change was largely originated from the unfolding of the soluble domain. Strengthening of trimer association through the introduction of an inter-subunit disulfide bond had little effect on the unfolding profile, suggesting that trimer dissociation was not the rate-limiting step in unfolding monitored by fluorescence emission. Under our experimental condition, AcrB unfolding was not reversible. Furthermore, we experimented with the refolding of a monomeric mutant, AcrBΔloop , from the SDS unfolded state. The CD spectrum of the refolded AcrBΔloop superimposed well onto the spectra of the original folded protein, while the fluorescence spectrum was not fully recovered. In summary, our results suggested that the unfolding of the trimeric AcrB started with a local structural rearrangement. While the refolding of secondary structure in individual monomers could be achieved, the re-association of the trimer might be the limiting factor to obtain folded wild-type AcrB. © 2014 The Protein Society.
Evolution and thermodynamics of the slow unfolding of hyperstable monomeric proteins
Directory of Open Access Journals (Sweden)
Koga Yuichi
2010-07-01
Full Text Available Abstract Background The unfolding speed of some hyperthermophilic proteins is dramatically lower than that of their mesostable homologs. Ribonuclease HII from the hyperthermophilic archaeon Thermococcus kodakaraensis (Tk-RNase HII is stabilized by its remarkably slow unfolding rate, whereas RNase HI from the thermophilic bacterium Thermus thermophilus (Tt-RNase HI unfolds rapidly, comparable with to that of RNase HI from Escherichia coli (Ec-RNase HI. Results To clarify whether the difference in the unfolding rate is due to differences in the types of RNase H or differences in proteins from archaea and bacteria, we examined the equilibrium stability and unfolding reaction of RNases HII from the hyperthermophilic bacteria Thermotoga maritima (Tm-RNase HII and Aquifex aeolicus (Aa-RNase HII and RNase HI from the hyperthermophilic archaeon Sulfolobus tokodaii (Sto-RNase HI. These proteins from hyperthermophiles are more stable than Ec-RNase HI over all the temperature ranges examined. The observed unfolding speeds of all hyperstable proteins at the different denaturant concentrations studied are much lower than those of Ec-RNase HI, which is in accordance with the familiar slow unfolding of hyperstable proteins. However, the unfolding rate constants of these RNases H in water are dispersed, and the unfolding rate constant of thermophilic archaeal proteins is lower than that of thermophilic bacterial proteins. Conclusions These results suggest that the nature of slow unfolding of thermophilic proteins is determined by the evolutionary history of the organisms involved. The unfolding rate constants in water are related to the amount of buried hydrophobic residues in the tertiary structure.
Application of long-range order to predict unfolding rates of two-state proteins.
Harihar, B; Selvaraj, S
2011-03-01
Predicting the experimental unfolding rates of two-state proteins and models describing the unfolding rates of these proteins is quite limited because of the complexity present in the unfolding mechanism and the lack of experimental unfolding data compared with folding data. In this work, 25 two-state proteins characterized by Maxwell et al. (Protein Sci 2005;14:602–616) using a consensus set of experimental conditions were taken, and the parameter long-range order (LRO) derived from their three-dimensional structures were related with their experimental unfolding rates ln(k(u)). From the total data set of 30 proteins used by Maxwell et al. (Protein Sci 2005;14:602–616), five slow-unfolding proteins with very low unfolding rates were considered to be outliers and were not included in our data set. Except all beta structural class, LRO of both the all-alpha and mixed-class proteins showed a strong inverse correlation of r = -0.99 and -0.88, respectively, with experimental ln(k(u)). LRO shows a correlation of -0.62 with experimental ln(k(u)) for all-beta proteins. For predicting the unfolding rates, a simple statistical method has been used and linear regression equations were developed for individual structural classes of proteins using LRO, and the results obtained showed a better agreement with experimental results. Copyright © 2010 Wiley-Liss, Inc.
Conformational dynamics of a protein in the folded and the unfolded state
Energy Technology Data Exchange (ETDEWEB)
Fitter, Joerg
2003-08-01
In a quasielastic neutron scattering experiment, the picosecond dynamics of {alpha}-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{sub 2}O-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 A; unfolded state, 1.8 A). 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.
Dry molten globule intermediates and the mechanism of protein unfolding.
Baldwin, Robert L; Frieden, Carl; Rose, George D
2010-10-01
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 (DMG) intermediates, an expanded form of the protein that lacks appreciable solvent. When an unfolding protein expands to the DMG state, side chains unlock and gain conformational entropy, while liquid-like van der Waals interactions persist. Four unrelated proteins are now known to form DMGs 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 DMG, 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 DMG. The absence of close packing at this barrier provides an explanation for why phi-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 DMG intermediates, a realization that has major implications for future experimental work on the mechanism of protein folding. 2010 Wiley-Liss, Inc.
History, rare, and multiple events of mechanical unfolding of repeat proteins
Sumbul, Fidan; Marchesi, Arin; Rico, Felix
2018-03-01
Mechanical unfolding of proteins consisting of repeat domains is an excellent tool to obtain large statistics. Force spectroscopy experiments using atomic force microscopy on proteins presenting multiple domains have revealed that unfolding forces depend on the number of folded domains (history) and have reported intermediate states and rare events. However, the common use of unspecific attachment approaches to pull the protein of interest holds important limitations to study unfolding history and may lead to discarding rare and multiple probing events due to the presence of unspecific adhesion and uncertainty on the pulling site. Site-specific methods that have recently emerged minimize this uncertainty and would be excellent tools to probe unfolding history and rare events. However, detailed characterization of these approaches is required to identify their advantages and limitations. Here, we characterize a site-specific binding approach based on the ultrastable complex dockerin/cohesin III revealing its advantages and limitations to assess the unfolding history and to investigate rare and multiple events during the unfolding of repeated domains. We show that this approach is more robust, reproducible, and provides larger statistics than conventional unspecific methods. We show that the method is optimal to reveal the history of unfolding from the very first domain and to detect rare events, while being more limited to assess intermediate states. Finally, we quantify the forces required to unfold two molecules pulled in parallel, difficult when using unspecific approaches. The proposed method represents a step forward toward more reproducible measurements to probe protein unfolding history and opens the door to systematic probing of rare and multiple molecule unfolding mechanisms.
Experimental parameterization of an energy function for the simulation of unfolded proteins
DEFF Research Database (Denmark)
Norgaard, A.B.; Ferkinghoff-Borg, Jesper; Lindorff-Larsen, K.
2008-01-01
The determination of conformational preferences in unfolded and disordered proteins is an important challenge in structural biology. We here describe an algorithm to optimize energy functions for the simulation of unfolded proteins. The procedure is based on the maximum likelihood principle and e...... and can be applied to a range of experimental data and energy functions including the force fields used in molecular dynamics simulations.......The determination of conformational preferences in unfolded and disordered proteins is an important challenge in structural biology. We here describe an algorithm to optimize energy functions for the simulation of unfolded proteins. The procedure is based on the maximum likelihood principle...
Del Galdo, Sara; Amadei, Andrea
2016-10-12
In this paper we apply the computational analysis recently proposed by our group to characterize the solvation properties of a native protein in aqueous solution, and to four model aqueous solutions of globular proteins in their unfolded states thus characterizing the protein unfolded state hydration shell and quantitatively evaluating the protein unfolded state partial molar volumes. Moreover, by using both the native and unfolded protein partial molar volumes, we obtain the corresponding variations (unfolding partial molar volumes) to be compared with the available experimental estimates. We also reconstruct the temperature and pressure dependence of the unfolding partial molar volume of Myoglobin dissecting the structural and hydration effects involved in the process.
Kinetics of protein unfolding at interfaces
International Nuclear Information System (INIS)
Yano, Yohko F
2012-01-01
The conformation of protein molecules is determined by a balance of various forces, including van der Waals attraction, electrostatic interaction, hydrogen bonding, and conformational entropy. When protein molecules encounter an interface, they are often adsorbed on the interface. The conformation of an adsorbed protein molecule strongly depends on the interaction between the protein and the interface. Recent time-resolved investigations have revealed that protein conformation changes during the adsorption process due to the protein-protein interaction increasing with increasing interface coverage. External conditions also affect the protein conformation. This review considers recent dynamic observations of protein adsorption at various interfaces and their implications for the kinetics of protein unfolding at interfaces. (topical review)
Measurement of the unfolded protein response (UPR) in monocytes.
LENUS (Irish Health Repository)
Carroll, Tomás P
2011-01-01
In mammalian cells, the primary function of the endoplasmic reticulum (ER) is to synthesize and assemble membrane and secreted proteins. As the main site of protein folding and posttranslational modification in the cell, the ER operates a highly conserved quality control system to ensure only correctly assembled proteins exit the ER and misfolded and unfolded proteins are retained for disposal. Any disruption in the equilibrium of the ER engages a multifaceted intracellular signaling pathway termed the unfolded protein response (UPR) to restore normal conditions in the cell. A variety of pathological conditions can induce activation of the UPR, including neurodegenerative disorders such as Parkinson\\'s disease, metabolic disorders such as atherosclerosis, and conformational disorders such as cystic fibrosis. Conformational disorders are characterized by mutations that modify the final structure of a protein and any cells that express abnormal protein risk functional impairment. The monocyte is an important and long-lived immune cell and acts as a key immunological orchestrator, dictating the intensity and duration of the host immune response. Monocytes expressing misfolded or unfolded protein may exhibit UPR activation and this can compromise the host immune system. Here, we describe in detail methods and protocols for the examination of UPR activation in peripheral blood monocytes. This guide should provide new investigators to the field with a broad understanding of the tools required to investigate the UPR in the monocyte.
Measurement of the unfolded protein response (UPR) in monocytes.
LENUS (Irish Health Repository)
Carroll, Tomas P
2012-02-01
In mammalian cells, the primary function of the endoplasmic reticulum (ER) is to synthesize and assemble membrane and secreted proteins. As the main site of protein folding and posttranslational modification in the cell, the ER operates a highly conserved quality control system to ensure only correctly assembled proteins exit the ER and misfolded and unfolded proteins are retained for disposal. Any disruption in the equilibrium of the ER engages a multifaceted intracellular signaling pathway termed the unfolded protein response (UPR) to restore normal conditions in the cell. A variety of pathological conditions can induce activation of the UPR, including neurodegenerative disorders such as Parkinson\\'s disease, metabolic disorders such as atherosclerosis, and conformational disorders such as cystic fibrosis. Conformational disorders are characterized by mutations that modify the final structure of a protein and any cells that express abnormal protein risk functional impairment. The monocyte is an important and long-lived immune cell and acts as a key immunological orchestrator, dictating the intensity and duration of the host immune response. Monocytes expressing misfolded or unfolded protein may exhibit UPR activation and this can compromise the host immune system. Here, we describe in detail methods and protocols for the examination of UPR activation in peripheral blood monocytes. This guide should provide new investigators to the field with a broad understanding of the tools required to investigate the UPR in the monocyte.
Towards data warehousing and mining of protein unfolding simulation data.
Berrar, Daniel; Stahl, Frederic; Silva, Candida; Rodrigues, J Rui; Brito, Rui M M; Dubitzky, Werner
2005-10-01
The prediction of protein structure and the precise understanding of protein folding and unfolding processes remains one of the greatest challenges in structural biology and bioinformatics. Computer simulations based on molecular dynamics (MD) are at the forefront of the effort to gain a deeper understanding of these complex processes. Currently, these MD simulations are usually on the order of tens of nanoseconds, generate a large amount of conformational data and are computationally expensive. More and more groups run such simulations and generate a myriad of data, which raises new challenges in managing and analyzing these data. Because the vast range of proteins researchers want to study and simulate, the computational effort needed to generate data, the large data volumes involved, and the different types of analyses scientists need to perform, it is desirable to provide a public repository allowing researchers to pool and share protein unfolding data. To adequately organize, manage, and analyze the data generated by unfolding simulation studies, we designed a data warehouse system that is embedded in a grid environment to facilitate the seamless sharing of available computer resources and thus enable many groups to share complex molecular dynamics simulations on a more regular basis. To gain insight into the conformational fluctuations and stability of the monomeric forms of the amyloidogenic protein transthyretin (TTR), molecular dynamics unfolding simulations of the monomer of human TTR have been conducted. Trajectory data and meta-data of the wild-type (WT) protein and the highly amyloidogenic variant L55P-TTR represent the test case for the data warehouse. Web and grid services, especially pre-defined data mining services that can run on or 'near' the data repository of the data warehouse, are likely to play a pivotal role in the analysis of molecular dynamics unfolding data.
Complete all-atom hydrodynamics of protein unfolding in uniform flow
International Nuclear Information System (INIS)
Wang, Guan M; Sandberg, William C
2010-01-01
The unfolding dynamics of a protein, ubiquitin, pinned in several uniform flows, was studied at low and high flow rates in an all-atom style through a non-equilibrium molecular dynamics approach with explicit water molecules included. Atomic hydrodynamic force components on individual amino acids, as a function of time, due to the collisional interactions with the flowing water molecules were calculated explicitly. The protein conformational change in response to those time-varying forces was computed completely at the high flow rate up to nanosecond until the fully stretched state was reached. The end-to-end length of the single ubiquitin protein molecule at high flow rate is smoothly increasing. The step-like jumps between metastable states that describe the μm ms -1 scale force pulling experiments conducted on polyubiquitins at low flow rates, are not seen at the high flow speeds necessary to computationally probe the ns nm -1 scale regime. No unfolding was observed in the low flow rate atomic computations at nanosecond scale while partial and complete unfolding was observed in the coarse-grained low flow rate computations at microsecond scale. Examination of the all-atom computation of the time variation of the hydrodynamic forces on, and the velocity components of, the protein molecule unveiled to some extent the details of the complexity of the hydrodynamic friction variation in the nm ns -1 regime of high rate flow-driven protein unfolding. This demonstrates quantitatively that all-atom computations are more suitable than the Langevin equation or Brownian dynamics methods for probing the interaction dynamics and resulting conformational dynamics of protein unfolding in strong flows on nm ns -1 time/length scales while the reverse is true for investigation of slow, diffusively driven systems.
The unfolded protein response has a protective role in yeast models of classic galactosemia
Directory of Open Access Journals (Sweden)
Evandro A. De-Souza
2014-01-01
Full Text Available Classic galactosemia is a human autosomal recessive disorder caused by mutations in the GALT gene (GAL7 in yeast, which encodes the enzyme galactose-1-phosphate uridyltransferase. Here we show that the unfolded protein response pathway is triggered by galactose in two yeast models of galactosemia: lithium-treated cells and the gal7Δ mutant. The synthesis of galactose-1-phosphate is essential to trigger the unfolded protein response under these conditions because the deletion of the galactokinase-encoding gene GAL1 completely abolishes unfolded protein response activation and galactose toxicity. Impairment of the unfolded protein response in both yeast models makes cells even more sensitive to galactose, unmasking its cytotoxic effect. These results indicate that endoplasmic reticulum stress is induced under galactosemic conditions and underscores the importance of the unfolded protein response pathway to cellular adaptation in these models of classic galactosemia.
Solvent Effects on Protein Folding/Unfolding
García, A. E.; Hillson, N.; Onuchic, J. N.
Pressure effects on the hydrophobic potential of mean force led Hummer et al. to postulate a model for pressure denaturation of proteins in which denaturation occurs by means of water penetration into the protein interior, rather than by exposing the protein hydrophobic core to the solvent --- commonly used to describe temperature denaturation. We study the effects of pressure in protein folding/unfolding kinetics in an off-lattice minimalist model of a protein in which pressure effects have been incorporated by means of the pair-wise potential of mean force of hydrophobic groups in water. We show that pressure slows down the kinetics of folding by decreasing the reconfigurational diffusion coefficient and moves the location of the folding transition state.
Comparison of intra-organellar chaperone capacity for dealing with stress-induced protein unfolding
Hageman, Jurre; Vos, Michel J.; van Waarde, Maria A. W. H.; Kampinga, Harm H.
2007-01-01
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
Plant transducers of the endoplasmic reticulum unfolded protein response
Iwata, Yuji; Koizumi, Nozomu
2012-01-01
The unfolded protein response (UPR) activates a set of genes to overcome accumulation of unfolded proteins in the endoplasmic reticulum (ER), a condition termed ER stress, and constitutes an essential part of ER protein quality control that ensures efficient maturation of secretory and membrane proteins in eukaryotes. Recent studies on Arabidopsis and rice identified the signaling pathway in which the ER membrane-localized ribonuclease IRE1 (inositol-requiring enzyme 1) catalyzes unconventional cytoplasmic splicing of mRNA, thereby producing the active transcription factor Arabidopsis bZIP60 (basic leucine zipper 60) and its ortholog in rice. Here we review recent findings identifying the molecular components of the plant UPR, including IRE1/bZIP60 and the membrane-bound transcription factors bZIP17 and bZIP28, and implicating its importance in several physiological phenomena such as pathogen response. © 2012 Elsevier Ltd.
Plant transducers of the endoplasmic reticulum unfolded protein response
Iwata, Yuji
2012-12-01
The unfolded protein response (UPR) activates a set of genes to overcome accumulation of unfolded proteins in the endoplasmic reticulum (ER), a condition termed ER stress, and constitutes an essential part of ER protein quality control that ensures efficient maturation of secretory and membrane proteins in eukaryotes. Recent studies on Arabidopsis and rice identified the signaling pathway in which the ER membrane-localized ribonuclease IRE1 (inositol-requiring enzyme 1) catalyzes unconventional cytoplasmic splicing of mRNA, thereby producing the active transcription factor Arabidopsis bZIP60 (basic leucine zipper 60) and its ortholog in rice. Here we review recent findings identifying the molecular components of the plant UPR, including IRE1/bZIP60 and the membrane-bound transcription factors bZIP17 and bZIP28, and implicating its importance in several physiological phenomena such as pathogen response. © 2012 Elsevier Ltd.
DEFF Research Database (Denmark)
Calcutta, Antonello; Jessen, Christian M; Behrens, Manja Annette
2012-01-01
induced by unfolding of an integral membrane protein, namely TFE-induced unfolding of KcsA solubilized by the n-dodecyl ß-d-maltoside (DDM) surfactant is investigated by the recently introduced GPS-NMR (Global Protein folding State mapping by multivariate NMR) (Malmendal et al., PlosONE 5, e10262 (2010......)) along with dynamic light scattering (DLS) and small-angle X-ray scattering (SAXS). GPS-NMR is used as a tool for fast analysis of the protein unfolding processes upon external perturbation, and DLS and SAXS are used for further structural characterization of the unfolding states. The combination allows...
A highly compliant protein native state with a spontaneous-like mechanical unfolding pathway
DEFF Research Database (Denmark)
Heiðarsson, Pétur Orri; Valpapuram, Immanuel; Camilloni, Carlo
2012-01-01
The mechanical properties of proteins and their force-induced structural changes play key roles in many biological processes. Previous studies have shown that natively folded proteins are brittle under tension, unfolding after small mechanical deformations, while partially folded intermediate...... states, such as molten globules, are compliant and can deform elastically a great amount before crossing the transition state barrier. Moreover, under tension proteins appear to unfold through a different sequence of events than during spontaneous unfolding. Here, we describe the response to force...... of the four-α-helix acyl-CoA binding protein (ACBP) in the low-force regime using optical tweezers and ratcheted molecular dynamics simulations. The results of our studies reveal an unprecedented mechanical behavior of a natively folded protein. ACBP displays an atypical compliance along two nearly orthogonal...
Wang, Tian; Chen, Jeannie
2014-10-17
Phototransduction is a G-protein signal transduction cascade that converts photon absorption to a change in current at the plasma membrane. Certain genetic mutations affecting the proteins in the phototransduction cascade cause blinding disorders in humans. Some of these mutations serve as a genetic source of "equivalent light" that activates the cascade, whereas other mutations lead to amplification of the light response. How constitutive phototransduction causes photoreceptor cell death is poorly understood. We showed that persistent G-protein signaling, which occurs in rod arrestin and rhodopsin kinase knock-out mice, caused a rapid and specific induction of the PERK pathway of the unfolded protein response. These changes were not observed in the cGMP-gated channel knock-out rods, an equivalent light condition that mimics light-stimulated channel closure. Thus transducin signaling, but not channel closure, triggers rapid cell death in light damage caused by constitutive phototransduction. Additionally, we show that in the albino light damage model cell death was not associated with increase in global protein ubiquitination or unfolded protein response induction. Taken together, these observations provide novel mechanistic insights into the cell death pathway caused by constitutive phototransduction and identify the unfolded protein response as a potential target for therapeutic intervention. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.
Stable intermediates determine proteins' primary unfolding sites in the presence of surfactants
DEFF Research Database (Denmark)
Petersen, Steen Vang; Andersen, Kell kleiner; Enghild, Jan J.
2009-01-01
Despite detailed knowledge of the overall structural changes and stoichiometries of surfactant binding, little is known about which protein regions constitute the preferred sites of attack for initial unfolding. Here we have exposed three proteins to limited proteolysis at anionic (SDS) and catio......Despite detailed knowledge of the overall structural changes and stoichiometries of surfactant binding, little is known about which protein regions constitute the preferred sites of attack for initial unfolding. Here we have exposed three proteins to limited proteolysis at anionic (SDS......) and cationic (DTAC) surfactant concentrations corresponding to specific conformational transitions, using the surfactant-robust broad-specificity proteases Savinase and Alcalase. Cleavage sites are identified by SDS-PAGE and N-terminal sequencing. We observe well-defined cleavage fragments, which suggest......, cleavage sites can be rationalized from the structure of the protein's folding transition state and the position of loops in the native state. Nevertheless, they are more sensitive to choice of surfactant and protease, probably reflecting a heterogeneous and fluctuating ensemble of partially unfolded...
Energy Technology Data Exchange (ETDEWEB)
Patananan, A.N.; Goheen, S.C.
2008-01-01
The adsorption and unfolding pathways of proteins on rigid surfaces are essential in numerous complex processes associated with biomedical engineering, nanotechnology, and chromatography. It is now well accepted that the kinetics of unfolding are characterized by chemical and physical interactions dependent on protein deformability and structure, as well as environmental pH, temperature, and surface chemistry. Although this fundamental process has broad implications in medicine and industry, little is known about the mechanism because of the atomic lengths and rapid time scales involved. Therefore, the unfolding kinetics of myoglobin, β-glucosidase, and ovalbumin were investigated by adsorbing the globular proteins to non-porous cationic polymer beads. The protein fractions were adsorbed at different residence times (0, 9, 10, 20, and 30 min) at near-physiological conditions using a gradient elution system similar to that in high-performance liquid chromatography. The elution profi les and retention times were obtained by ultraviolet/visible spectrophotometry. A decrease in recovery was observed with time for almost all proteins and was attributed to irreversible protein unfolding on the non-porous surfaces. These data, and those of previous studies, fi t a positively increasing linear trend between percent unfolding after a fi xed (9 min) residence time (71.8%, 31.1%, and 32.1% of myoglobin, β-glucosidase, and ovalbumin, respectively) and molecular weight. Of all the proteins examined so far, only myoglobin deviated from this trend with higher than predicted unfolding rates. Myoglobin also exhibited an increase in retention time over a wide temperature range (0°C and 55°C, 4.39 min and 5.74 min, respectively) whereas ovalbumin and β-glucosidase did not. Further studies using a larger set of proteins are required to better understand the physiological and physiochemical implications of protein unfolding kinetics. This study confi rms that surface
The Unfolded Protein Response in Homeostasis and Modulation of Mammalian Immune Cells.
Martins, Ana Sofia; Alves, Inês; Helguero, Luisa; Domingues, Maria Rosário; Neves, Bruno Miguel
2016-11-01
The endoplasmic reticulum (ER) plays important roles in eukaryotic protein folding and lipid biosynthesis. Several exogenous and endogenous cellular sources of stress can perturb ER homeostasis leading to the accumulation of unfolded proteins in the lumen. Unfolded protein accumulation triggers a signal-transduction cascade known as the unfolded protein response (UPR), an adaptive mechanism which aims to protect cells from protein aggregates and to restore ER functions. Further to this protective mechanism, in immune cells, UPR molecular effectors have been shown to participate in a wide range of biological processes such as cell differentiation, survival and immunoglobulin and cytokine production. Recent findings also highlight the involvement of the UPR machinery in the maturational program and antigen presentation capacities of dendritic cells. UPR is therefore a key element in immune system homeostasis with direct implications on both adaptive and innate immune responses. The present review summarizes the knowledge on the emerging roles of UPR signaling cascades in mammalian immune cells as well as the consequences of their dysregulation in relation to the pathogenesis of several diseases.
Žoldák, Gabriel; Jancura, Daniel; Sedlák, Erik
2017-06-01
Monitoring the fluorescence of proteins, particularly the fluorescence of intrinsic tryptophan residues, is a popular method often used in the analysis of unfolding transitions (induced by temperature, chemical denaturant, and pH) in proteins. The tryptophan fluorescence provides several suitable parameters, such as steady-state fluorescence intensity, apparent quantum yield, mean fluorescence lifetime, position of emission maximum that are often utilized for the observation of the conformational/unfolding transitions of proteins. In addition, the fluorescence intensities ratio at different wavelengths (usually at 330 nm and 350 nm) is becoming an increasingly popular parameter for the evaluation of thermal transitions. We show that, under certain conditions, the use of this parameter for the analysis of unfolding transitions leads to the incorrect determination of thermodynamic parameters characterizing unfolding transitions in proteins (e.g., melting temperature) and, hence, can compromise the hit identification during high-throughput drug screening campaigns. © 2017 The Protein Society.
Reversibility and two state behaviour in the thermal unfolding of oligomeric TIM barrel proteins.
Romero-Romero, Sergio; Costas, Miguel; Rodríguez-Romero, Adela; Alejandro Fernández-Velasco, D
2015-08-28
Temperature is one of the main variables that modulate protein function and stability. Thermodynamic studies of oligomeric proteins, the dominant protein natural form, have been often hampered because irreversible aggregation and/or slow reactions are common. There are no reports on the reversible equilibrium thermal unfolding of proteins composed of (β/α)8 barrel subunits, albeit this "TIM barrel" topology is one of the most abundant and versatile in nature. We studied the eponymous TIM barrel, triosephosphate isomerase (TIM), belonging to five species of different bacterial taxa. All of them were found to be catalytically efficient dimers. The three-dimensional structure of four enzymes was solved at high/medium resolution. Irreversibility and kinetic control were observed in the thermal unfolding of two TIMs, while for the other three the thermal unfolding was found to follow a two-state equilibrium reversible process. Shifts in the global stability curves of these three proteins are related to the organismal temperature range of optimal growth and modulated by variations in maximum stability temperature and in the enthalpy change at that temperature. Reversibility appears to correlate with the low isoelectric point, the absence of a residual structure in the unfolded state, small cavity volume in the native state, low conformational stability and a low melting temperature. Furthermore, the strong coupling between dimer dissociation and monomer unfolding may reduce aggregation and favour reversibility. It is therefore very thought-provoking to find that a common topological ensemble, such as the TIM barrel, can unfold/refold in the Anfinsen way, i.e. without the help of the cellular machinery.
Amyloid protein unfolding and insertion kinetics on neuronal membrane mimics
Qiu, Liming; Buie, Creighton; Vaughn, Mark; Cheng, Kwan
2010-03-01
Atomistic details of beta-amyloid (Aβ ) protein unfolding and lipid interaction kinetics mediated by the neuronal membrane surface are important for developing new therapeutic strategies to prevent and cure Alzheimer's disease. Using all-atom MD simulations, we explored the early unfolding and insertion kinetics of 40 and 42 residue long Aβ in binary lipid mixtures with and without cholesterol that mimic the cholesterol-depleted and cholesterol-enriched lipid nanodomains of neurons. The protein conformational transition kinetics was evaluated from the secondary structure profile versus simulation time plot. The extent of membrane disruption was examined by the calculated order parameters of lipid acyl chains and cholesterol fused rings as well as the density profiles of water and lipid headgroups at defined regions across the lipid bilayer from our simulations. Our results revealed that both the cholesterol content and the length of the protein affect the protein-insertion and membrane stability in our model lipid bilayer systems.
PPARγ Ligand-Induced Unfolded Protein Responses in Monocytes
African Journals Online (AJOL)
High levels of oxLDL lead to cell dysfunction and apoptosis, a phenomenon known as lipotoxicity. Disturbing endoplasmic reticulum (ER) function results in ER stress and unfolded protein response (UPR), which tends to restore ER homeostasis but switches to apoptosis when ER stress is prolonged. In the present study the ...
PPARγ Ligand-Induced Unfolded Protein Responses in Monocytes ...
African Journals Online (AJOL)
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Disturbing endoplasmic reticulum (ER) function results in ER stress and unfolded protein response. (UPR), which tends to ... in mnocyte/macrophage cell lines as evident of the activation/up-regulation of ER stress/UPR genes. Cholesterol does not seem to exert ... inflammation (Tiwari et al., 2008). One prominent feature of ...
Unfolded protein response and activated degradative pathways regulation in GNE myopathy.
Directory of Open Access Journals (Sweden)
Honghao Li
Full Text Available Although intracellular beta amyloid (Aβ accumulation is known as an early upstream event in the degenerative course of UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (GNE myopathy, the process by which Aβdeposits initiate various degradative pathways, and their relationship have not been fully clarified. We studied the possible secondary responses after amyloid beta precursor protein (AβPP deposition including unfolded protein response (UPR, ubiquitin proteasome system (UPS activation and its correlation with autophagy system. Eight GNE myopathy patients and five individuals with normal muscle morphology were included in this study. We performed immunofluorescence and immunoblotting to investigate the expression of AβPP, phosphorylated tau (p-tau and endoplasmic reticulum molecular chaperones. Proteasome activities were measured by cleavage of fluorogenic substrates. The expression of proteasome subunits and linkers between proteasomal and autophagy systems were also evaluated by immunoblotting and relative quantitative real-time RT-PCR. Four molecular chaperones, glucose-regulated protein 94 (GRP94, glucose-regulated protein 78 (GRP78, calreticulin and calnexin and valosin containing protein (VCP were highly expressed in GNE myopathy. 20S proteasome subunits, three main proteasome proteolytic activities, and the factors linking UPS and autophagy system were also increased. Our study suggests that AβPP deposition results in endoplasmic reticulum stress (ERS and highly expressed VCP deliver unfolded proteins from endoplasmic reticulum to proteosomal system which is activated in endoplasmic reticulum associated degradation (ERAD in GNE myopathy. Excessive ubiquitinated unfolded proteins are exported by proteins that connect UPS and autophagy to autophagy system, which is activated as an alternative pathway for degradation.
Motional properties of unfolded ubiquitin: a model for a random coil protein
Energy Technology Data Exchange (ETDEWEB)
Wirmer, Julia [Johann Wolfgang GoeUniversityFrankfurt, Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance (Germany); Peti, Wolfgang [Brown University, Department of Molecular Pharmacology, Physiology and Biotechnology (United States); Schwalbe, Harald [Johann Wolfgang GoeUniversityFrankfurt, Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance (Germany)], E-mail: schwalbe@nmr.uni-frankfurt.de
2006-07-15
The characterization of unfolded states of proteins has recently attracted considerable interest, as the residual structure present in these states may play a crucial role in determining their folding and misfolding behavior. Here, we investigated the dynamics in the denatured state of ubiquitin in 8 M urea at pH2. Under these conditions, ubiquitin does not have any detectable local residual structure, and uniform {sup 15}N relaxation rates along the sequence indicate the absence of motional restrictions caused by residual secondary structure and/or long-range interactions. A comparison of different models to predict relaxation data in unfolded proteins suggests that the subnanosecond dynamics in unfolded states depend on segmental motions only and do not show a dependence on the residue type but for proline and glycine residues.
Oxidative stress impairs the heat stress response and delays unfolded protein recovery.
Directory of Open Access Journals (Sweden)
Masaaki Adachi
2009-11-01
Full Text Available 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.Pretreatment of hydrogen peroxide (H(2O(2 specifically and highly sensitized cells to heat stress, and enhanced loss of mitochondrial membrane potential. H(2O(2 exposure impaired the HSP40/HSP70 induction as heat shock response (HSR and the unfolded protein recovery, and enhanced eIF2alpha phosphorylation and/or XBP1 splicing, land marks of ER stress. These H(2O(2-mediated effects mimicked enhanced heat sensitivity in HSF1 knockdown or knockout cells. Importantly, thermal preconditioning blocked H(2O(2-mediated inhibitory effects on refolding activity and rescued HSF1 +/+ MEFs, but neither blocked the effects nor rescued HSF1 -/- MEFs. These data strongly suggest that inhibition of HSR and refolding activity is crucial for H(2O(2-mediated enhanced heat sensitivity.H(2O(2 blocks HSR and refolding activity under heat stress, thereby leading to insufficient quality control and enhancing ER stress. These uncontrolled stress responses may enhance cell death. Our data thus highlight oxidative stress as a crucial factor affecting heat tolerance.
Malhotra, Pooja; Udgaonkar, Jayant B
2014-06-10
A protein unfolding reaction usually appears to be so dominated by a large free energy barrier that identifying and characterizing high-energy intermediates and, hence, dissecting the unfolding reaction into multiple structural transitions have proven to be a challenge. In particular, it has been difficult to identify any detected high-energy intermediate with the dry (DMG) and wet (WMG) molten globules that have been implicated in the unfolding reactions of at least some proteins. In this study, a native-state thiol labeling methodology was used to identify high-energy intermediates, as well as to delineate the barriers to the disruption of side chain packing interactions and to site-specific solvent exposure in different regions of the small protein, single-chain monellin (MNEI). Labeling studies of four single-cysteine-containing variants of MNEI have identified three high-energy intermediates, populated to very low extents under nativelike conditions. A significant dispersion in the opening rates of the cysteine side chains has allowed multiple steps, leading to the loss of side chain packing, to be resolved temporally. A detailed structural analysis of the positions of the four cysteine residue positions, which are buried to different depths within the protein, has suggested a direct correlation with the structure of a DMG, detected in previous studies. It is observed that side chain packing within the core of the protein is maintained, while that at the surface is disrupted, in the DMG. The core of the protein becomes solvent-exposed only in a WMG populated after the rate-limiting step of unfolding at high denaturant concentrations.
Inter-regulation of the unfolded protein response and auxin signaling
Czech Academy of Sciences Publication Activity Database
Chen, Y.N.; Aung, K.; Rolčík, Jakub; Walicki, K.; Friml, J.; Brandizzi, F.
2014-01-01
Roč. 77, č. 1 (2014), s. 97-107 ISSN 0960-7412 Institutional support: RVO:61389030 Keywords : endoplasmic reticulum stress * unfolded protein response * auxin response Subject RIV: ED - Physiology Impact factor: 5.972, year: 2014
Lighezan, Liliana; Georgieva, Ralitsa; Neagu, Adrian
2016-09-01
Surface layer (S-layer) proteins have been identified in the cell envelope of many organisms, such as bacteria and archaea. They self-assemble, forming monomolecular crystalline arrays. Isolated S-layer proteins are able to recrystallize into regular lattices, which proved useful in biotechnology. Here we investigate the structure and thermal unfolding of the S-layer protein isolated from Lactobacillus salivarius 16 strain of human origin. Using circular dichroism (CD) spectroscopy, and the software CDSSTR from DICHROWEB, CONTINLL from CDPro, as well as CDNN, we assess the fractions of the protein's secondary structural elements at temperatures ranging between 10 and 90 °C, and predict the tertiary class of the protein. To study the thermal unfolding of the protein, we analyze the temperature dependence of the CD signal in the far- and near-UV domains. Fitting the experimental data by two- and three-state models of thermal unfolding, we infer the midpoint temperatures, the temperature dependence of the changes in Gibbs free energy, enthalpy, and entropy of the unfolding transitions in standard conditions, and the temperature dependence of the equilibrium constant. We also estimate the changes in heat capacity at constant pressure in standard conditions. The results indicate that the thermal unfolding of the S-layer protein from L. salivarius is highly cooperative, since changes in the secondary and tertiary structures occur simultaneously. The thermodynamic analysis predicts a "cold" transition, at about -3 °C, of both the secondary and tertiary structures. Our findings may be important for the use of S-layer proteins in biotechnology and in biomedical applications.
Microsecond simulations of the folding/unfolding thermodynamics of the Trp-cage mini protein
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 μs per replica timescale. The total simulation time employed in the calculations exceeds 100 μs. 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, ΔG(P,T), over the whole region of temperature and pressures sampled in the simulations. The ΔG(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
The structural basis of urea-induced protein unfolding in β-catenin
Wang, Chao; Chen, Zhongzhou; Hong, Xia; Ning, Fangkun; Liu, Haolin; Zang, Jianye; Yan, Xiaoxue; Kemp, Jennifer; Musselman, Catherine A.; Kutateladze, Tatinna G.; Zhao, Rui; Jiang, Chengyu; Zhang, Gongyi
2014-01-01
Although urea and guanidine hydrochloride are commonly used to denature proteins, the molecular underpinnings of this process have remained unclear for a century. To address this question, crystal structures of β-catenin were determined at various urea concentrations. These structures contained at least 105 unique positions that were occupied by urea molecules, each of which interacted with the protein primarily via hydrogen bonds. Hydrogen-bond competition experiments showed that the denaturing effects of urea were neutralized when polyethylene glycol was added to the solution. These data suggest that urea primarily causes proteins to unfold by competing and disrupting hydrogen bonds in proteins. Moreover, circular-dichroism spectra and nuclear magnetic resonance (NMR) analysis revealed that a similar mechanism caused protein denaturation in the absence of urea at pH levels greater than 12. Taken together, the results led to the conclusion that the disruption of hydrogen bonds is a general mechanism of unfolding induced by urea, high pH and potentially other denaturing agents such as guanidine hydrochloride. Traditionally, the disruption of hydrophobic interactions instead of hydrogen bonds has been thought to be the most important cause of protein denaturation. PMID:25372676
Directory of Open Access Journals (Sweden)
Deiana Antonio
2010-04-01
Full Text Available Abstract Background Natively unfolded proteins lack a well defined three dimensional structure but have important biological functions, suggesting a re-assignment of the structure-function paradigm. To assess that a given protein is natively unfolded requires laborious experimental investigations, then reliable sequence-only methods for predicting whether a sequence corresponds to a folded or to an unfolded protein are of interest in fundamental and applicative studies. Many proteins have amino acidic compositions compatible both with the folded and unfolded status, and belong to a twilight zone between order and disorder. This makes difficult a dichotomic classification of protein sequences into folded and natively unfolded ones. In this work we propose an operational method to identify proteins belonging to the twilight zone by combining into a consensus score good performing single predictors of folding. Results In this methodological paper dichotomic folding indexes are considered: hydrophobicity-charge, mean packing, mean pairwise energy, Poodle-W and a new global index, that is called here gVSL2, based on the local disorder predictor VSL2. The performance of these indexes is evaluated on different datasets, in particular on a new dataset composed by 2369 folded and 81 natively unfolded proteins. Poodle-W, gVSL2 and mean pairwise energy have good performance and stability in all the datasets considered and are combined into a strictly unanimous combination score SSU, that leaves proteins unclassified when the consensus of all combined indexes is not reached. The unclassified proteins: i belong to an overlap region in the vector space of amino acidic compositions occupied by both folded and unfolded proteins; ii are composed by approximately the same number of order-promoting and disorder-promoting amino acids; iii have a mean flexibility intermediate between that of folded and that of unfolded proteins. Conclusions Our results show that
β-sheet-like formation during the mechanical unfolding of prion protein
Tao, Weiwei; Yoon, Gwonchan; Cao, Penghui; Eom, Kilho; Park, Harold S.
2015-09-01
Single molecule experiments and simulations have been widely used to characterize the unfolding and folding pathways of different proteins. However, with few exceptions, these tools have not been applied to study prion protein, PrPC, whose misfolded form PrPSc can induce a group of fatal neurodegenerative diseases. Here, we apply novel atomistic modeling based on potential energy surface exploration to study the constant force unfolding of human PrP at time scales inaccessible with standard molecular dynamics. We demonstrate for forces around 100 pN, prion forms a stable, three-stranded β-sheet-like intermediate configuration containing residues 155-214 with a lifetime exceeding hundreds of nanoseconds. A mutant without the disulfide bridge shows lower stability during the unfolding process but still forms the three-stranded structure. The simulations thus not only show the atomistic details of the mechanically induced structural conversion from the native α-helical structure to the β-rich-like form but also lend support to the structural theory that there is a core of the recombinant PrP amyloid, a misfolded form reported to induce transmissible disease, mapping to C-terminal residues ≈160-220.
β-sheet-like formation during the mechanical unfolding of prion protein
Energy Technology Data Exchange (ETDEWEB)
Tao, Weiwei; Cao, Penghui; Park, Harold S., E-mail: parkhs@bu.edu [Department of Mechanical Engineering, Boston University, Boston, Massachusetts 02215 (United States); Yoon, Gwonchan [Department of Mechanical Engineering, Boston University, Boston, Massachusetts 02215 (United States); Department of Mechanical Engineering, Korea University, Seoul 136-701 (Korea, Republic of); Eom, Kilho [Biomechanics Laboratory, College of Sport Science, Sungkyunkwan University, Suwon 16419 (Korea, Republic of)
2015-09-28
Single molecule experiments and simulations have been widely used to characterize the unfolding and folding pathways of different proteins. However, with few exceptions, these tools have not been applied to study prion protein, PrP{sup C}, whose misfolded form PrP{sup Sc} can induce a group of fatal neurodegenerative diseases. Here, we apply novel atomistic modeling based on potential energy surface exploration to study the constant force unfolding of human PrP at time scales inaccessible with standard molecular dynamics. We demonstrate for forces around 100 pN, prion forms a stable, three-stranded β-sheet-like intermediate configuration containing residues 155-214 with a lifetime exceeding hundreds of nanoseconds. A mutant without the disulfide bridge shows lower stability during the unfolding process but still forms the three-stranded structure. The simulations thus not only show the atomistic details of the mechanically induced structural conversion from the native α-helical structure to the β-rich-like form but also lend support to the structural theory that there is a core of the recombinant PrP amyloid, a misfolded form reported to induce transmissible disease, mapping to C-terminal residues ≈160-220.
β-sheet-like formation during the mechanical unfolding of prion protein
International Nuclear Information System (INIS)
Tao, Weiwei; Cao, Penghui; Park, Harold S.; Yoon, Gwonchan; Eom, Kilho
2015-01-01
Single molecule experiments and simulations have been widely used to characterize the unfolding and folding pathways of different proteins. However, with few exceptions, these tools have not been applied to study prion protein, PrP C , whose misfolded form PrP Sc can induce a group of fatal neurodegenerative diseases. Here, we apply novel atomistic modeling based on potential energy surface exploration to study the constant force unfolding of human PrP at time scales inaccessible with standard molecular dynamics. We demonstrate for forces around 100 pN, prion forms a stable, three-stranded β-sheet-like intermediate configuration containing residues 155-214 with a lifetime exceeding hundreds of nanoseconds. A mutant without the disulfide bridge shows lower stability during the unfolding process but still forms the three-stranded structure. The simulations thus not only show the atomistic details of the mechanically induced structural conversion from the native α-helical structure to the β-rich-like form but also lend support to the structural theory that there is a core of the recombinant PrP amyloid, a misfolded form reported to induce transmissible disease, mapping to C-terminal residues ≈160-220
Methods for monitoring endoplasmic reticulum stress and the unfolded protein response.
LENUS (Irish Health Repository)
Samali, Afshin
2010-01-01
The endoplasmic reticulum (ER) is the site of folding of membrane and secreted proteins in the cell. Physiological or pathological processes that disturb protein folding in the endoplasmic reticulum cause ER stress and activate a set of signaling pathways termed the Unfolded Protein Response (UPR). The UPR can promote cellular repair and sustained survival by reducing the load of unfolded proteins through upregulation of chaperones and global attenuation of protein synthesis. Research into ER stress and the UPR continues to grow at a rapid rate as many new investigators are entering the field. There are also many researchers not working directly on ER stress, but who wish to determine whether this response is activated in the system they are studying: thus, it is important to list a standard set of criteria for monitoring UPR in different model systems. Here, we discuss approaches that can be used by researchers to plan and interpret experiments aimed at evaluating whether the UPR and related processes are activated. We would like to emphasize that no individual assay is guaranteed to be the most appropriate one in every situation and strongly recommend the use of multiple assays to verify UPR activation.
Methods for Monitoring Endoplasmic Reticulum Stress and the Unfolded Protein Response
Directory of Open Access Journals (Sweden)
Afshin Samali
2010-01-01
Full Text Available The endoplasmic reticulum (ER is the site of folding of membrane and secreted proteins in the cell. Physiological or pathological processes that disturb protein folding in the endoplasmic reticulum cause ER stress and activate a set of signaling pathways termed the Unfolded Protein Response (UPR. The UPR can promote cellular repair and sustained survival by reducing the load of unfolded proteins through upregulation of chaperones and global attenuation of protein synthesis. Research into ER stress and the UPR continues to grow at a rapid rate as many new investigators are entering the field. There are also many researchers not working directly on ER stress, but who wish to determine whether this response is activated in the system they are studying: thus, it is important to list a standard set of criteria for monitoring UPR in different model systems. Here, we discuss approaches that can be used by researchers to plan and interpret experiments aimed at evaluating whether the UPR and related processes are activated. We would like to emphasize that no individual assay is guaranteed to be the most appropriate one in every situation and strongly recommend the use of multiple assays to verify UPR activation.
The Unfolded Protein Response in Chronic Obstructive Pulmonary Disease.
Kelsen, Steven G
2016-04-01
Accumulation of nonfunctional and potentially cytotoxic, misfolded proteins in chronic obstructive pulmonary disease (COPD) is believed to contribute to lung cell apoptosis, inflammation, and autophagy. Because of its fundamental role as a quality control system in protein metabolism, the "unfolded protein response" (UPR) is of potential importance in the pathogenesis of COPD. The UPR comprises a series of transcriptional, translational, and post-translational processes that decrease protein synthesis while enhancing protein folding capacity and protein degradation. Several studies have suggested that the UPR contributes to lung cell apoptosis and lung inflammation in at least some subjects with human COPD. However, information on the prevalence of the UPR in subjects with COPD, the lung cells that manifest a UPR, and the role of the UPR in the pathogenesis of COPD is extremely limited and requires additional study.
Osmolyte Effects on the Unfolding Pathway of β-Lactoglobulin
International Nuclear Information System (INIS)
Meng Wei; Pan Hai; Qin Meng; Cao Yi; Wang Wei
2013-01-01
There are large amounts of osmolytes inside cells, which impact many physiological processes by complicated mechanisms. The osmolyte effects on the stability and folding of proteins have been studied in detail using simple two-state folding proteins. However, many important functional proteins fold in complex pathways involving various intermediates. Little is known about the osmolyte effects on the folding and unfolding of these proteins. It is noted that β-lactoglobulin (BLG) is an example of such proteins, whose unfolding involves an obvious intermediate state. Using equilibrium chemical denaturation and stopped-flow kinetics, we investigate the unfolding of BLG in the presence of different osmolytes, e.g., glycerol, ethylene glycol (EG) and poly(ethylene glycol)400 (PEG400). It is found that all these osmolytes can stabilize the unfolding intermediate by modulating the relative unfolding kinetics of the native and the intermediate states. The stabilization effects are similar for EG and PEG400 but distinct for glycerol. Since the unfolding intermediates of many proteins are directly related to protein misfolding diseases, evaluation of the osmolyte effects for the unfolding of these proteins in vitro should be beneficial for the understanding of the occurrence of the related diseases in vivo
Hentilä, Jaakko; Ahtiainen, Juha P; Paulsen, Gøran; Raastad, Truls; Häkkinen, Keijo; Mero, Antti A; Hulmi, Juha J
2018-04-02
Autophagy and unfolded protein response (UPR) appear to be important for skeletal muscle homeostasis and may be altered by exercise. Our aim was to investigate the effects of resistance exercise and training on indicators of UPR and autophagy in healthy untrained young men (n = 12, 27 ± 4 years) and older men (n = 8, 61 ± 6 years) as well as in resistance-trained individuals (n = 15, 25 ± 5 years). Indicators of autophagy and UPR were investigated from the muscle biopsies after a single resistance exercise bout and after 21 weeks of resistance training. Lipidated LC3II as an indicator of autophagosome content increased at 48 hours post resistance exercise (P resistance-training period (P resistance exercise in untrained young and older men (P resistance-training period regardless of age. UPR was unchanged within the first few hours after the resistance exercise bout regardless of the training status. Changes in autophagy and UPR ER indicators did not correlate with a resistance-training-induced increase in muscle strength and size. Autophagosome content is increased by resistance training in young previously untrained men, but this response may be blunted by aging. However, unfolded protein response is induced by an unaccustomed resistance exercise bout in a delayed manner regardless of age. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
DEFF Research Database (Denmark)
Mörck, Catarina; Elmelund-Præstekær, Louise Cathrine Braun; Kurth, Caroline
2009-01-01
of lipid moieties for protein prenylation. The nematode Caenorhabditis elegans possesses a mevalonate pathway that lacks the branch leading to cholesterol synthesis, and thus represents an ideal organism to specifically study the noncholesterol roles of the pathway. Inhibiting HMG-CoA reductase in C....... elegans using statins or RNAi leads to developmental arrest and loss of membrane association of a GFP-based prenylation reporter. The unfolded protein response (UPR) is also strongly activated, suggesting that impaired prenylation of small GTPases leads to the accumulation of unfolded proteins and ER...... and fatty acid composition were unaffected in statin-treated worms, even though they showed reduced staining with Nile red. We conclude that inhibitors of HMG-CoA reductase or of farnesyl transferases induce the UPR by inhibiting the prenylation of M57.2 substrates, resulting in developmental arrest in C...
Newcomer, Rebecca L.; Fraser, LaTasha C.R.; Teschke, Carolyn M.; Alexandrescu, Andrei T.
2015-01-01
The I-domain is an insertion domain of the bacteriophage P22 coat protein that drives rapid folding and accounts for over half of the stability of the full-length protein. We sought to determine the role of hydrogen bonds (H-bonds) in the unfolding of the I-domain by examining 3JNC’ couplings transmitted through H-bonds, the temperature and urea-concentration dependence of 1HN and 15N chemical shifts, and native-state hydrogen exchange at urea concentrations where the domain is predominantly folded. The native-state hydrogen-exchange data suggest that the six-stranded β-barrel core of the I-domain is more stable against unfolding than a smaller subdomain comprised of a short α-helix and three-stranded β-sheet. H-bonds, separately determined from solvent protection and 3JNC’ H-bond couplings, are identified with an accuracy of 90% by 1HN temperature coefficients. The accuracy is improved to 95% when 15N temperature coefficients are also included. In contrast, the urea dependence of 1HN and 15N chemical shifts is unrelated to H-bonding. The protein segments with the largest chemical-shift changes in the presence of urea show curved or sigmoidal titration curves suggestive of direct urea binding. Nuclear Overhauser effects to urea for these segments are also consistent with specific urea-binding sites in the I-domain. Taken together, the results support a mechanism of urea unfolding in which denaturant binds to distinct sites in the I-domain. Disordered segments bind urea more readily than regions in stable secondary structure. The locations of the putative urea-binding sites correlate with the lower stability of the structure against solvent exchange, suggesting that partial unfolding of the structure is related to urea accessibility. PMID:26682823
Digital force-feedback for protein unfolding experiments using atomic force microscopy
Bippes, Christian A.; Janovjak, Harald; Kedrov, Alexej; Muller, Daniel J.
2007-01-01
Since its invention in the 1990s single-molecule force spectroscopy has been increasingly applied to study protein (un-)folding, cell adhesion, and ligand-receptor interactions. In most force spectroscopy studies, the cantilever of an atomic force microscope (AFM) is separated from a surface at a constant velocity, thus applying an increasing force to folded bio-molecules or bio-molecular bonds. Recently, Fernandez and co-workers introduced the so-called force-clamp technique. Single proteins were subjected to a defined constant force allowing their life times and life time distributions to be directly measured. Up to now, the force-clamping was performed by analogue PID controllers, which require complex additional hardware and might make it difficult to combine the force-feedback with other modes such as constant velocity. These points may be limiting the applicability and versatility of this technique. Here we present a simple, fast, and all-digital (software-based) PID controller that yields response times of a few milliseconds in combination with a commercial AFM. We demonstrate the performance of our feedback loop by force-clamp unfolding of single Ig27 domains of titin and the membrane proteins bacteriorhodopsin (BR) and the sodium/proton antiporter NhaA.
Digital force-feedback for protein unfolding experiments using atomic force microscopy
International Nuclear Information System (INIS)
Bippes, Christian A; Janovjak, Harald; Kedrov, Alexej; Muller, Daniel J
2007-01-01
Since its invention in the 1990s single-molecule force spectroscopy has been increasingly applied to study protein (un-)folding, cell adhesion, and ligand-receptor interactions. In most force spectroscopy studies, the cantilever of an atomic force microscope (AFM) is separated from a surface at a constant velocity, thus applying an increasing force to folded bio-molecules or bio-molecular bonds. Recently, Fernandez and co-workers introduced the so-called force-clamp technique. Single proteins were subjected to a defined constant force allowing their life times and life time distributions to be directly measured. Up to now, the force-clamping was performed by analogue PID controllers, which require complex additional hardware and might make it difficult to combine the force-feedback with other modes such as constant velocity. These points may be limiting the applicability and versatility of this technique. Here we present a simple, fast, and all-digital (software-based) PID controller that yields response times of a few milliseconds in combination with a commercial AFM. We demonstrate the performance of our feedback loop by force-clamp unfolding of single Ig27 domains of titin and the membrane proteins bacteriorhodopsin (BR) and the sodium/proton antiporter NhaA
Thermal unfolding of barstar and the properties of interfacial water around the unfolded forms
Energy Technology Data Exchange (ETDEWEB)
Pal, Somedatta; Bandyopadhyay, Sanjoy, E-mail: sanjoy@chem.iitkgp.ernet.in [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 α-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.
Inhibition of the Unfolded Protein Response Mechanism Prevents Cardiac Fibrosis.
Directory of Open Access Journals (Sweden)
Jody Groenendyk
Full Text Available Cardiac fibrosis attributed to excessive deposition of extracellular matrix proteins is a major cause of heart failure and death. Cardiac fibrosis is extremely difficult and challenging to treat in a clinical setting due to lack of understanding of molecular mechanisms leading to cardiac fibrosis and effective anti-fibrotic therapies. The objective in this study was to examine whether unfolded protein response (UPR pathway mediates cardiac fibrosis and whether a pharmacological intervention to modulate UPR can prevent cardiac fibrosis and preserve heart function.We demonstrate here that the mechanism leading to development of fibrosis in a mouse with increased expression of calreticulin, a model of heart failure, stems from impairment of endoplasmic reticulum (ER homeostasis, transient activation of the unfolded protein response (UPR pathway and stimulation of the TGFβ1/Smad2/3 signaling pathway. Remarkably, sustained pharmacologic inhibition of the UPR pathway by tauroursodeoxycholic acid (TUDCA is sufficient to prevent cardiac fibrosis, and improved exercise tolerance.We show that the mechanism leading to development of fibrosis in a mouse model of heart failure stems from transient activation of UPR pathway leading to persistent remodelling of cardiac tissue. Blocking the activation of the transiently activated UPR pathway by TUDCA prevented cardiac fibrosis, and improved prognosis. These findings offer a window for additional interventions that can preserve heart function.
Thermal unfolding of a Ca- and Lanthanide-binding protein
Energy Technology Data Exchange (ETDEWEB)
Fahmy, Karim [Helmholtz-Zentrum Dresden-Rossendorf e.V., Dresden (Germany). Biophysics; Goettfert, M. [Technische Univ. Dresden (Germany); Knoeppel, J.
2017-06-01
The MIIA (metal ion-induced autocleavage)-domain of the protein Vic001052 from the pathogen Vibrio coralliilyticus, comprises 173 amino acids and exhibits Ca-dependent autoproteolytic activity. It shows homology to nodulation proteins which are secreted by Rhizobiacea into plant host cells where they exert Ca-dependent functions. We have studied the structural and energetic aspects of metal protein interactions of the MIIA domain which appear attractive for engineering metal-binding synthetic peptides. Using a non-cleavable MIIA domain construct, we detected very similar structural changes upon binding to Ca{sup 2+} and Eu{sup 3+}. The thermal denaturation of the Ca-bound state was studied by circular dichroism spectroscopy. The metal-bound folded state unfolds reversibly into an unstructured metal-free state similar to the metal-free state at room temperature.
Respiratory epithelial cell responses to cigarette smoke: the unfolded protein response.
Kelsen, Steven G
2012-12-01
Cigarette smoking exposes the respiratory epithelium to highly toxic, reactive oxygen nitrogen species which damage lung proteins in the endoplasmic reticulum (ER), the cell organelle in which all secreted and membrane proteins are processed. Accumulation of damaged or misfolded proteins in the ER, a condition termed ER stress, activates a complex cellular process termed the unfolded protein responses (UPR). The UPR acts to restore cellular protein homeostasis by regulating all aspects of protein metabolism including: protein translation and syntheses; protein folding; and protein degradation. However, activation of the UPR may also induce signaling pathways which induce inflammation and cell apoptosis. This review discusses the role of UPR in the respiratory epithelial cell response to cigarette smoke and the pathogenesis of lung diseases like COPD. Copyright © 2012 Elsevier Ltd. All rights reserved.
Ham, Hyeilin; Woolery, Andrew R.; Tracy, Charles; Stenesen, Drew; Krämer, Helmut; Orth, Kim
2014-01-01
Drosophila Fic (dFic) mediates AMPylation, a covalent attachment of adenosine monophosphate (AMP) from ATP to hydroxyl side chains of protein substrates. Here, we identified the endoplasmic reticulum (ER) chaperone BiP as a substrate for dFic and mapped the modification site to Thr-366 within the ATPase domain. The level of AMPylated BiP in Drosophila S2 cells is high during homeostasis, whereas the level of AMPylated BiP decreases upon the accumulation of misfolded proteins in the ER. Both dFic and BiP are transcriptionally activated upon ER stress, supporting the role of dFic in the unfolded protein response pathway. The inactive conformation of BiP is the preferred substrate for dFic, thus endorsing a model whereby AMPylation regulates the function of BiP as a chaperone, allowing acute activation of BiP by deAMPylation during an ER stress response. These findings not only present the first substrate of eukaryotic AMPylator but also provide a target for regulating the unfolded protein response, an emerging avenue for cancer therapy. PMID:25395623
Unfolded protein response in filamentous fungi-implications in biotechnology.
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.
Directory of Open Access Journals (Sweden)
J. M. Dick
2006-01-01
Full Text Available Thermodynamic calculations can be used to quantify environmental constraints on the speciation of proteins, such as the pH and temperature dependence of ionization state, and the relative chemical stabilities of proteins in different biogeochemical settings. These calculations depend in part on values of the standard molal Gibbs energies of proteins and their ionization reactions as a function of temperature and pressure. Because these values are not generally available, we calculated values of the standard molal thermodynamic properties at 25°C and 1 bar as well as the revised Helgeson-Kirkham-Flowers equations of state parameters of neutral and charged zwitterionic reference model compounds including aqueous amino acids, polypeptides, and unfolded proteins. The experimental calorimetric and volumetric data for these species taken from the literature were combined with group additivity algorithms to calculate the properties and parameters of neutral and ionized sidechain and backbone groups in unfolded proteins. The resulting set of group contributions enables the calculation of the standard molal Gibbs energy, enthalpy, entropy, isobaric heat capacity, volume, and isothermal compressibility of unfolded proteins in a range of proton ionization states to temperatures and pressures exceeding 100°C and 1000 bar. This approach provides a useful frame of reference for thermodynamic studies of protein folding and complexation reactions. It can also be used to assign provisional values of the net charge and Gibbs energy of ionized proteins as a function of temperature and pH. Using these values, an Eh-pH diagram for a reaction representing the speciation of extracellular proteins from Pyrococcus furiosus and Bacillus subtilis was generated. The predicted predominance limits of these proteins correspond with the different electrochemical conditions of hydrothermal vents and soils. More comprehensive calculations of this kind may reveal pervasive
The Unfolded Protein Response and Cell Fate Control.
Hetz, Claudio; Papa, Feroz R
2018-01-18
The secretory capacity of a cell is constantly challenged by physiological demands and pathological perturbations. To adjust and match the protein-folding capacity of the endoplasmic reticulum (ER) to changing secretory needs, cells employ a dynamic intracellular signaling pathway known as the unfolded protein response (UPR). Homeostatic activation of the UPR enforces adaptive programs that modulate and augment key aspects of the entire secretory pathway, whereas maladaptive UPR outputs trigger apoptosis. Here, we discuss recent advances into how the UPR integrates information about the intensity and duration of ER stress stimuli in order to control cell fate. These findings are timely and significant because they inform an evolving mechanistic understanding of a wide variety of human diseases, including diabetes mellitus, neurodegeneration, and cancer, thus opening up the potential for new therapeutic modalities to treat these diverse diseases. Copyright © 2017 Elsevier Inc. All rights reserved.
Judith A. Smith; Judith A. Smith
2018-01-01
Protein folding in the endoplasmic reticulum (ER) is an essential cell function. To safeguard this process in the face of environmental threats and internal stressors, cells mount an evolutionarily conserved response known as the unfolded protein response (UPR). Invading pathogens induce cellular stress that impacts protein folding, thus the UPR is well situated to sense danger and contribute to immune responses. Cytokines (inflammatory cytokines and interferons) critically mediate host defen...
The unfolded protein response is required for dendrite morphogenesis
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
Directory of Open Access Journals (Sweden)
Cheng Ji
2015-06-01
Full Text Available 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.
Energy Technology Data Exchange (ETDEWEB)
Furuno, Taiji, E-mail: t_furuno@a8.keio.jp
2016-04-01
A Langmuir monolayer of carbonic anhydrase (CA) unfolded at an air/water interface was transferred onto the hydrophobic surface of a silicon wafer by means of the Langmuir–Schaefer technique. The transferred CA film was biotinylated and was incubated in a streptavidin (SAv) solution to obtain a densely packed SAv layer by biotin–SAv linkage. Biotinylated proteins including ferritin, catalase, alcohol dehydrogenase, and carbonic anhydrase were incubated with the SAv layer and binding of these proteins was examined by atomic force microscopy. High-density binding of the biotinylated proteins was observed, whereas the amount of adsorbed non-biotinylated proteins was low or negligible. The SAv layer on the Langmuir–Schaefer film of unfolded protein could become a basic architecture for protein immobilization studies. - Highlights: • Langmuir–Schaefer film of carbonic anhydrase (LSF-CA) was biotinylated. • A densely packed streptavidin (SAv) layer was formed on the biotinylated LSF-CA. • Biotinylated proteins were bound to the SAv layer at high density. • Nonspecific adsorption of intact proteins to the SAv layer was weak. • Atomic force microscopy showed the binding of proteins at molecular resolution.
Directory of Open Access Journals (Sweden)
Yejing Wang
Full Text Available 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.
DEFF Research Database (Denmark)
Dasmeh, Pouria; Kepp, Kasper Planeta
2013-01-01
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......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...
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. © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands.
Nagpal, Suhani; Tiwari, Satyam; Mapa, Koyeli; Thukral, Lipi
2015-01-01
Many proteins comprising of complex topologies require molecular chaperones to achieve their unique three-dimensional folded structure. The E.coli chaperone, GroEL binds with a large number of unfolded and partially folded proteins, to facilitate proper folding and prevent misfolding and aggregation. Although the major structural components of GroEL are well defined, scaffolds of the non-native substrates that determine chaperone-mediated folding have been difficult to recognize. Here we performed all-atomistic and replica-exchange molecular dynamics simulations to dissect non-native ensemble of an obligate GroEL folder, DapA. Thermodynamics analyses of unfolding simulations revealed populated intermediates with distinct structural characteristics. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native β-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spectrometry (HDX-MS). Further, we constructed network graphs to elucidate long-range intra-protein connectivity of native and intermediate topologies, demonstrating regions that serve as central "hubs". Overall, our results implicate that genomic variations (or mutations) in the distinct regions of protein structures might disrupt these topological signatures disabling chaperone-mediated folding, leading to formation of aggregates.
Gray, Harry B.; Winkler, Jay R.; Kozak, John J.
2011-03-01
A geometrical model has been developed to describe the early stages of unfolding of cytochromes c‧ and c-b562 . Calculations are based on a step-wise extension of the polypeptide chain subject to the constraint that the spatial relationship among the residues of each triplet is fixed by the native-state crystallographic data. The response of each protein to these structural perturbations allows the evolution of each of the four helices in these two proteins to be differentiated. It is found that the two external helices in c‧ unfold before its two internal helices, whereas exactly the opposite behaviour is demonstrated by c-b562 . Each of these cytochromes has an extended, internal, non-helical ('turning') region that initially lags behind the most labile helix but then, at a certain stage (identified for each cytochrome), unravels before any of the four helices present in the native structure. It is believed that these predictions will be useful in guiding future experimental studies on the unfolding of these two cytochromes.
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Tony Ibnu Sumaryada
2018-02-01
Full Text Available Molecular dynamics simulations of the B1 fragment of protein G (56 residues have been performed at 325, 350, 375, 400, 450 and 500 K for 10 ns. An analysis of its structural and energetic parameters has indicated that the unfolding process of the GB1 protein begins at 900 ps of a 500-K simulation. The unfolding process is initiated when hydrogen bonds in the hydrophobic core region are broken; it continues with the α-helix transformation into coils and turns and ends with the destruction of the β-hairpins. These unfolding events are consistent with the hybrid model of the protein folding/unfolding mechanism, which is a compromise between the hydrophobic core collapse model and the zipper model. Salt-bridge pairs were found to play an important role in the unfolding process by maintaining the integrity of the tertiary structure of the protein. The breaking (or disappearance of the salt-bridge pairs E27–K31 (in the α-helix and E56–K10 (connecting β4 and β1 has resulted in the destruction of secondary structures and indicates the beginning of the unfolding process. Our results also suggest that the unfolding process in this simulation was not a complete denaturation of the protein because some β-hairpins remained
Directory of Open Access Journals (Sweden)
Surinder M Singh
Full Text Available Genetic mutations in a vital muscle protein dystrophin trigger X-linked dilated cardiomyopathy (XLDCM. However, disease mechanisms at the fundamental protein level are not understood. Such molecular knowledge is essential for developing therapies for XLDCM. Our main objective is to understand the effect of disease-causing mutations on the structure and function of dystrophin. This study is on a missense mutation K18N. The K18N mutation occurs in the N-terminal actin binding domain (N-ABD. We created and expressed the wild-type (WT N-ABD and its K18N mutant, and purified to homogeneity. Reversible folding experiments demonstrated that both mutant and WT did not aggregate upon refolding. Mutation did not affect the protein's overall secondary structure, as indicated by no changes in circular dichroism of the protein. However, the mutant is thermodynamically less stable than the WT (denaturant melts, and unfolds faster than the WT (stopped-flow kinetics. Despite having global secondary structure similar to that of the WT, mutant showed significant local structural changes at many amino acids when compared with the WT (heteronuclear NMR experiments. These structural changes indicate that the effect of mutation is propagated over long distances in the protein structure. Contrary to these structural and stability changes, the mutant had no significant effect on the actin-binding function as evident from co-sedimentation and depolymerization assays. These results summarize that the K18N mutation decreases thermodynamic stability, accelerates unfolding, perturbs protein structure, but does not affect the function. Therefore, K18N is a stability defect rather than a functional defect. Decrease in stability and increase in unfolding decrease the net population of dystrophin molecules available for function, which might trigger XLDCM. Consistently, XLDCM patients have decreased levels of dystrophin in cardiac muscle.
Exploring the Unfolding Pathway of Maltose Binding Proteins: An Integrated Computational Approach
Guardiani, Carlo; Marino, Daniele Di; Tramontano, Anna; Chinappi, Mauro; Cecconi, Fabio
2014-01-01
© 2014 American Chemical Society. Recent single-molecule force spectroscopy experiments on the Maltose Binding Proteins (MBPs) identified four stable structural units, termed unfoldons, that resist mechanical stress and determine the intermediates of the unfolding pathway. In this work, we analyze the topological origin and the dynamical role of the unfoldons using an integrated approach which combines a graph-theoretical analysis of the interaction network of the MBP native-state with steered molecular dynamics simulations. The topological analysis of the native state, while revealing the structural nature of the unfoldons, provides a framework to interpret the MBP mechanical unfolding pathway. Indeed, the experimental pathway can be effectively predicted by means of molecular dynamics simulations with a simple topology-based and low-resolution model of the MBP. The results obtained from the coarse-grained approach are confirmed and further refined by all-atom molecular dynamics.
Exploring the Unfolding Pathway of Maltose Binding Proteins: An Integrated Computational Approach
Guardiani, Carlo
2014-09-09
© 2014 American Chemical Society. Recent single-molecule force spectroscopy experiments on the Maltose Binding Proteins (MBPs) identified four stable structural units, termed unfoldons, that resist mechanical stress and determine the intermediates of the unfolding pathway. In this work, we analyze the topological origin and the dynamical role of the unfoldons using an integrated approach which combines a graph-theoretical analysis of the interaction network of the MBP native-state with steered molecular dynamics simulations. The topological analysis of the native state, while revealing the structural nature of the unfoldons, provides a framework to interpret the MBP mechanical unfolding pathway. Indeed, the experimental pathway can be effectively predicted by means of molecular dynamics simulations with a simple topology-based and low-resolution model of the MBP. The results obtained from the coarse-grained approach are confirmed and further refined by all-atom molecular dynamics.
Protein unfolding versus β-sheet separation in spider silk nanocrystals
International Nuclear Information System (INIS)
Alam, Parvez
2014-01-01
In this communication a mechanism for spider silk strain hardening is proposed. Shear failure of β-sheet nanocrystals is the first failure mode that gives rise to the creation of smaller nanocrystals, which are of higher strength and stiffness. β-sheet unfolding requires more energy than nanocrystal separation in a shear mode of failure. As a result, unfolding occurs after the nanocrystals separate in shear. β-sheet unfolding yields a secondary strain hardening effect once the β-sheet conformation is geometrically stable and acts like a unidirectional fibre in a fibre reinforced composite. The mechanism suggested herein is based on molecular dynamics calculations of residual inter-β-sheet separation strengths against residual intra-β-sheet unfolding strengths. (paper)
Cheng, Ryan R.; Hawk, Alexander T.; Makarov, Dmitrii E.
2013-02-01
Recent experiments showed that the reconfiguration dynamics of unfolded proteins are often adequately described by simple polymer models. In particular, the Rouse model with internal friction (RIF) captures internal friction effects as observed in single-molecule fluorescence correlation spectroscopy (FCS) studies of a number of proteins. Here we use RIF, and its non-free draining analog, Zimm model with internal friction, to explore the effect of internal friction on the rate with which intramolecular contacts can be formed within the unfolded chain. Unlike the reconfiguration times inferred from FCS experiments, which depend linearly on the solvent viscosity, the first passage times to form intramolecular contacts are shown to display a more complex viscosity dependence. We further describe scaling relationships obeyed by contact formation times in the limits of high and low internal friction. Our findings provide experimentally testable predictions that can serve as a framework for the analysis of future studies of contact formation in proteins.
Reversible Unfolding of Rhomboid Intramembrane Proteases.
Panigrahi, Rashmi; Arutyunova, Elena; Panwar, Pankaj; Gimpl, Katharina; Keller, Sandro; Lemieux, M Joanne
2016-03-29
Denaturant-induced unfolding of helical membrane proteins provides insights into their mechanism of folding and domain organization, which take place in the chemically heterogeneous, anisotropic environment of a lipid membrane. Rhomboid proteases are intramembrane proteases that play key roles in various diseases. Crystal structures have revealed a compact helical bundle with a buried active site, which requires conformational changes for the cleavage of transmembrane substrates. A dimeric form of the rhomboid protease has been shown to be important for activity. In this study, we examine the mechanism of refolding for two distinct rhomboids to gain insight into their secondary structure-activity relationships. Although helicity is largely abolished in the unfolded states of both proteins, unfolding is completely reversible for HiGlpG but only partially reversible for PsAarA. Refolding of both proteins results in reassociation of the dimer, with a 90% regain of catalytic activity for HiGlpG but only a 70% regain for PsAarA. For both proteins, a broad, gradual transition from the native, folded state to the denatured, partly unfolded state was revealed with the aid of circular dichroism spectroscopy as a function of denaturant concentration, thus arguing against a classical two-state model as found for many globular soluble proteins. Thermal denaturation has irreversible destabilizing effects on both proteins, yet reveals important functional details regarding substrate accessibility to the buried active site. This concerted biophysical and functional analysis demonstrates that HiGlpG, with a simple six-transmembrane-segment organization, is more robust than PsAarA, which has seven predicted transmembrane segments, thus rendering HiGlpG amenable to in vitro studies of membrane-protein folding. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Becoming a Peroxidase: Cardiolipin-Induced Unfolding of Cytochrome c
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
Shah, Riddhi; Ohashi, Tomoo; Erickson, Harold P; Oas, Terrence G
2017-01-20
Globular proteins are not permanently folded but spontaneously unfold and refold on time scales that can span orders of magnitude for different proteins. A longstanding debate in the protein-folding field is whether unfolding rates or folding rates correlate to the stability of a protein. In the present study, we have determined the unfolding and folding kinetics of 10 FNIII domains. FNIII domains are one of the most common protein folds and are present in 2% of animal proteins. FNIII domains are ideal for this study because they have an identical seven-strand β-sandwich structure, but they vary widely in sequence and thermodynamic stability. We assayed thermodynamic stability of each domain by equilibrium denaturation in urea. We then assayed the kinetics of domain opening and closing by a technique known as thiol exchange. For this we introduced a buried Cys at the identical location in each FNIII domain and measured the kinetics of labeling with DTNB over a range of urea concentrations. A global fit of the kinetics data gave the kinetics of spontaneous unfolding and refolding in zero urea. We found that the folding rates were relatively similar, ∼0.1-1 s -1 , for the different domains. The unfolding rates varied widely and correlated with thermodynamic stability. Our study is the first to address this question using a set of domains that are structurally homologous but evolved with widely varying sequence identity and thermodynamic stability. These data add new evidence that thermodynamic stability correlates primarily with unfolding rate rather than folding rate. The study also has implications for the question of whether opening of FNIII domains contributes to the stretching of fibronectin matrix fibrils. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Effects of ubiquilin 1 on the unfolded protein response.
Lu, Alice; Hiltunen, Mikko; Romano, Donna M; Soininen, Hilkka; Hyman, Bradley T; Bertram, Lars; Tanzi, Rudolph E
2009-05-01
Previous studies have implicated the unfolded protein response (UPR) in the pathogenesis of Alzheimer's disease (AD). We previously reported that DNA variants in the ubiquilin 1 (UBQLN1) gene increase the risk for AD. Since UBQLN1 has been shown to play a role in the UPR, we assessed the effects of overexpression and downregulation of UBQLN1 splice variants during tunicamycin-induced ER stress. In addition to previously described transcript variants, TV1 and TV2, we identified two novel transcript variants of UBQLN1 in brain: TV3 (lacking exons 2-4) and TV4 (lacking exon 4). Overexpression of TV1-3, but not TV4 significantly decreased the mRNA induction of UPR-inducible genes, C/EBP homologous protein (CHOP), BiP/GRP78, and protein disulfide isomerase (PDI) during the UPR. Stable overexpression of TV1-3, but not TV4, also significantly decreased the induction of CHOP protein and increased cell viability during the UPR. In contrast, downregulation of UBQLN1 did not affect CHOP mRNA induction, but instead increased PDI mRNA levels. These findings suggest that overexpression UBQLN1 transcript variants TV1-3, but not TV4, exert a protective effect during the UPR by attenuating CHOP induction and potentially increasing cell viability.
Nagpal, Suhani; Tiwari, Satyam; Mapa, Koyeli; Thukral, Lipi
2015-01-01
Many proteins comprising of complex topologies require molecular chaperones to achieve their unique three-dimensional folded structure. The E.coli chaperone, GroEL binds with a large number of unfolded and partially folded proteins, to facilitate proper folding and prevent misfolding and aggregation. Although the major structural components of GroEL are well defined, scaffolds of the non-native substrates that determine chaperone-mediated folding have been difficult to recognize. Here we performed all-atomistic and replica-exchange molecular dynamics simulations to dissect non-native ensemble of an obligate GroEL folder, DapA. Thermodynamics analyses of unfolding simulations revealed populated intermediates with distinct structural characteristics. We found that surface exposed hydrophobic patches are significantly increased, primarily contributed from native and non-native β-sheet elements. We validate the structural properties of these conformers using experimental data, including circular dichroism (CD), 1-anilinonaphthalene-8-sulfonic acid (ANS) binding measurements and previously reported hydrogen-deutrium exchange coupled to mass spectrometry (HDX-MS). Further, we constructed network graphs to elucidate long-range intra-protein connectivity of native and intermediate topologies, demonstrating regions that serve as central “hubs”. Overall, our results implicate that genomic variations (or mutations) in the distinct regions of protein structures might disrupt these topological signatures disabling chaperone-mediated folding, leading to formation of aggregates. PMID:26394388
Microscopic dynamics of water around unfolded structures of barstar at room temperature
Energy Technology Data Exchange (ETDEWEB)
Pal, Somedatta; Chakraborty, Kaushik; Khatua, Prabir; Bandyopadhyay, Sanjoy, E-mail: sanjoy@chem.iitkgp.ernet.in [Molecular Modeling Laboratory, Department of Chemistry, Indian Institute of Technology, Kharagpur 721302 (India)
2015-02-07
The breaking of the native structure of a protein and its influences on the dynamic response of the surrounding solvent is an important issue in protein folding. In this work, we have carried out atomistic molecular dynamics simulations to unfold the protein barstar at two different temperatures (400 K and 450 K). The two unfolded forms obtained at such high temperatures are further studied at room temperature to explore the effects of nonuniform unfolding of the protein secondary structures along two different pathways on the microscopic dynamical properties of the surface water molecules. It is demonstrated that though the structural transition of the protein in general results in less restricted water motions around its segments, but there are evidences of formation of new conformational motifs upon unfolding with increasingly confined environment around them, thereby resulting in further restricted water mobility in their hydration layers. Moreover, it is noticed that the effects of nonuniform unfolding of the protein segments on the relaxation times of the protein–water (PW) and the water–water (WW) hydrogen bonds are correlated with hindered hydration water motions. However, the kinetics of breaking and reformation of such hydrogen bonds are found to be influenced differently at the interface. It is observed that while the effects of unfolding on the PW hydrogen bond kinetics seem to be minimum, but the kinetics involving the WW hydrogen bonds around the protein segments exhibit noticeably heterogeneous characteristics. We believe that this is an important observation, which can provide valuable insights on the origin of heterogeneous influence of unfolding of a protein on the microscopic properties of its hydration water.
Manga, Prashiela; Bis, Sabina; Knoll, Kristen; Perez, Beremis; Orlow, Seth J
2010-10-01
Accumulation of proteins in the endoplasmic reticulum (ER) triggers the unfolded protein response (UPR), comprising three signaling pathways initiated by Ire1, Perk and Atf6 respectively. Unfolded protein response activation was compared in chemically stressed murine wildtype melanocytes and mutant melanocytes that retain tyrosinase in the ER. Thapsigargin, an ER stressor, activated all pathways in wildtype melanocytes, triggering Caspase 12-mediated apoptosis at toxic doses. Albino melanocytes expressing mutant tyrosinase showed evidence of ER stress with increased Ire1 expression, but the downstream effector, Xbp1, was not activated even following thapsigargin treatment. Attenuation of Ire1 signaling was recapitulated in wildtype melanocytes treated with thapsigargin for 8 days, with diminished Xbp1 activation observed after 4 days. Atf6 was also activated in albino melanocytes, with no response to thapsigargin, while the Perk pathway was not activated and thapsigargin treatment elicited robust expression of the downstream effector CCAAT-enhancer-binding protein homologous protein. Thus, melanocytes adapt to ER stress by attenuating two UPR pathways.
International Nuclear Information System (INIS)
Ishii, Takaaki; Murayama, Yoshihiro; Katano, Atsuto; Maki, Kosuke; Kuwajima, Kunihiro; Sano, Masaki
2008-01-01
Single-molecule manipulation techniques have given experimental access to unfolding intermediates of proteins that are inaccessible in conventional experiments. A detailed characterization of the intermediates is a challenging problem that provides new possibilities for directly probing the energy landscape of proteins. We investigated single-molecule mechanical unfolding of a small globular protein, staphylococcal nuclease (SNase), using atomic force microscopy. The unfolding trajectories of the protein displayed sub-molecular and stochastic behavior with typical lengths corresponding to the size of the unfolded substructures. Our results support the view that the single protein unfolds along multiple pathways as suggested in recent theoretical studies. Moreover, we found the drastic change, caused by the ligand and inhibitor bindings, in the mechanical unfolding dynamics
DEFF Research Database (Denmark)
Treiman, Marek
2002-01-01
for the protein folding pathway require Ca(2+) binding for their activity. A number of factors, including Ca(2+) depletion, may interfere with the folding pathway within the ER, with a potential for cell injury through an accumulation of malfolded protein aggregates. The Unfolded Protein Response involves...... a transcriptional upregulation of a number of the ER-resident folding helper proteins and becomes triggered when the folding pathway is blocked. To be effective, these upregulated proteins require a sufficient supply of Ca(2+) cofactor within the ER lumen. In tissue ischemia, where the availablity of this cofactor...
Takatori, Osamu; Usui, Soichiro; Okajima, Masaki; Kaneko, Shuichi; Ootsuji, Hiroshi; Takashima, Shin-Ichiro; Kobayashi, Daisuke; Murai, Hisayoshi; Furusho, Hiroshi; Takamura, Masayuki
2017-05-01
The unfolded protein response (UPR) plays a pivotal role in ischemia-reperfusion (I/R) injury in various organs such as heart, brain, and liver. Sodium 4-phenylbutyrate (PBA) reportedly acts as a chemical chaperone that reduces UPR. In the present study, we evaluated the effect of PBA on reducing the UPR and protecting against myocardial I/R injury in mice. Male C57BL/6 mice were subjected to 30-minute myocardial I/R, and were treated with phosphate-buffered saline (as a vehicle) or PBA. At 4 hours after reperfusion, mice treated with PBA had reduced serum cardiac troponin I levels and numbers of apoptotic cells in left ventricles (LVs) in myocardial I/R. Infarct size had also reduced in mice treated with PBA at 48 hours after reperfusion. At 2 hours after reperfusion, UPR markers, including eukaryotic initiation of the factor 2α-subunit, activating transcription factor-6, inositol-requiring enzyme-1, glucose-regulated protein 78, CCAAT/enhancer-binding protein (C/EBP) homologous protein, and caspase-12, were significantly increased in mice treated with vehicle compared to sham-operated mice. Administration of PBA significantly reduced the I/R-induced increases of these markers. Cardiac function and dimensions were assessed at 21 days after I/R. Sodium 4-phenylbutyrate dedicated to the improvement of cardiac parameters deterioration including LV end-diastolic diameter and LV fractional shortening. Consistently, PBA reduced messenger RNA expression levels of cardiac remodeling markers such as collagen type 1α1, brain natriuretic peptide, and α skeletal muscle actin in LV at 21 days after I/R. Unfolded protein response mediates myocardial I/R injury. Administration of PBA reduces the UPR, apoptosis, infarct size, and preserved cardiac function. Hence, PBA may be a therapeutic option to attenuate myocardial I/R injury in clinical practice.
The Unfolded Protein Response in Amelogenesis and Enamel Pathologies
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Steven J. Brookes
2017-09-01
Full Text Available During the secretory phase of their life-cycle, ameloblasts are highly specialized secretory cells whose role is to elaborate an extracellular matrix that ultimately confers both form and function to dental enamel, the most highly mineralized of all mammalian tissues. In common with many other “professional” secretory cells, ameloblasts employ the unfolded protein response (UPR to help them cope with the large secretory cargo of extracellular matrix proteins transiting their ER (endoplasmic reticulum/Golgi complex and so minimize ER stress. However, the UPR is a double-edged sword, and, in cases where ER stress is severe and prolonged, the UPR switches from pro-survival to pro-apoptotic mode. The purpose of this review is to consider the role of the ameloblast UPR in the biology and pathology of amelogenesis; specifically in respect of amelogenesis imperfecta (AI and fluorosis. Some forms of AI appear to correspond to classic proteopathies, where pathological intra-cellular accumulations of protein tip the UPR toward apoptosis. Fluorosis also involves the UPR and, while not of itself a classic proteopathic disease, shares some common elements through the involvement of the UPR. The possibility of therapeutic intervention by pharmacological modulation of the UPR in AI and fluorosis is also discussed.
The Unfolded Protein Response in Amelogenesis and Enamel Pathologies.
Brookes, Steven J; Barron, Martin J; Dixon, Michael J; Kirkham, Jennifer
2017-01-01
During the secretory phase of their life-cycle, ameloblasts are highly specialized secretory cells whose role is to elaborate an extracellular matrix that ultimately confers both form and function to dental enamel, the most highly mineralized of all mammalian tissues. In common with many other "professional" secretory cells, ameloblasts employ the unfolded protein response (UPR) to help them cope with the large secretory cargo of extracellular matrix proteins transiting their ER (endoplasmic reticulum)/Golgi complex and so minimize ER stress. However, the UPR is a double-edged sword, and, in cases where ER stress is severe and prolonged, the UPR switches from pro-survival to pro-apoptotic mode. The purpose of this review is to consider the role of the ameloblast UPR in the biology and pathology of amelogenesis; specifically in respect of amelogenesis imperfecta (AI) and fluorosis. Some forms of AI appear to correspond to classic proteopathies, where pathological intra-cellular accumulations of protein tip the UPR toward apoptosis. Fluorosis also involves the UPR and, while not of itself a classic proteopathic disease, shares some common elements through the involvement of the UPR. The possibility of therapeutic intervention by pharmacological modulation of the UPR in AI and fluorosis is also discussed.
Shechtman, Caryn F.; Henneberry, Annette L.; Seimon, Tracie A.; Tinkelenberg, Arthur H.; Wilcox, Lisa J.; Lee, Eunjee; Fazlollahi, Mina; Munkacsi, Andrew B.; Bussemaker, Harmen J.; Tabas, Ira; Sturley, Stephen L.
2011-01-01
The ARV1-encoded protein mediates sterol transport from the endoplasmic reticulum (ER) to the plasma membrane. Yeast ARV1 mutants accumulate multiple lipids in the ER and are sensitive to pharmacological modulators of both sterol and sphingolipid metabolism. Using fluorescent and electron microscopy, we demonstrate sterol accumulation, subcellular membrane expansion, elevated lipid droplet formation, and vacuolar fragmentation in ARV1 mutants. Motif-based regression analysis of ARV1 deletion transcription profiles indicates activation of Hac1p, an integral component of the unfolded protein response (UPR). Accordingly, we show constitutive splicing of HAC1 transcripts, induction of a UPR reporter, and elevated expression of UPR targets in ARV1 mutants. IRE1, encoding the unfolded protein sensor in the ER lumen, exhibits a lethal genetic interaction with ARV1, indicating a viability requirement for the UPR in cells lacking ARV1. Surprisingly, ARV1 mutants expressing a variant of Ire1p defective in sensing unfolded proteins are viable. Moreover, these strains also exhibit constitutive HAC1 splicing that interacts with DTT-mediated perturbation of protein folding. These data suggest that a component of UPR induction in arv1Δ strains is distinct from protein misfolding. Decreased ARV1 expression in murine macrophages also results in UPR induction, particularly up-regulation of activating transcription factor-4, CHOP (C/EBP homologous protein), and apoptosis. Cholesterol loading or inhibition of cholesterol esterification further elevated CHOP expression in ARV1 knockdown cells. Thus, loss or down-regulation of ARV1 disturbs membrane and lipid homeostasis, resulting in a disruption of ER integrity, one consequence of which is induction of the UPR. PMID:21266578
Krüger, Dennis M; Rathi, Prakash Chandra; Pfleger, Christopher; Gohlke, Holger
2013-07-01
The Constraint Network Analysis (CNA) web server provides a user-friendly interface to the CNA approach developed in our laboratory for linking results from rigidity analyses to biologically relevant characteristics of a biomolecular structure. The CNA web server provides a refined modeling of thermal unfolding simulations that considers the temperature dependence of hydrophobic tethers and computes a set of global and local indices for quantifying biomacromolecular stability. From the global indices, phase transition points are identified where the structure switches from a rigid to a floppy state; these phase transition points can be related to a protein's (thermo-)stability. Structural weak spots (unfolding nuclei) are automatically identified, too; this knowledge can be exploited in data-driven protein engineering. The local indices are useful in linking flexibility and function and to understand the impact of ligand binding on protein flexibility. The CNA web server robustly handles small-molecule ligands in general. To overcome issues of sensitivity with respect to the input structure, the CNA web server allows performing two ensemble-based variants of thermal unfolding simulations. The web server output is provided as raw data, plots and/or Jmol representations. The CNA web server, accessible at http://cpclab.uni-duesseldorf.de/cna or http://www.cnanalysis.de, is free and open to all users with no login requirement.
Thermal dissociation and unfolding of insulin
DEFF Research Database (Denmark)
Huus, Kasper; Havelund, Svend; Olsen, Helle B
2005-01-01
The thermal stability of human insulin was studied by differential scanning microcalorimetry and near-UV circular dichroism as a function of zinc/protein ratio, to elucidate the dissociation and unfolding processes of insulin in different association states. Zinc-free insulin, which is primarily...... dimeric at room temperature, unfolded at approximately 70 degrees C. The two monomeric insulin mutants Asp(B28) and Asp(B9),Glu(B27) unfolded at higher temperatures, but with enthalpies of unfolding that were approximately 30% smaller. Small amounts of zinc caused a biphasic thermal denaturation pattern...... of insulin. The biphasic denaturation is caused by a redistribution of zinc ions during the heating process and results in two distinct transitions with T(m)'s of approximately 70 and approximately 87 degrees C corresponding to monomer/dimer and hexamer, respectively. At high zinc concentrations (>or=5 Zn(2...
Augustyniak, Rafal; Kay, Lewis E
2018-05-22
Protein remodeling by AAA+ enzymes is central for maintaining proteostasis in a living cell. However, a detailed structural description of how this is accomplished at the level of the substrate molecules that are acted upon is lacking. Here, we combine chemical cross-linking and methyl transverse relaxation-optimized NMR spectroscopy to study, at atomic resolution, the stepwise unfolding and subsequent refolding of the two-domain substrate calmodulin by the VAT AAA+ unfoldase from Thermoplasma acidophilum By engineering intermolecular disulphide bridges between the substrate and VAT we trap the substrate at different stages of translocation, allowing structural studies throughout the translocation process. Our results show that VAT initiates substrate translocation by pulling on intrinsically unstructured N or C termini of substrate molecules without showing specificity for a particular amino acid sequence. Although the B1 domain of protein G is shown to unfold cooperatively, translocation of calmodulin leads to the formation of intermediates, and these differ on an individual domain level in a manner that depends on whether pulling is from the N or C terminus. The approach presented generates an atomic resolution picture of substrate unfolding and subsequent refolding by unfoldases that can be quite different from results obtained via in vitro denaturation experiments.
The unfolded protein response and the role of protein disulphide isomerase in neurodegeneration.
Directory of Open Access Journals (Sweden)
Emma ePerri
2016-01-01
Full Text Available The maintenance and regulation of proteostasis is a critical function for post-mitotic neurons and dysregulation of proteostasis is increasingly implicated in neurodegenerative diseases. Despite having different clinical manifestations, these disorders share similar pathology; an accumulation of misfolded proteins in neurons and subsequent disruption to cellular proteostasis. The endoplasmic reticulum (ER is an important component of proteostasis, and when the accumulation of misfolded proteins occurs within the ER, this disturbs ER homeostasis, giving rise to ER stress. This triggers the unfolded protein response (UPR, distinct signalling pathways that whilst initially protective, are pro-apoptotic if ER stress is prolonged. ER stress is increasingly implicated in neurodegenerative diseases, and emerging evidence highlights the complexity of the UPR in these disorders, with both protective and detrimental components being described. Protein Disulphide Isomerase (PDI is an ER chaperone induced during ER stress that is responsible for the formation of disulphide bonds in proteins. Whilst initially considered to be protective, recent studies have revealed unconventional roles for PDI in neurodegenerative diseases, distinct from its normal function in the UPR and the ER, although these mechanisms remain poorly defined. However specific aspects of PDI function may offer the potential to be exploited therapeutically in the future. This review will focus on the evidence linking ER stress and the UPR to neurodegenerative diseases, with particular emphasis on the emerging functions ascribed to PDI in these conditions.
Salt bridge as a gatekeeper against partial unfolding.
Hinzman, Mark W; Essex, Morgan E; Park, Chiwook
2016-05-01
Salt bridges are frequently observed in protein structures. Because the energetic contribution of salt bridges is strongly dependent on the environmental context, salt bridges are believed to contribute to the structural specificity rather than the stability. To test the role of salt bridges in enhancing structural specificity, we investigated the contribution of a salt bridge to the energetics of native-state partial unfolding in a cysteine-free version of Escherichia coli ribonuclease H (RNase H*). Thermolysin cleaves a protruding loop of RNase H(*) through transient partial unfolding under native conditions. Lys86 and Asp108 in RNase H(*) form a partially buried salt bridge that tethers the protruding loop. Investigation of the global stability of K86Q/D108N RNase H(*) showed that the salt bridge does not significantly contribute to the global stability. However, K86Q/D108N RNase H(*) is greatly more susceptible to proteolysis by thermolysin than wild-type RNase H(*) is. The free energy for partial unfolding determined by native-state proteolysis indicates that the salt bridge significantly increases the energy for partial unfolding by destabilizing the partially unfolded form. Double mutant cycles with single and double mutations of the salt bridge suggest that the partially unfolded form is destabilized due to a significant decrease in the interaction energy between Lys86 and Asp108 upon partial unfolding. This study demonstrates that, even in the case that a salt bridge does not contribute to the global stability, the salt bridge may function as a gatekeeper against partial unfolding that disturbs the optimal geometry of the salt bridge. © 2016 The Protein Society.
Dysregulation of the unfolded protein response in db/db mice with diet induced steatohepatitis
Rinella, Mary E.; Siddiqui, M. Shaddab; Gardikiotes, Konstantina; Gottstein, Jeanne; Elias, Marc; Green, Richard M.
2011-01-01
In humans with non-alcoholic fatty liver, diabetes is associated with more advanced disease. We have previously shown that diabetic db/db mice are highly susceptible to methionine choline deficient diet (MCD) induced hepatic injury. Since activation of the unfolded protein response (UPR) is an important adaptive cellular mechanism in diabetes, obesity and fatty liver, we hypothesized that dysregulation of the UPR may partially explain how diabetes could promote liver injury.
Wang, Zhao V; Deng, Yingfeng; Gao, Ningguo; Pedrozo, Zully; Li, Dan L; Morales, Cyndi R; Criollo, Alfredo; Luo, Xiang; Tan, Wei; Jiang, Nan; Lehrman, Mark A; Rothermel, Beverly A; Lee, Ann-Hwee; Lavandero, Sergio; Mammen, Pradeep P A; Ferdous, Anwarul; Gillette, Thomas G; Scherer, Philipp E; Hill, Joseph A
2014-03-13
The hexosamine biosynthetic pathway (HBP) generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) for glycan synthesis and O-linked GlcNAc (O-GlcNAc) protein modifications. Despite the established role of the HBP in metabolism and multiple diseases, regulation of the HBP remains largely undefined. Here, we show that spliced X-box binding protein 1 (Xbp1s), the most conserved signal transducer of the unfolded protein response (UPR), is a direct transcriptional activator of the HBP. We demonstrate that the UPR triggers HBP activation via Xbp1s-dependent transcription of genes coding for key, rate-limiting enzymes. We further establish that this previously unrecognized UPR-HBP axis is triggered in a variety of stress conditions. Finally, we demonstrate a physiologic role for the UPR-HBP axis by showing that acute stimulation of Xbp1s in heart by ischemia/reperfusion confers robust cardioprotection in part through induction of the HBP. Collectively, these studies reveal that Xbp1s couples the UPR to the HBP to protect cells under stress. Copyright © 2014 Elsevier Inc. All rights reserved.
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 (eIF2α) 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.
Unfolding of cytochrome c immobilized on self-assembled monolayers. An electrochemical study
International Nuclear Information System (INIS)
Monari, Stefano; Ranieri, Antonio; Bortolotti, Carlo Augusto; Peressini, Silvia; Tavagnacco, Claudio; Borsari, Marco
2011-01-01
Highlights: → Denaturation involves intermediate and partially unfolded forms. → An unfolded species displaying the haem with Fe coordinated by two His is observed. → Under unfolding conditions the nature of the SAM influences conformation of protein. → Concentration of the unfolding agent affects redox properties of immobilized protein. - Abstract: The electron transfer (ET) process of progressively unfolded bovine cytochrome c immobilized on different self-assembled monolayers (SAMs) was investigated. Insight is gained on the role of the SAM surface on the functionality of the partially unfolded and non-native forms of the adsorbed protein. Direct electrochemical measurements were performed on cytochrome c adsorbed on mercaptopyridine (MP) and mixed 11-mercapto-1-undecanoic acid/11-mercapto-1-undecanol (MUA/MU) at varying temperature, in the presence of urea as unfolding agent. Under strongly unfolding conditions, a non-native form of cytochrome c, in which the methionine ligand is replaced by a histidine, was observed on both MP and MUA/MU SAMs. The E o ' of the native form, in which the haem is axially coordinated by methionine and histidine, slightly shifts to negative values upon increasing urea concentration. However, the non-native bis-histidinate species shows a much lower E o ' value (by approximately 0.4 V) which is by far enthalpic in origin and largely determined by axial ligand swapping. Analysis of the reduction enthalpies and entropies and of the ET rate constants indicate that the nature of the SAM (hydrophilic or anionic) results in changes in the conformational rearrangement of the cytochrome c under unfolding conditions.
Unfolding of cytochrome c immobilized on self-assembled monolayers. An electrochemical study
Energy Technology Data Exchange (ETDEWEB)
Monari, Stefano; Ranieri, Antonio; Bortolotti, Carlo Augusto; Peressini, Silvia [Department of Chemistry, University of Modena and Reggio Emilia, via Campi 183, 41125 Modena (Italy); Tavagnacco, Claudio [Department of Chemistry, University of Trieste, via Giorgieri 1, 34127 Trieste (Italy); Borsari, Marco, E-mail: marco.borsari@unimore.it [Department of Chemistry, University of Modena and Reggio Emilia, via Campi 183, 41125 Modena (Italy)
2011-08-01
Highlights: > Denaturation involves intermediate and partially unfolded forms. > An unfolded species displaying the haem with Fe coordinated by two His is observed. > Under unfolding conditions the nature of the SAM influences conformation of protein. > Concentration of the unfolding agent affects redox properties of immobilized protein. - Abstract: The electron transfer (ET) process of progressively unfolded bovine cytochrome c immobilized on different self-assembled monolayers (SAMs) was investigated. Insight is gained on the role of the SAM surface on the functionality of the partially unfolded and non-native forms of the adsorbed protein. Direct electrochemical measurements were performed on cytochrome c adsorbed on mercaptopyridine (MP) and mixed 11-mercapto-1-undecanoic acid/11-mercapto-1-undecanol (MUA/MU) at varying temperature, in the presence of urea as unfolding agent. Under strongly unfolding conditions, a non-native form of cytochrome c, in which the methionine ligand is replaced by a histidine, was observed on both MP and MUA/MU SAMs. The E{sup o}' of the native form, in which the haem is axially coordinated by methionine and histidine, slightly shifts to negative values upon increasing urea concentration. However, the non-native bis-histidinate species shows a much lower E{sup o}' value (by approximately 0.4 V) which is by far enthalpic in origin and largely determined by axial ligand swapping. Analysis of the reduction enthalpies and entropies and of the ET rate constants indicate that the nature of the SAM (hydrophilic or anionic) results in changes in the conformational rearrangement of the cytochrome c under unfolding conditions.
Directory of Open Access Journals (Sweden)
Claudia Rodríguez-Almazán
Full Text Available The intracellular concentration of protein may be as high as 400 mg per ml; thus it seems inevitable that within the cell, numerous protein-protein contacts are constantly occurring. A basic biochemical principle states that the equilibrium of an association reaction can be shifted by ligand binding. This indicates that if within the cell many protein-protein interactions are indeed taking place, some fundamental characteristics of proteins would necessarily differ from those observed in traditional biochemical systems. Accordingly, we measured the effect of eight different proteins on the formation of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM from guanidinium chloride unfolded monomers. The eight proteins at concentrations of micrograms per ml induced an important increase on active dimer formation. Studies on the mechanism of this phenomenon showed that the proteins stabilize the dimeric structure of TbTIM, and that this is the driving force that promotes the formation of active dimers. Similar data were obtained with TIM from three other species. The heat changes that occur when TbTIM is mixed with lysozyme were determined by isothermal titration calorimetry; the results provided direct evidence of the weak interaction between apparently unrelated proteins. The data, therefore, are strongly suggestive that the numerous protein-protein interactions that occur in the intracellular space are an additional control factor in the formation and stability of proteins.
Bacteria, the endoplasmic reticulum and the unfolded protein response: friends or foes?
Celli, Jean; Tsolis, Renée M
2015-02-01
The unfolded protein response (UPR) is a cytoprotective response that is aimed at restoring cellular homeostasis following physiological stress exerted on the endoplasmic reticulum (ER), which also invokes innate immune signalling in response to invading microorganisms. Although it has been known for some time that the UPR is modulated by various viruses, recent evidence indicates that it also has multiple roles during bacterial infections. In this Review, we describe how bacteria interact with the ER, including how bacteria induce the UPR, how subversion of the UPR promotes bacterial proliferation and how the UPR contributes to innate immune responses against invading bacteria.
Tunicamycin-induced unfolded protein response in the developing mouse brain
International Nuclear Information System (INIS)
Wang, Haiping; Wang, Xin; Ke, Zun-Ji; Comer, Ashley L.; Xu, Mei; Frank, Jacqueline A.; Zhang, Zhuo; Shi, Xianglin; Luo, Jia
2015-01-01
Accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) causes ER stress, resulting in the activation of the unfolded protein response (UPR). ER stress and UPR are associated with many neurodevelopmental and neurodegenerative disorders. The developing brain is particularly susceptible to environmental insults which may cause ER stress. We evaluated the UPR in the brain of postnatal mice. Tunicamycin, a commonly used ER stress inducer, was administered subcutaneously to mice of postnatal days (PDs) 4, 12 and 25. Tunicamycin caused UPR in the cerebral cortex, hippocampus and cerebellum of mice of PD4 and PD12, which was evident by the upregulation of ATF6, XBP1s, p-eIF2α, GRP78, GRP94 and MANF, but failed to induce UPR in the brain of PD25 mice. Tunicamycin-induced UPR in the liver was observed at all stages. In PD4 mice, tunicamycin-induced caspase-3 activation was observed in layer II of the parietal and optical cortex, CA1–CA3 and the subiculum of the hippocampus, the cerebellar external germinal layer and the superior/inferior colliculus. Tunicamycin-induced caspase-3 activation was also shown on PD12 but to a much lesser degree and mainly located in the dentate gyrus of the hippocampus, deep cerebellar nuclei and pons. Tunicamycin did not activate caspase-3 in the brain of PD25 mice and the liver of all stages. Similarly, immature cerebellar neurons were sensitive to tunicamycin-induced cell death in culture, but became resistant as they matured in vitro. These results suggest that the UPR is developmentally regulated and the immature brain is more susceptible to ER stress. - Highlights: • Tunicamycin caused a development-dependent UPR in the mouse brain. • Immature brain was more susceptible to tunicamycin-induced endoplasmic reticulum stress. • Tunicamycin caused more neuronal death in immature brain than mature brain. • Tunicamycin-induced neuronal death is region-specific
Tunicamycin-induced unfolded protein response in the developing mouse brain
Energy Technology Data Exchange (ETDEWEB)
Wang, Haiping; Wang, Xin [Department of Pharmacology and Nutritional Sciences, University of Kentucky College of Medicine, Lexington, KY 40536 (United States); Ke, Zun-Ji [Department of Biochemistry, Shanghai University of Traditional Chinese Medicine, 1200 Cailun Road, Shanghai 201203 (China); Comer, Ashley L.; Xu, Mei; Frank, Jacqueline A. [Department of Pharmacology and Nutritional Sciences, University of Kentucky College of Medicine, Lexington, KY 40536 (United States); Zhang, Zhuo; Shi, Xianglin [Graduate Center for Toxicology, University of Kentucky College of Medicine, Lexington, KY 40536 (United States); Luo, Jia, E-mail: jialuo888@uky.edu [Department of Pharmacology and Nutritional Sciences, University of Kentucky College of Medicine, Lexington, KY 40536 (United States)
2015-03-15
Accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) causes ER stress, resulting in the activation of the unfolded protein response (UPR). ER stress and UPR are associated with many neurodevelopmental and neurodegenerative disorders. The developing brain is particularly susceptible to environmental insults which may cause ER stress. We evaluated the UPR in the brain of postnatal mice. Tunicamycin, a commonly used ER stress inducer, was administered subcutaneously to mice of postnatal days (PDs) 4, 12 and 25. Tunicamycin caused UPR in the cerebral cortex, hippocampus and cerebellum of mice of PD4 and PD12, which was evident by the upregulation of ATF6, XBP1s, p-eIF2α, GRP78, GRP94 and MANF, but failed to induce UPR in the brain of PD25 mice. Tunicamycin-induced UPR in the liver was observed at all stages. In PD4 mice, tunicamycin-induced caspase-3 activation was observed in layer II of the parietal and optical cortex, CA1–CA3 and the subiculum of the hippocampus, the cerebellar external germinal layer and the superior/inferior colliculus. Tunicamycin-induced caspase-3 activation was also shown on PD12 but to a much lesser degree and mainly located in the dentate gyrus of the hippocampus, deep cerebellar nuclei and pons. Tunicamycin did not activate caspase-3 in the brain of PD25 mice and the liver of all stages. Similarly, immature cerebellar neurons were sensitive to tunicamycin-induced cell death in culture, but became resistant as they matured in vitro. These results suggest that the UPR is developmentally regulated and the immature brain is more susceptible to ER stress. - Highlights: • Tunicamycin caused a development-dependent UPR in the mouse brain. • Immature brain was more susceptible to tunicamycin-induced endoplasmic reticulum stress. • Tunicamycin caused more neuronal death in immature brain than mature brain. • Tunicamycin-induced neuronal death is region-specific.
Emerging Role of the Unfolded Protein Response in Tumor Immunosurveillance.
Vanacker, Hélène; Vetters, Jessica; Moudombi, Lyvia; Caux, Christophe; Janssens, Sophie; Michallet, Marie-Cécile
2017-07-01
Disruption of endoplasmic reticulum (ER) homeostasis results in ER stress and activation of the unfolded protein response (UPR). This response alleviates cell stress, and is activated in both tumor cells and tumor infiltrating immune cells. The UPR plays a dual function in cancer biology, acting as a barrier to tumorigenesis at the premalignant stage, while fostering cancer maintenance in established tumors. In infiltrating immune cells, the UPR has been involved in both immunosurveillance and immunosuppressive functions. This review aims to decipher the role of the UPR at different stages of tumorigenesis and how the UPR shapes the balance between immunosurveillance and immune escape. This knowledge may improve existing UPR-targeted therapies and the design of novel strategies for cancer treatment. Copyright © 2017 Elsevier Inc. All rights reserved.
International Nuclear Information System (INIS)
Honma, Yuichi; Harada, Masaru
2013-01-01
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
Directory of Open Access Journals (Sweden)
Xi Ling Fu
Full Text Available Dormancy mechanisms in seeds and buds arrest growth until environmental conditions are optimal for development. A genotype-specific period of chilling is usually required to release dormancy, but the underlying molecular mechanisms are still not fully understood. To discover transcriptional pathways associated with dormancy release common to seed stratification and bud endodormancy, we explored the chilling-dependent expression of 11 genes involved in endoplasmic reticulum stress and the unfolded protein response signal pathways. We propose that endoplasmic reticulum stress and the unfolded protein response impact on seed as well as bud germination and development by chilling-dependent mechanisms. The emerging discovery of similarities between seed stratification and bud endodormancy status indicate that these two processes are probably regulated by common endoplasmic reticulum stress and unfolded protein response signalling pathways. Clarification of regulatory pathways common to both seed and bud dormancy may enhance understanding of the mechanisms underlying dormancy and breeding programs may benefit from earlier prediction of chilling requirements for uniform blooming of novel genotypes of deciduous fruit tree species.
Comparison of intra-organellar chaperone capacity for dealing with stress-induced protein unfolding.
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.
Mechanical unfolding reveals stable 3-helix intermediates in talin and α-catenin.
Directory of Open Access Journals (Sweden)
Vasyl V Mykuliak
2018-04-01
Full Text Available Mechanical stability is a key feature in the regulation of structural scaffolding proteins and their functions. Despite the abundance of α-helical structures among the human proteome and their undisputed importance in health and disease, the fundamental principles of their behavior under mechanical load are poorly understood. Talin and α-catenin are two key molecules in focal adhesions and adherens junctions, respectively. In this study, we used a combination of atomistic steered molecular dynamics (SMD simulations, polyprotein engineering, and single-molecule atomic force microscopy (smAFM to investigate unfolding of these proteins. SMD simulations revealed that talin rod α-helix bundles as well as α-catenin α-helix domains unfold through stable 3-helix intermediates. While the 5-helix bundles were found to be mechanically stable, a second stable conformation corresponding to the 3-helix state was revealed. Mechanically weaker 4-helix bundles easily unfolded into a stable 3-helix conformation. The results of smAFM experiments were in agreement with the findings of the computational simulations. The disulfide clamp mutants, designed to protect the stable state, support the 3-helix intermediate model in both experimental and computational setups. As a result, multiple discrete unfolding intermediate states in the talin and α-catenin unfolding pathway were discovered. Better understanding of the mechanical unfolding mechanism of α-helix proteins is a key step towards comprehensive models describing the mechanoregulation of proteins.
Cooperative unfolding of apolipoprotein A-1 induced by chemical denaturation.
Eckhardt, D; Li-Blatter, X; Schönfeld, H-J; Heerklotz, H; Seelig, J
2018-05-25
Apolipoprotein A-1 (Apo A-1) plays an important role in lipid transfer and obesity. Chemical unfolding of α-helical Apo A-1 is induced with guanidineHCl and monitored with differential scanning calorimetry (DSC) and CD spectroscopy. The unfolding enthalpy and the midpoint temperature of unfolding decrease linearly with increasing guanidineHCl concentration, caused by the weak binding of denaturant. At room temperature, binding of 50-60 molecules guanidineHCl leads to a complete Apo A-1 unfolding. The entropy of unfolding decreases to a lesser extent than the unfolding enthalpy. Apo A-1 chemical unfolding is a dynamic multi-state equilibrium that is analysed with the Zimm-Bragg theory modified for chemical unfolding. The chemical Zimm-Bragg theory predicts the denaturant binding constant K D and the protein cooperativity σ. Chemical unfolding of Apo A-1 is two orders of magnitude less cooperative than thermal unfolding. The free energy of thermal unfolding is ~0.2 kcal/mol per amino acid residue and ~1.0 kcal/mol for chemical unfolding at room temperature. The Zimm-Bragg theory calculates conformational probabilities and the chemical Zimm-Bragg theory predicts stretches of α-helical segments in dynamic equilibrium, unfolding and refolding independently and fast. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.
Highly Perturbed pKa Values in the Unfolded State of Hen Egg White Lysozyme
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 separa...
Directory of Open Access Journals (Sweden)
Martin Hampel
Full Text Available The unfolded protein response (UPR, a conserved eukaryotic signaling pathway to ensure protein homeostasis in the endoplasmic reticulum (ER, coordinates biotrophic development in the corn smut fungus Ustilago maydis. Exact timing of UPR activation is required for virulence and presumably connected to the elevated expression of secreted effector proteins during infection of the host plant Zea mays. In the baker's yeast Saccharomyces cerevisiae, expression of UPR target genes is induced upon binding of the central regulator Hac1 to unfolded protein response elements (UPREs in their promoters. While a role of the UPR in effector secretion has been described previously, we investigated a potential UPR-dependent regulation of genes encoding secreted effector proteins. In silico prediction of UPREs in promoter regions identified the previously characterized effector genes pit2 and tin1-1, as bona fide UPR target genes. Furthermore, direct binding of the Hac1-homolog Cib1 to the UPRE containing promoter fragments of both genes was confirmed by quantitative chromatin immunoprecipitation (qChIP analysis. Targeted deletion of the UPRE abolished Cib1-dependent expression of pit2 and significantly affected virulence. Furthermore, ER stress strongly increased Pit2 expression and secretion. This study expands the role of the UPR as a signal hub in fungal virulence and illustrates, how biotrophic fungi can coordinate cellular physiology, development and regulation of secreted virulence factors.
Tanaka, Mizuki; Shintani, Takahiro; Gomi, Katsuya
2015-12-01
Unfolded protein response (UPR) is an intracellular signaling pathway for adaptation to endoplasmic reticulum (ER) stress. In yeast UPR, Ire1 cleaves the unconventional intron of HAC1 mRNA, and the functional Hac1 protein translated from the spliced HAC1 mRNA induces the expression of ER chaperone genes and ER-associated degradation genes for the refolding or degradation of unfolded proteins. In this study, we constructed an ireA (IRE1 ortholog) conditionally expressing strain of Aspergillus oryzae, a filamentous fungus producing a large amount of amylolytic enzymes, and examined the contribution of UPR to ER stress adaptation under physiological conditions. Repression of ireA completely blocked A. oryzae growth under conditions inducing the production of hydrolytic enzymes, such as amylases and proteases. This growth defect was restored by the introduction of unconventional intronless hacA (hacA-i). Furthermore, UPR was observed to be induced by amylolytic gene expression, and the disruption of the transcriptional activator for amylolytic genes resulted in partial growth restoration of the ireA-repressing strain. In addition, a homokaryotic ireA disruption mutant was successfully generated using the strain harboring hacA-i as a parental host. These results indicated that UPR is required for A. oryzae growth to alleviate ER stress induced by excessive production of hydrolytic enzymes. Copyright © 2015 Elsevier Inc. All rights reserved.
Li, Changzhao; Xu, Jianmin; Li, Fugui; Chaudhary, Sandeep C.; Weng, Zhiping; Wen, Jianming; Elmets, Craig A.; Ahsan, Habibul; Athar, Mohammad
2011-01-01
Arsenic exposure through drinking water is a major global public health problem and is associated with an enhanced risk of various cancers including skin cancer. In human skin, arsenic induces precancerous melanosis and keratosis, which may progress to basal cell and squamous cell carcinoma. However, the mechanism by which these pathophysiological alterations occur remains elusive. In this study, we showed that sub-chronic arsenic exposure to SKH-1 mice induced unfolded protein response (UPR)...
Wierenga, P.A.; Egmond, M.R.; Voragen, A.G.J.; Jongh, H.H.J.de
2006-01-01
Unfolding of proteins has often been mentioned as an important factor during the adsorption process at air-water interfaces and in the increase of surface pressure at later stages of the adsorption process. This work focuses on the question whether the folding state of the adsorbed protein depends
mtDNA, Metastasis, and the Mitochondrial Unfolded Protein Response (UPRmt).
Kenny, Timothy C; Germain, Doris
2017-01-01
While several studies have confirmed a link between mitochondrial DNA (mtDNA) mutations and cancer cell metastasis, much debate remains regarding the nature of the alternations in mtDNA leading to this effect. Meanwhile, the mitochondrial unfolded protein response (UPR mt ) has gained much attention in recent years, with most studies of this pathway focusing on its role in aging. However, the UPR mt has also been studied in the context of cancer. More recent work suggests that rather than a single mutation or alternation, specific combinatorial mtDNA landscapes able to activate the UPR mt may be those that are selected by metastatic cells, while mtDNA landscapes unable to activate the UPR mt do not. This review aims at offering an overview of the confusing literature on mtDNA mutations and metastasis and the more recent work on the UPR mt in this setting.
Unfolding energetics and stability of banana lectin.
Gupta, Garima; Sinha, Sharmistha; Surolia, Avadhesha
2008-08-01
The unfolding pathway of banana lectin from Musa paradisiaca was determined by isothermal denaturation induced by the chaotrope GdnCl. The unfolding was found to be a reversible process. The data obtained by isothermal denaturation provided information on conformational stability of banana lectin. The high values of DeltaG of unfolding at various temperatures indicated the strength of intersubunit interactions. It was found that banana lectin is a very stable and denatures at high chaotrope concentrations only. The basis of the stability may be attributed to strong hydrogen bonds of the order 2.5-3.1 A at the dimeric interface along with the presence of water bridges. This is perhaps very unique example in proteins where subunit association is not a consequence of the predominance of hydrophobic interactions. (c) 2008 Wiley-Liss, Inc.
Structural dynamics of the MecA-ClpC complex: a type II AAA+ protein unfolding machine.
Liu, Jing; Mei, Ziqing; Li, Ningning; Qi, Yutao; Xu, Yanji; Shi, Yigong; Wang, Feng; Lei, Jianlin; Gao, Ning
2013-06-14
The MecA-ClpC complex is a bacterial type II AAA(+) molecular machine responsible for regulated unfolding of substrates, such as transcription factors ComK and ComS, and targeting them to ClpP for degradation. The six subunits of the MecA-ClpC complex form a closed barrel-like structure, featured with three stacked rings and a hollow passage, where substrates are threaded and translocated through successive pores. Although the general concepts of how polypeptides are unfolded and translocated by internal pore loops of AAA(+) proteins have long been conceived, the detailed mechanistic model remains elusive. With cryoelectron microscopy, we captured four different structures of the MecA-ClpC complexes. These complexes differ in the nucleotide binding states of the two AAA(+) rings and therefore might presumably reflect distinctive, representative snapshots from a dynamic unfolding cycle of this hexameric complex. Structural analysis reveals that nucleotide binding and hydrolysis modulate the hexameric complex in a number of ways, including the opening of the N-terminal ring, the axial and radial positions of pore loops, the compactness of the C-terminal ring, as well as the relative rotation between the two nucleotide-binding domain rings. More importantly, our structural and biochemical data indicate there is an active allosteric communication between the two AAA(+) rings and suggest that concerted actions of the two AAA(+) rings are required for the efficiency of the substrate unfolding and translocation. These findings provide important mechanistic insights into the dynamic cycle of the MecA-ClpC unfoldase and especially lay a foundation toward the complete understanding of the structural dynamics of the general type II AAA(+) hexamers.
International Nuclear Information System (INIS)
Born, E J; Hartman, S V; Holstein, S A
2013-01-01
Multiple myeloma is characterized by the production of substantial quantities of monoclonal protein. We have previously demonstrated that select inhibitors of the isoprenoid biosynthetic pathway (IBP) induce apoptosis of myeloma cells via inhibition of Rab geranylgeranylation, leading to disruption of monoclonal protein trafficking and induction of the unfolded protein response (UPR) pathway. Heat-shock protein 90 (HSP90) inhibitors disrupt protein folding and are currently under clinical investigation in myeloma. The effects of combining IBP and HSP90 inhibitors on cell death, monoclonal protein trafficking, the UPR and chaperone regulation were investigated in monoclonal protein-producing cells. An enhanced induction of cell death was observed following treatment with IBP and HSP90 inhibitors, which occurred through both ER stress and non-ER stress pathways. The HSP90 inhibitor 17-AAG abrogated the effects of the IBP inhibitors on intracellular monoclonal protein levels and localization as well as induction of the UPR in myeloma cells. Disparate effects on chaperone expression were observed in myeloma vs amyloid light chain cells. Here we demonstrate that the novel strategy of targeting MP trafficking in concert with HSP90 enhances myeloma cell death via a complex modulation of ER stress, UPR, and cell death pathways
Nagelkerke, A.; Bussink, J.; Mujcic, H.; Wouters, B.G.; Lehmann, S.A.; Sweep, F.C.; Span, P.N.
2013-01-01
ABSTRACT: INTRODUCTION: The hypoxia-inducible factor (HIF)-1 pathway can stimulate tumor cell migration and metastasis. Furthermore, hypoxic tumors are associated with a poor prognosis. Besides the HIF-1 pathway, the unfolded protein response (UPR) is also induced by hypoxic conditions. The PKR-like
Scrimale, Thomas; Didone, Louis; de Mesy Bentley, Karen L.; Krysan, Damian J.
2009-01-01
The yeast cell wall is an extracellular structure that is dependent on secretory and membrane proteins for its construction. We investigated the role of protein quality control mechanisms in cell wall integrity and found that the unfolded protein response (UPR) and, to a lesser extent, endoplasmic reticulum (ER)-associated degradation (ERAD) pathways are required for proper cell wall construction. Null mutation of IRE1, double mutation of ERAD components (hrd1Δ and ubc7Δ) and ire1Δ, or expres...
Shaheen, Alaa
2018-05-05
The unfolded protein response (UPR) is an adaptive cellular response that aims to relieve endoplasmic reticulum (ER) stress via several mechanisms, including inhibition of protein synthesis and enhancement of protein folding and degradation. There is a controversy over the effect of the UPR on ER protein export. While some investigators suggested that ER export is inhibited during ER stress, others suggested the opposite. In this article, their conflicting studies are analyzed and compared in attempt to solve this controversy. The UPR appears indeed to enhance ER export, possibly via multiple mechanisms. However, another factor, which is the integrity of the folding machinery/environment inside ER, determines whether ER export will appear increased or decreased during experimentation. Also, different methods of stress induction appear to have different effects on ER export. Thus, improvement of ER export may represent a new mechanism by which the UPR alleviates ER stress. This may help researchers to understand how the UPR works inside cells and how to manipulate it to alter cell fate during stress, either to promote cell survival or death. This may open up new approaches for the treatment of ER stress-related diseases.
Immobilized unfolded cytochrome c acts as a catalyst for dioxygen reduction.
Tavagnacco, Claudio; Monari, Stefano; Ranieri, Antonio; Bortolotti, Carlo Augusto; Peressini, Silvia; Borsari, Marco
2011-10-21
Unfolding turns immobilized cytochrome c into a His-His ligated form endowed with catalytic activity towards O(2), which is absent in the native protein. Dioxygen could be used by naturally occurring unfolded cytochrome c as a substrate for the production of partially reduced oxygen species (PROS) contributing to the cell oxidative stress.
Hombach-Klonisch, Sabine; Mehrpour, Maryam; Shojaei, Shahla; Harlos, Craig; Pitz, Marshall; Hamai, Ahmed; Siemianowicz, Krzysztof; Likus, Wirginia; Wiechec, Emilia; Toyota, Brian D; Hoshyar, Reyhane; Seyfoori, Amir; Sepehri, Zahra; Ande, Sudharsana R; Khadem, Forough; Akbari, Mohsen; Gorman, Adrienne M; Samali, Afshin; Klonisch, Thomas; Ghavami, Saeid
2018-04-01
Despite advances in neurosurgical techniques and radio-/chemotherapy, the treatment of brain tumors remains a challenge. This is particularly true for the most frequent and fatal adult brain tumor, glioblastoma (GB). Upon diagnosis, the average survival time of GB patients remains only approximately 15months. The alkylating drug temozolomide (TMZ) is routinely used in brain tumor patients and induces apoptosis, autophagy and unfolded protein response (UPR). Here, we review these cellular mechanisms and their contributions to TMZ chemoresistance in brain tumors, with a particular emphasis on TMZ chemoresistance in glioma stem cells and GB. Copyright © 2017 Elsevier Inc. All rights reserved.
Puri, Sarita; Chaudhuri, Tapan K
2017-03-01
The conformation and thermodynamic stability of monomeric GroEL were studied by CD and fluorescence spectroscopy. GroEL denaturation with urea and dilution in buffer leads to formation of a folded GroEL monomer. The monomeric nature of this protein was verified by size-exclusion chromatography and native PAGE. It has a well-defined secondary and tertiary structure, folding activity (prevention of aggregation) for substrate protein and is resistant to proteolysis. Being a properly folded and reversibly refoldable, monomeric GroEL is amenable for the study of thermodynamic stability by unfolding transition methods. We present the equilibrium unfolding of monomeric GroEL as studied by urea and heat mediated unfolding processes. The urea mediated unfolding shows two transitions and a single transition in the heat mediated unfolding process. In the case of thermal unfolding, some residual structure unfolds at a higher temperature (70-75°C). The process of folding/unfolding is reversible in both cases. Analysis of folding/unfolding data provides a measure of ΔG NU H 2 O , T m , ΔH van and ΔS van of monomeric GroEL. The thermodynamic stability parameter ΔG NU H 2 O is similar with both CD and intrinsic fluorescence i.e. 7.10±1.0kcal/mol. The calculated T m , ΔH van and ΔS van from the thermal unfolding transition is 46±0.5°C, 43.3±0.1kcal/mol and 143.9±0.1cal/mol/k respectively. Copyright © 2016 Elsevier B.V. All rights reserved.
Thermal, chemical and pH induced unfolding of turmeric root lectin: modes of denaturation.
Directory of Open Access Journals (Sweden)
Himadri Biswas
Full Text Available Curcuma longa rhizome lectin, of non-seed origin having antifungal, antibacterial and α-glucosidase inhibitory activities, forms a homodimer with high thermal stability as well as acid tolerance. Size exclusion chromatography and dynamic light scattering show it to be a dimer at pH 7, but it converts to a monomer near pH 2. Circular dichroism spectra and fluorescence emission maxima are virtually indistinguishable from pH 7 to 2, indicating secondary and tertiary structures remain the same in dimer and monomer within experimental error. The tryptophan environment as probed by acrylamide quenching data yielded very similar data at pH 2 and pH 7, implying very similar folding for monomer and dimer. Differential scanning calorimetry shows a transition at 350.3 K for dimer and at 327.0 K for monomer. Thermal unfolding and chemical unfolding induced by guanidinium chloride for dimer are both reversible and can be described by two-state models. The temperatures and the denaturant concentrations at which one-half of the protein molecules are unfolded, are protein concentration-dependent for dimer but protein concentration-independent for monomer. The free energy of unfolding at 298 K was found to be 5.23 Kcal mol-1 and 14.90 Kcal mol-1 for the monomer and dimer respectively. The value of change in excess heat capacity upon protein denaturation (ΔCp is 3.42 Kcal mol-1 K-1 for dimer. The small ΔCp for unfolding of CLA reflects a buried hydrophobic core in the folded dimeric protein. These unfolding experiments, temperature dependent circular dichroism and dynamic light scattering for the dimer at pH 7 indicate its higher stability than for the monomer at pH 2. This difference in stability of dimeric and monomeric forms highlights the contribution of inter-subunit interactions in the former.
Verification of unfold error estimates in the unfold operator code
International Nuclear Information System (INIS)
Fehl, D.L.; Biggs, F.
1997-01-01
Spectral unfolding is an inverse mathematical operation that attempts to obtain spectral source information from a set of response functions and data measurements. Several unfold algorithms have appeared over the past 30 years; among them is the unfold operator (UFO) code written at Sandia National Laboratories. In addition to an unfolded spectrum, the UFO code also estimates the unfold uncertainty (error) induced by estimated random uncertainties in the data. In UFO the unfold uncertainty is obtained from the error matrix. This built-in estimate has now been compared to error estimates obtained by running the code in a Monte Carlo fashion with prescribed data distributions (Gaussian deviates). In the test problem studied, data were simulated from an arbitrarily chosen blackbody spectrum (10 keV) and a set of overlapping response functions. The data were assumed to have an imprecision of 5% (standard deviation). One hundred random data sets were generated. The built-in estimate of unfold uncertainty agreed with the Monte Carlo estimate to within the statistical resolution of this relatively small sample size (95% confidence level). A possible 10% bias between the two methods was unresolved. The Monte Carlo technique is also useful in underdetermined problems, for which the error matrix method does not apply. UFO has been applied to the diagnosis of low energy x rays emitted by Z-pinch and ion-beam driven hohlraums. copyright 1997 American Institute of Physics
The Myocardial Unfolded Protein Response during Ischemic Cardiovascular Disease
Directory of Open Access Journals (Sweden)
Edward B. Thorp
2012-01-01
Full Text Available Heart failure is a progressive and disabling disease. The incidence of heart failure is also on the rise, particularly in the elderly of industrialized societies. This is in part due to an increased ageing population, whom initially benefits from improved, and life-extending cardiovascular therapy, yet ultimately succumb to myocardial failure. A major cause of heart failure is ischemia secondary to the sequence of events that is dyslipidemia, atherosclerosis, and myocardial infarction. In the case of heart failure postmyocardial infarction, ischemia can lead to myocardial cell death by both necrosis and apoptosis. The extent of myocyte death postinfarction is associated with adverse cardiac remodeling that can contribute to progressive heart chamber dilation, ventricular wall thinning, and the onset of loss of cardiac function. In cardiomyocytes, recent studies indicate that myocardial ischemic injury activates the unfolded protein stress response (UPR and this is associated with increased apoptosis. This paper focuses on the intersection of ischemia, the UPR, and cell death in cardiomyocytes. Targeting of the myocardial UPR may prove to be a viable target for the prevention of myocyte cell loss and the progression of heart failure due to ischemic injury.
Gupta, Amar Nath; Neupane, Krishna; Rezajooei, Negar; Cortez, Leonardo M.; Sim, Valerie L.; Woodside, Michael T.
2016-06-01
The development of small-molecule pharmacological chaperones as therapeutics for protein misfolding diseases has proven challenging, partly because their mechanism of action remains unclear. Here we study Fe-TMPyP, a tetrapyrrole that binds to the prion protein PrP and inhibits misfolding, examining its effects on PrP folding at the single-molecule level with force spectroscopy. Single PrP molecules are unfolded with and without Fe-TMPyP present using optical tweezers. Ligand binding to the native structure increases the unfolding force significantly and alters the transition state for unfolding, making it more brittle and raising the barrier height. Fe-TMPyP also binds the unfolded state, delaying native refolding. Furthermore, Fe-TMPyP binding blocks the formation of a stable misfolded dimer by interfering with intermolecular interactions, acting in a similar manner to some molecular chaperones. The ligand thus promotes native folding by stabilizing the native state while also suppressing interactions driving aggregation.
Hakin, A W; Hedwig, G R
2001-02-15
A recent paper in this journal [Amend and Helgeson, Biophys. Chem. 84 (2000) 105] presented a new group additivity model to calculate various thermodynamic properties of unfolded proteins in aqueous solution. The parameters given for the revised Helgeson-Kirkham-Flowers (HKF) equations of state for all the constituent groups of unfolded proteins can be used, in principle, to calculate the partial molar heat capacity, C(o)p.2, and volume, V2(0), at infinite dilution of any polypeptide. Calculations of the values of C(o)p.2 and V2(0) for several polypeptides have been carried out to test the predictive utility of the HKF group additivity model. The results obtained are in very poor agreement with experimental data, and also with results calculated using a peptide-based group additivity model. A critical assessment of these two additivity models is presented.
Sweezy, J; Veinot, K
2002-01-01
A new multisphere neutron spectrometer unfolding package, Bonner sphere Unfolding Made Simple (BUMS) has been developed that uses an HTML interface to simplify data input and code execution for the novice and the advanced user. This new unfolding package combines the unfolding algorithms contained in other popular unfolding codes under one easy to use interface. The interface makes use of web browsing software to provide a graphical user interface to the unfolding algorithms. BUMS integrates the SPUNIT, BON, MAXIET, and SAND-II unfolding algorithms into a single package. This package also includes a library of 14 response matrices, 58 starting spectra, and 24 dose and detector responses. BUMS has several improvements beyond the addition of unfolding algorithms. It has the ability to search for the most appropriate starting spectra. Also, plots of the unfolded neutron spectra are automatically generated. The BUMS package runs via a web server and may be accessed by any computer with access to the Internet at h...
International Nuclear Information System (INIS)
Sweezy, Jeremy; Hertel, Nolan; Veinot, Ken
2002-01-01
A new multisphere neutron spectrometer unfolding package, Bonner sphere Unfolding Made Simple (BUMS) has been developed that uses an HTML interface to simplify data input and code execution for the novice and the advanced user. This new unfolding package combines the unfolding algorithms contained in other popular unfolding codes under one easy to use interface. The interface makes use of web browsing software to provide a graphical user interface to the unfolding algorithms. BUMS integrates the SPUNIT, BON, MAXIET, and SAND-II unfolding algorithms into a single package. This package also includes a library of 14 response matrices, 58 starting spectra, and 24 dose and detector responses. BUMS has several improvements beyond the addition of unfolding algorithms. It has the ability to search for the most appropriate starting spectra. Also, plots of the unfolded neutron spectra are automatically generated. The BUMS package runs via a web server and may be accessed by any computer with access to the Internet at http://nukeisit.gatech.edu/bums
RPA-mediated unfolding of systematically varying G-quadruplex structures.
Ray, Sujay; Qureshi, Mohammad H; Malcolm, Dominic W; Budhathoki, Jagat B; Celik, Uğur; Balci, Hamza
2013-05-21
G-quadruplex (GQ) is a noncanonical nucleic acid structure that is formed by guanine rich sequences. Unless it is destabilized by proteins such as replication protein A (RPA), GQ could interfere with DNA metabolic functions, such as replication or repair. We studied RPA-mediated GQ unfolding using single-molecule FRET on two groups of GQ structures that have different loop lengths and different numbers of G-tetrad layers. We observed a linear increase in the steady-state stability of the GQ against RPA-mediated unfolding with increasing number of layers or decreasing loop length. The stability demonstrated by different GQ structures varied by at least three orders of magnitude. Those with shorter loops (less than three nucleotides long) or a greater number of layers (more than three layers) maintained a significant folded population even at physiological RPA concentration (≈1 μM), raising the possibility of physiological viability of such GQ structures. Finally, we measured the transition time between the start and end of the RPA-mediated GQ unfolding process to be 0.35 ± 0.10 s for all GQ constructs we studied, despite significant differences in their steady-state stabilities. We propose a two-step RPA-mediated GQ unfolding mechanism that is consistent with our observations. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Wiley, Sandra E; Andreyev, Alexander Y; Divakaruni, Ajit S; Karisch, Robert; Perkins, Guy; Wall, Estelle A; van der Geer, Peter; Chen, Yi-Fan; Tsai, Ting-Fen; Simon, Melvin I; Neel, Benjamin G; Dixon, Jack E; Murphy, Anne N
2013-06-01
Miner1 is a redox-active 2Fe2S cluster protein. Mutations in Miner1 result in Wolfram Syndrome, a metabolic disease associated with diabetes, blindness, deafness, and a shortened lifespan. Embryonic fibroblasts from Miner1(-/-) mice displayed ER stress and showed hallmarks of the unfolded protein response. In addition, loss of Miner1 caused a depletion of ER Ca(2+) stores, a dramatic increase in mitochondrial Ca(2+) load, increased reactive oxygen and nitrogen species, an increase in the GSSG/GSH and NAD(+)/NADH ratios, and an increase in the ADP/ATP ratio consistent with enhanced ATP utilization. Furthermore, mitochondria in fibroblasts lacking Miner1 displayed ultrastructural alterations, such as increased cristae density and punctate morphology, and an increase in O2 consumption. Treatment with the sulphydryl anti-oxidant N-acetylcysteine reversed the abnormalities in the Miner1 deficient cells, suggesting that sulphydryl reducing agents should be explored as a treatment for this rare genetic disease. Copyright © 2013 The Authors. Published by John Wiley and Sons, Ltd on behalf of EMBO.
Equilibrium unfolding of A. niger RNase: pH dependence of chemical and thermal denaturation.
Kumar, Gundampati Ravi; Sharma, Anurag; Kumari, Moni; Jagannadham, Medicherla V; Debnath, Mira
2011-08-01
Equilibrium unfolding of A. niger RNase with chemical denaturants, for example GuHCl and urea, and thermal unfolding have been studied as a function of pH using fluorescence, far-UV, near-UV, and absorbance spectroscopy. Because of their ability to affect electrostatic interactions, pH and chemical denaturants have a marked effect on the stability, structure, and function of many globular proteins. ANS binding studies have been conducted to enable understanding of the folding mechanism of the protein in the presence of the denaturants. Spectroscopic studies by absorbance, fluorescence, and circular dichroism and use of K2D software revealed that the enzyme has α + β type secondary structure with approximately 29% α-helix, 24% β-sheet, and 47% random coil. Under neutral conditions the enzyme is stable in urea whereas GuHCl-induced equilibrium unfolding was cooperative. A. niger RNase has little ANS binding even under neutral conditions. Multiple intermediates were populated during the pH-induced unfolding of A. niger RNase. Urea and temperature-induced unfolding of A. niger RNase into the molten globule-like state is non-cooperative, in contrast to the cooperativity seen with the native protein, suggesting the presence of two parts/domains, in the molecular structure of A. niger RNase, with different stability that unfolds in steps. Interestingly, the GuHCl-induced unfolding of the A state (molten globule state) of A. niger RNase is unique, because a low concentration of denaturant not only induces structural change but also facilitates transition from one molten globule like state (A(MG1)) into another (I(MG2)).
DEFF Research Database (Denmark)
Alm, Petter S; de Castro Barbosa, Thais; Barrès, Romain
2017-01-01
OBJECTIVE: Parental nutrition and lifestyle impact the metabolic phenotype of the offspring. We have reported that grandpaternal chronic high-fat diet (HFD) transgenerationally impairs glucose metabolism in subsequent generations. Here we determined whether grandpaternal diet transgenerationally....... Gene set enrichment analysis (GSEA) was performed to determine pathways reprogrammed by grandpaternal diet. RESULTS: GSEA revealed an enrichment of the unfolded protein response pathway in skeletal muscle of grand-offspring from HFD-fed grandfathers compared to grand-offspring of chow-fed males....... Activation of the stress sensor (ATF6α), may be a pivotal point whereby this pathway is activated. Interestingly, skeletal muscle from F1-offspring was not affected in a similar manner. No major changes were observed in the skeletal muscle lipidome profile due to grandpaternal diet. CONCLUSIONS...
DEFF Research Database (Denmark)
Saad-Sulonen, Joanna; Halskov, Kim; Eriksson, Eva
2015-01-01
The aim of the Unfolding Participation workshop is to outline an agenda for the next 10 years of participatory design (PD) and participatory human computer interaction (HCI) research. We will do that through a double strategy: 1) by critically interrogating the concept of participation (unfolding...... the concept itself), while at the same time, 2) reflecting on the way that participation unfolds across different participatory configurations. We invite researchers and practitioners from PD and HCI and fields in which information technology mediated participation is embedded (e.g. in political studies......, urban planning, participatory arts, business, science and technology studies) to bring a plurality of perspectives and expertise related to participation....
Many channel spectrum unfolding
International Nuclear Information System (INIS)
Najzer, M.; Glumac, B.; Pauko, M.
1980-01-01
The principle of the ITER unfolding code as used for the many channel spectrum unfolding is described. Its unfolding ability is tested on seven typical neutron spectra. The effect of the initial spectrum approximation upon the solution is discussed
Geometrical analysis of cytochrome c unfolding
Urie, Kristopher G.; Pletneva, Ekaterina; Gray, Harry B.; Winkler, Jay R.; Kozak, John J.
2011-01-01
A geometrical model has been developed to study the unfolding of iso-1 cytochrome c. The model draws on the crystallographic data reported for this protein. These data were used to calculate the distance between specific residues in the folded state, and in a sequence of extended states defined by n = 3, 5, 7, 9, 11, 13, and 15 residue units. Exact calculations carried out for each of the 103 residues in the polypeptide chain demonstrate that different regions of the chain have different unfolding histories. Regions where there is a persistence of compact structures can be identified, and this geometrical characterization is fully consistent with analyses of time-resolved fluorescence energy-transfer (TrFET) data using dansyl-derivatized cysteine side-chain probes at positions 39, 50, 66, 85, and 99. The calculations were carried out assuming that different regions of the polypeptide chain unfold synchronously. To test this assumption, lattice Monte Carlo simulations were performed to study systematically the possible importance of asynchronicity. Calculations show that small departures from synchronous dynamics can arise if displacements of residues in the main body of the chain are much more sluggish than near-terminal residues.
Contribution of long-range interactions to the secondary structure of an unfolded globin.
Fedyukina, Daria V; Rajagopalan, Senapathy; Sekhar, Ashok; Fulmer, Eric C; Eun, Ye-Jin; Cavagnero, Silvia
2010-09-08
This work explores the effect of long-range tertiary contacts on the distribution of residual secondary structure in the unfolded state of an alpha-helical protein. N-terminal fragments of increasing length, in conjunction with multidimensional nuclear magnetic resonance, were employed. A protein representative of the ubiquitous globin fold was chosen as the model system. We found that, while most of the detectable alpha-helical population in the unfolded ensemble does not depend on the presence of the C-terminal region (corresponding to the native G and H helices), specific N-to-C long-range contacts between the H and A-B-C regions enhance the helical secondary structure content of the N terminus (A-B-C regions). The simple approach introduced here, based on the evaluation of N-terminal polypeptide fragments of increasing length, is of general applicability to identify the influence of long-range interactions in unfolded proteins. Copyright 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Prediction and analysis of structure, stability and unfolding of thermolysin-like proteases
Vriend, Gert; Eijsink, Vincent
1993-08-01
Bacillus neutral proteases (NPs) form a group of well-characterized homologous enzymes, that exhibit large differences in thermostability. The three-dimensional (3D) structures of several of these enzymes have been modelled on the basis of the crystal structures of the NPs of B. thermoproteolyticus (thermolysin) and B. cercus. Several new techniques have been developed to improve the model-building procedures. Also a model-building by mutagenesis' strategy was used, in which mutants were designed just to shed light on parts of the structures that were particularly hard to model. The NP models have been used for the prediction of site-directed mutations aimed at improving the thermostability of the enzymes. Predictions were made using several novel computational techniques, such as position-specific rotamer searching, packing quality analysis and property-profile database searches. Many stabilizing mutations were predicted and produced: improvement of hydrogen bonding, exclusion of buried water molecules, capping helices, improvement of hydrophobic interactions and entropic stabilization have been applied successfully. At elevated temperatures NPs are irreversibly inactivated as a result of autolysis. It has been shown that this denaturation process is independent of the protease activity and concentration and that the inactivation follows first-order kinetics. From this it has been conjectured that local unfolding of (surface) loops, which renders the protein susceptible to autolysis, is the rate-limiting step. Despite the particular nature of the thermal denaturation process, normal rules for protein stability can be applied to NPs. However, rather than stabilizing the whole protein against global unfolding, only a small region has to be protected against local unfolding. In contrast to proteins in general, mutational effects in proteases are not additive and their magnitude is strongly dependent on the location of the mutation. Mutations that alter the stability
Activation of the Unfolded Protein Response Contributes toward the Antitumor Activity of Vorinostat
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Soumen Kahali
2010-01-01
Full Text Available Histone deacetylase (HDAC inhibitors represent an emerging class of anticancer agents progressing through clinical trials. Although their primary target is thought to involve acetylation of core histones, several nonhistone substrates have been identified, including heat shock protein (HSP 90, which may contribute towards their antitumor activity. Glucose-regulated protein 78 (GRP78 is a member of the HSP family of molecular chaperones and plays a central role in regulating the unfolded protein response (UPR. Emerging data suggest that GRP78 is critical in cellular adaptation and survival associated with oncogenesis and may serve as a cancer-specific therapeutic target. On the basis of shared homology with HSP family proteins, we sought to determine whether GRP78 could serve as a molecular target of the HDAC inhibitor vorinostat. Vorinostat treatment led to GRP78 acetylation, dissociation, and subsequent activation of its client protein double-stranded RNA-activated protein-like endoplasmic reticulum kinase (PERK. Investigations in a panel of cancer cell lines identified that UPR activation after vorinostat exposure is specific to certain lines. Mass spectrometry performed on immunoprecipitated GRP78 identified lysine-585 as a specific vorinostat-induced acetylation site of GRP78. Downstream activation of the UPR was confirmed, including eukaryotic initiating factor 2α phosphorylation and increase in ATF4 and C/EBP homologous protein expression. To determine the biologic relevance of UPR activation after vorinostat, RNA interference of PERK was performed, demonstrating significantly decreased sensitivity to vorinostat-induced cytotoxicity. Collectively, these findings indicate that GRP78 is a biologic target of vorinostat, and activation of the UPR through PERK phosphorylation contributes toward its antitumor activity.
Murciano-Calles, Javier; Güell-Bosch, Jofre; Villegas, Sandra; Martinez, Jose C
2016-01-12
PDZ domains are protein-protein interaction modules sharing the same structural arrangement. To discern whether they display common features in their unfolding/misfolding behaviour we have analyzed in this work the unfolding thermodynamics, together with the misfolding kinetics, of the PDZ fold using three archetypical examples: the second and third PDZ domains of the PSD95 protein and the Erbin PDZ domain. Results showed that all domains passed through a common intermediate, which populated upon unfolding, and that this in turn drove the misfolding towards worm-like fibrillar structures. Thus, the unfolding/misfolding behaviour appears to be shared within these domains. We have also analyzed how this landscape can be modified upon the inclusion of extra-elements, as it is in the nNOS PDZ domain, or the organization of swapped species, as happens in the second PDZ domain of the ZO2 protein. Although the intermediates still formed upon thermal unfolding, the misfolding was prevented to varying degrees.
Vandewynckel, Yves-Paul; Coucke, Céline; Laukens, Debby; Devisscher, Lindsey; Paridaens, Annelies; Bogaerts, Eliene; Vandierendonck, Astrid; Raevens, Sarah; Verhelst, Xavier; Van Steenkiste, Christophe; Libbrecht, Louis; Geerts, Anja; Van Vlierberghe, Hans
2016-06-07
Hepatocellular carcinoma (HCC) responds poorly to conventional systemic therapies. The first-in-class proteasome inhibitor bortezomib has been approved in clinical use for hematologic malignancies and has shown modest activity in solid tumors, including HCC. However, a considerable proportion of patients fail to respond and experience important adverse events. Recently, the next-generation orally bioavailable irreversible proteasome inhibitor oprozomib was developed. Here, we assessed the efficacy of oprozomib and its effects on the unfolded protein response (UPR), a signaling cascade activated through the ATF6, PERK and IRE1 pathways by accumulation of unfolded proteins in the endoplasmic reticulum, in HCC. The effects of oprozomib and the role of the UPR were evaluated in HCC cell lines and in diethylnitrosamine-induced and xenograft mouse models for HCC. Oprozomib dose-dependently reduced the viability and proliferation of human HCC cells. Unexpectedly, oprozomib-treated cells displayed diminished cytoprotective ATF6-mediated signal transduction as well as unaltered PERK and IRE1 signaling. However, oprozomib increased pro-apoptotic UPR-mediated protein levels by prolonging their half-life, implying that the proteasome acts as a negative UPR regulator. Supplementary boosting of UPR activity synergistically improved the sensitivity to oprozomib via the PERK pathway. Oral oprozomib displayed significant antitumor effects in the orthotopic and xenograft models for HCC, and importantly, combining oprozomib with different UPR activators enhanced the antitumor efficacy by stimulating UPR-induced apoptosis without cumulative toxicity. In conclusion, next-generation proteasome inhibition by oprozomib results in dysregulated UPR activation in HCC. This finding can be exploited to enhance the antitumor efficacy by combining oprozomib with clinically applicable UPR activators.
A new paradigm: innate immune sensing of viruses via the Unfolded Protein Response
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Judith A Smith
2014-05-01
Full Text Available The immune system depends upon combinations of signals to mount appropriate responses: pathogen specific signals in the context of co-stimulatory danger signals drive immune strength and accuracy. Viral infections trigger anti-viral type I interferon (IFN responses by stimulating endosomal and cytosolic pattern recognition receptors (PRRs. However, viruses have also evolved many strategies to counteract IFN responses. Are there intracellular danger signals that enhance immune responses to viruses? During infection, viruses place a heavy demand on the protein folding machinery of the host endoplasmic reticulum (ER. To survive ER stress, host cells mount an Unfolded Protein Response (UPR to decrease ER protein load and enhance protein-folding capacity. Viruses also directly elicit the UPR to enhance their replication. Increasing evidence supports an intersection between the host UPR and inflammation, in particular the production of pro-inflammatory cytokines and type I IFN. The UPR directly activates pro-inflammatory cytokine transcription factors and dramatically enhances cytokine production in response to viral PRR engagement. Additionally, viral PRR engagement may stimulate specific pathways within the UPR to enhance cytokine production. Through these mechanisms, viral detection via the UPR and inflammatory cytokine production are intertwined. Consequently, the UPR response is perfectly poised to act as an infection-triggered danger signal. The UPR may serve as an internal co-stimulatory signal that 1 provides specificity and 2 critically augments responses to overcome viral subterfuge. Further work is needed to test this hypothesis during viral infections.
Roy, Sourav; Basu, Sankar; Dasgupta, Dipak; Bhattacharyya, Dhananjay; Banerjee, Rahul
2015-01-01
Currently, considerable interest exists with regard to the dissociation of close packed aminoacids within proteins, in the course of unfolding, which could result in either wet or dry moltenglobules. The progressive disjuncture of residues constituting the hydrophobic core ofcyclophilin from L. donovani (LdCyp) has been studied during the thermal unfolding of the molecule, by molecular dynamics simulations. LdCyp has been represented as a surface contactnetwork (SCN) based on the surface complementarity (Sm) of interacting residues within themolecular interior. The application of Sm to side chain packing within proteins make it a very sensitive indicator of subtle perturbations in packing, in the thermal unfolding of the protein. Network based metrics have been defined to track the sequential changes in the disintegration ofthe SCN spanning the hydrophobic core of LdCyp and these metrics prove to be highly sensitive compared to traditional metrics in indicating the increased conformational (and dynamical) flexibility in the network. These metrics have been applied to suggest criteria distinguishing DMG, WMG and transition state ensembles and to identify key residues involved in crucial conformational/topological events during the unfolding process. PMID:26545107
Yano, Yohko F; Uruga, Tomoya; Tanida, Hajime; Toyokawa, Hidenori; Terada, Yasuko; Takagaki, Masafumi; Yamada, Hironari
2009-01-06
Time-resolved X-ray reflectivity measurements for lysozyme (LSZ) adsorbed at an air/water interface were performed to study the mechanism of adsorption-induced protein unfolding. The time dependence of the density profile at the air/water interface revealed that the molecular conformation changed significantly during adsorption. Taking into account previous work using Fourier transform infrared (FTIR) spectroscopy, we propose that the LSZ molecules initially adsorbed on the air/water interface have a flat unfolded structure, forming antiparallel beta-sheets as a result of hydrophobic interactions with the gas phase. In contrast, as adsorption continues, a second layer forms in which the molecules have a very loose structure having random coils as a result of hydrophilic interactions with the hydrophilic groups that protrude from the first layer.
Sun, Shengrong; Wang, Xiong; Wang, Changhua; Nawaz, Ahmed; Wei, Wen; Li, Juanjuan; Wang, Lijun; Yu, De-Hua
2011-01-01
The involvement of unfolded protein response (UPR) activation in tumor survival and resistance to chemotherapies suggests a new anticancer strategy targeting UPR pathway. Arctigenin, a natural product, has been recently identified for its antitumor activity with selective toxicity against cancer cells under glucose starvation with unknown mechanism. Here we found that arctigenin specifically blocks the transcriptional induction of two potential anticancer targets, namely glucose-regulated protein-78 (GRP78) and its analog GRP94, under glucose deprivation, but not by tunicamycin. The activation of other UPR pathways, e.g., XBP-1 and ATF4, by glucose deprivation was also suppressed by arctigenin. A further transgene experiment showed that ectopic expression of GRP78 at least partially rescued arctigenin/glucose starvation-mediated cell growth inhibition, suggesting the causal role of UPR suppression in arctigenin-mediated cytotoxicity under glucose starvation. These observations bring a new insight into the mechanism of action of arctigenin and may lead to the design of new anticancer therapeutics. © Georg Thieme Verlag KG Stuttgart · New York.
DEFF Research Database (Denmark)
Holm, Jan; Babol, Linnea N.; Markova, Natalia
2014-01-01
The present study utilized a combination of DLS (dynamic light scattering) and DSC (differential scanning calorimetry) to address thermostability of high-affinity folate binding protein (FBP), a transport protein and cellular receptor for the vitamin folate. At pH7.4 (pI=7-8) ligand binding......, intermolecular forces involved in concentration-dependent multimerization thus contribute to the thermostability of holo-FBP. Hence, thermal unfolding and dissociation of holo-FBP multimers occur simultaneously consistent with a gradual decrease from octameric to monomeric holo-FBP (10μM) in DLS after a step-wise...
Directory of Open Access Journals (Sweden)
Lifeng Yang
2017-05-01
Full Text Available Prion diseases are neurodegenerative pathologies characterized by the accumulation of a protease-resistant form of the cellular prion protein named prion protein scrapie (PrPSc in the brain. PrPSc accumulation in the endoplasmic reticulum (ER result in a dysregulated calcium (Ca2+ homeostasis and subsequent initiation of unfolded protein response (UPR leading to neuronal dysfunction and apoptosis. The molecular mechanisms for the transition between adaptation to ER stress and ER stress-induced apoptosis are still unclear. Mitogen-activated protein kinases (MAPKs are serine/threonine protein kinases that rule the signaling of many extracellular stimuli from plasma membrane to the nucleus. However the identification of numerous points of cross talk between the UPR and MAPK signaling pathways may contribute to our understanding of the consequences of ER stress in prion diseases. Indeed the MAPK signaling network is known to regulate cell cycle progression and cell survival or death responses following a variety of stresses including misfolded protein response stress. In this article, we review the UPR signaling in prion diseases and discuss the triad of MAPK signaling pathways. We also describe the role played by MAPK signaling cascades in Alzheimer’s (AD and Parkinson’s disease (PD. We will also overview the mechanisms of cell death and the role of MAPK signaling in prion disease progression and highlight potential avenues for therapeutic intervention.
Directory of Open Access Journals (Sweden)
Emily F A van 't Wout
2015-06-01
Full Text Available Pseudomonas aeruginosa infection can be disastrous in chronic lung diseases such as cystic fibrosis and chronic obstructive pulmonary disease. Its toxic effects are largely mediated by secreted virulence factors including pyocyanin, elastase and alkaline protease (AprA. Efficient functioning of the endoplasmic reticulum (ER is crucial for cell survival and appropriate immune responses, while an excess of unfolded proteins within the ER leads to "ER stress" and activation of the "unfolded protein response" (UPR. Bacterial infection and Toll-like receptor activation trigger the UPR most likely due to the increased demand for protein folding of inflammatory mediators. In this study, we show that cell-free conditioned medium of the PAO1 strain of P. aeruginosa, containing secreted virulence factors, induces ER stress in primary bronchial epithelial cells as evidenced by splicing of XBP1 mRNA and induction of CHOP, GRP78 and GADD34 expression. Most aspects of the ER stress response were dependent on TAK1 and p38 MAPK, except for the induction of GADD34 mRNA. Using various mutant strains and purified virulence factors, we identified pyocyanin and AprA as inducers of ER stress. However, the induction of GADD34 was mediated by an ER stress-independent integrated stress response (ISR which was at least partly dependent on the iron-sensing eIF2α kinase HRI. Our data strongly suggest that this increased GADD34 expression served to protect against Pseudomonas-induced, iron-sensitive cell cytotoxicity. In summary, virulence factors from P. aeruginosa induce ER stress in airway epithelial cells and also trigger the ISR to improve cell survival of the host.
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 eIF2α kinase HRI. Our data strongly suggest that this increased GADD34 expression served to protect against Pseudomonas-induced, iron-sensitive cell cytotoxicity. In summary, virulence factors from P. aeruginosa induce ER stress in airway epithelial cells and also trigger the ISR to improve cell survival of the host. PMID:26083346
Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis.
Why do total-body decay curves of iodine-labeled proteins begin with a delay
International Nuclear Information System (INIS)
Regoeczi, E.
1987-01-01
The initial delay that occurs in total-body radiation curves reaching their single-exponential slopes was analyzed from 106 experiments involving several mammalian species (guinea pig, mouse, rabbit, and rat) and plasma proteins (alpha 1-acid glycoprotein, antithrombin III, fibrinogen, immunoglobulin G, and transferrin) in 14 different combinations. The time interval (Td) between injection and the intercept of the slope with the full-dose value was adopted as a measure of curve nonideality. The overall mean Td was 6.6 h, but individual values showed a significant correlation to protein half-lives, whereby proteins of unequal metabolic properties exhibited different mean Td values. Targeting protein to the liver abolished delay. Choice of the isotope ( 125 I or 131 I) and size of the labeled protein had no influence on the magnitude of delay. Whole-body radiation curves of animals that received [ 125 I]iodotyrosines, Na 131 I, or 131 I-polyvinylpyrrolidone exhibited no initial delays. These results do not support the earlier notion that delay is caused by a redistribution of the labeled protein in the body to radiometrically more favorable sites. However, they are compatible with the assumption that delayed passage of a protein dose through the extracellular matrix and/or retarded transfer of proteolytic products from extravascular catabolic sites to plasma may be responsible for the phenomenon
Rana, T; Shinde, V M; Starr, C R; Kruglov, A A; Boitet, E R; Kotla, P; Zolotukhin, S; Gross, A K; Gorbatyuk, M S
2014-12-18
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-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α), 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-1β, 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-1β was capable of inducing retinal degeneration by injecting C57BL6 mice with a recombinant IL-1β. We observed ~19% reduction in ERG a-wave amplitudes and a 29% loss of photoreceptor cells compared with
Komoike, Yuta; Matsuoka, Masato
2013-10-15
Tributyltin (TBT) is a major marine contaminant and causes endocrine disruption, hepatotoxicity, immunotoxicity, and neurotoxicity. However, the molecular mechanisms underlying the toxicity of TBT have not been fully elucidated. We examined whether exposure to TBT induces the endoplasmic reticulum (ER) stress response in zebrafish, a model organism. Zebrafish-derived BRF41 fibroblast cells were exposed to 0.5 or 1 μM TBT for 0.5-16 h and subsequently lysed and immunoblotted to detect ER stress-related proteins. Zebrafish embryos, grown until 32 h post fertilization (hpf), were exposed to 1 μM TBT for 16 h and used in whole mount in situ hybridization and immunohistochemistry to visualize the expression of ER chaperones and an ER stress-related apoptosis factor. Exposure of the BRF41 cells to TBT caused phosphorylation of the zebrafish homolog of protein kinase RNA-activated-like ER kinase (PERK), eukaryotic translation initiation factor 2 alpha (eIF2α), and inositol-requiring enzyme 1 (IRE1), characteristic splicing of X-box binding protein 1 (XBP1) mRNA, and enhanced expression of activating transcription factor 4 (ATF4) protein. In TBT-exposed zebrafish embryos, ectopic expression of the gene encoding zebrafish homolog of the 78 kDa glucose-regulating protein (GRP78) and gene encoding CCAAT/enhancer-binding protein homologous protein (CHOP) was detected in the precursors of the neuromast, which is a sensory organ for detecting water flow and vibration. Our in vitro and in vivo studies revealed that exposure of zebrafish to TBT induces the ER stress response via activation of both the PERK-eIF2α and IRE1-XBP1 pathways of the unfolded protein response (UPR) in an organ-specific manner. Copyright © 2013 Elsevier B.V. All rights reserved.
Detection and characterization of partially unfolded oligomers of the SH3 domain of α-Spectrin
Casares, S.; Sadqi, M.; López-Mayorga, O.; Conejero-Lara, F.; van Nuland, N.A.J.
2004-01-01
For the purpose of equilibrium and kinetic folding-unfolding studies, the SH3 domain of α-spectrin (spc-SH3) has long been considered a classic two-state folding protein. In this work we have indeed observed that the thermal unfolding curves of spc-SH3 measured at pH 3.0 by differential scanning
Cigarette smoke induces an unfolded protein response in the human lung: a proteomic approach.
Kelsen, Steven G; Duan, Xunbao; Ji, Rong; Perez, Oscar; Liu, Chunli; Merali, Salim
2008-05-01
Cigarette smoking, which exposes the lung to high concentrations of reactive oxidant species (ROS) is the major risk factor for chronic obstructive pulmonary disease (COPD). Recent studies indicate that ROS interfere with protein folding in the endoplasmic reticulum and elicit a compensatory response termed the "unfolded protein response" (UPR). The importance of the UPR lies in its ability to alter expression of a variety of genes involved in antioxidant defense, inflammation, energy metabolism, protein synthesis, apoptosis, and cell cycle regulation. The present study used comparative proteomic technology to test the hypothesis that chronic cigarette smoking induces a UPR in the human lung. Studies were performed on lung tissue samples obtained from three groups of human subjects: nonsmokers, chronic cigarette smokers, and ex-smokers. Proteomes of lung samples from chronic cigarette smokers demonstrated 26 differentially expressed proteins (20 were up-regulated, 5 were down-regulated, and 1 was detected only in the smoking group) compared with nonsmokers. Several UPR proteins were up-regulated in smokers compared with nonsmokers and ex-smokers, including the chaperones, glucose-regulated protein 78 (GRP78) and calreticulin; a foldase, protein disulfide isomerase (PDI); and enzymes involved in antioxidant defense. In cultured human airway epithelial cells, GRP78 and the UPR-regulated basic leucine zipper, transcription factors, ATF4 and Nrf2, which enhance expression of important anti-oxidant genes, increased rapidly (< 24 h) with cigarette smoke extract. These data indicate that cigarette smoke induces a UPR response in the human lung that is rapid in onset, concentration dependent, and at least partially reversible with smoking cessation. We speculate that activation of a UPR by cigarette smoke may protect the lung from oxidant injury and the development of COPD.
Directory of Open Access Journals (Sweden)
Paolo Dametto
Full Text Available 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 (PrPΔ141-225, 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 eIF2α and upregulation of CHOP ultimately leading to neurodegeration similar to what was observed in prion infection.
Stress responses in flavivirus-infected cells: activation of unfolded protein response and autophagy
Directory of Open Access Journals (Sweden)
Ana-Belén eBlázquez
2014-06-01
Full Text Available 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.
Endoplasmic Reticulum Stress, Unfolded Protein Response, and Cancer Cell Fate
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Marco Corazzari
2017-04-01
Full Text Available Perturbation of endoplasmic reticulum (ER homeostasis results in a stress condition termed “ER stress” determining the activation of a finely regulated program defined as unfolded protein response (UPR and whose primary aim is to restore this organelle’s physiological activity. Several physiological and pathological stimuli deregulate normal ER activity causing UPR activation, such as hypoxia, glucose shortage, genome instability, and cytotoxic compounds administration. Some of these stimuli are frequently observed during uncontrolled proliferation of transformed cells, resulting in tumor core formation and stage progression. Therefore, it is not surprising that ER stress is usually induced during solid tumor development and stage progression, becoming an hallmark of such malignancies. Several UPR components are in fact deregulated in different tumor types, and accumulating data indicate their active involvement in tumor development/progression. However, although the UPR program is primarily a pro-survival process, sustained and/or prolonged stress may result in cell death induction. Therefore, understanding the mechanism(s regulating the cell survival/death decision under ER stress condition may be crucial in order to specifically target tumor cells and possibly circumvent or overcome tumor resistance to therapies. In this review, we discuss the role played by the UPR program in tumor initiation, progression and resistance to therapy, highlighting the recent advances that have improved our understanding of the molecular mechanisms that regulate the survival/death switch.
Structural changes during the unfolding of Bovine serum albumin
Indian Academy of Sciences (India)
The native form of serum albumin is the most important soluble protein in the body plasma. In order to investigate the structural changes of Bovine serum albumin (BSA) during its unfolding in the presence of urea, a small-angle neutron scattering (SANS) study was performed. The scattering curves of dilute solutions of BSA ...
High-Temperature unfolding of a trp-Cage mini-protein: a molecular dynamics simulation study
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Seshasayee Aswin Sai Narain
2005-03-01
Full Text Available Abstract Background Trp cage is a recently-constructed fast-folding miniprotein. It consists of a short helix, a 3,10 helix and a C-terminal poly-proline that packs against a Trp in the alpha helix. It is known to fold within 4 ns. Results High-temperature unfolding molecular dynamics simulations of the Trp cage miniprotein have been carried out in explicit water using the OPLS-AA force-field incorporated in the program GROMACS. The radius of gyration (Rg and Root Mean Square Deviation (RMSD have been used as order parameters to follow the unfolding process. Distributions of Rg were used to identify ensembles. Conclusion Three ensembles could be identified. While the native-state ensemble shows an Rg distribution that is slightly skewed, the second ensemble, which is presumably the Transition State Ensemble (TSE, shows an excellent fit. The denatured ensemble shows large fluctuations, but a Gaussian curve could be fitted. This means that the unfolding process is two-state. Representative structures from each of these ensembles are presented here.
Selective induction of cyclin B protein abrogates the G2 delay after irradiation
International Nuclear Information System (INIS)
Kao, G.; Muschel, R.J.; Maity, A.; Kunig, A.; McKenna, W.G.
1996-01-01
Purpose/Objective: Irradiation of tumor cells commonly results in G2 delay, which has been postulated to allow DNA repair and cell survival. The G2 delay after irradiation is also often marked in some cell lines by delayed expression of cyclin B protein, suggesting a role for cyclin B regulation. Investigations of these hypotheses however has been hampered by the inability to selectively perturb the G2 delay in a physiologic manner. Materials and Methods: We have devised a system, with which we are able to selectively induce cyclin B protein expression in vivo at specific points in the cell cycle, by transfecting Hela cells with an expression vector under control of a dexamethasone-inducible promoter. Experiments were subsequently performed by synchronizing, releasing, irradiating, inducing, and harvesting these cells through the cell cycle. Results: Irradiation with 5 Gy led to a pronounced G2 delay, reflected by markedly slowed progression into mitosis, concomitant with reduced expression of cyclin B protein. Induction of cyclin B after radiation in these cells abrogated the G2 delay by approximately doubling the rate at which the cells re-enter mitosis. Treatment of irradiated untransfected control cells with dexamethasone, in which cyclin B is not induced, led to minimal changes. Studies of effects of cyclin B induction on cyclin B localization (using immunofluorescence), cdc2 phosphorylation and activation will also be presented. Conclusion: This system should allow further investigations into fundamental mechanisms of cell cycle regulation after irradiation and DNA damage. This also provides direct evidence for the first time that cyclin B protein regulation may play a role in the G2 delay following irradiation in Hela cells, perhaps complementing phosphorylation events
LENUS (Irish Health Repository)
Carroll, Tomás P
2010-04-15
The hereditary disorder alpha-1 antitrypsin (AAT) deficiency results from mutations in the SERPINA1 gene and presents with emphysema in young adults and liver disease in childhood. The most common form of AAT deficiency occurs because of the Z mutation, causing the protein to fold aberrantly and accumulate in the endoplasmic reticulum (ER). This leads to ER stress and contributes significantly to the liver disease associated with the condition. In addition to hepatocytes, AAT is also synthesized by monocytes, neutrophils, and epithelial cells. In this study we show for the first time that the unfolded protein response (UPR) is activated in quiescent monocytes from ZZ individuals. Activating transcription factor 4, X-box binding protein 1, and a subset of genes involved in the UPR are increased in monocytes from ZZ compared with MM individuals. This contributes to an inflammatory phenotype with ZZ monocytes exhibiting enhanced cytokine production and activation of the NF-kappaB pathway when compared with MM monocytes. In addition, we demonstrate intracellular accumulation of AAT within the ER of ZZ monocytes. These are the first data showing that Z AAT protein accumulation induces UPR activation in peripheral blood monocytes. These findings change the current paradigm regarding lung inflammation in AAT deficiency, which up until now was derived from the protease-anti-protease hypothesis, but which now must include the exaggerated inflammatory response generated by accumulated aberrantly folded AAT in circulating blood cells.
Directory of Open Access Journals (Sweden)
Zsolt Mártonfalvi
Full Text Available 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.
International Nuclear Information System (INIS)
Rahaman, Hamidur; Khan, Md. Khurshid Alam; Hassan, Md. Imtaiyaz; Islam, Asimul; Moosavi-Movahedi, Ali Akbar; Ahmad, Faizan
2013-01-01
Highlights: ► Non-coincidence of normalized sigmoidal curves of two different structural properties is consistence with the two-state protein folding/unfolding. ► DSC measurements of denaturation show a two-state behavior of g-cyt-c at pH 6.0. ► Urea-induced denaturation of g-cyt-c is a variable two- state process at pH 6.0. ► GdmCl-induced denaturation of g-cyt-c is a fixed two- state process at pH 6.0. -- Abstract: In practice, the observation of non-coincidence of normalized sigmoidal transition curves measured by two different structural properties constitutes a proof of existence of thermodynamically stable intermediate(s) on the folding ↔ unfolding pathway of a protein. Here we give first experimental evidence that this non-coincidence is also observed for a two-state protein denaturation. Proof of this evidence comes from our studies of denaturation of goat cytochrome-c (g-cyt-c) at pH 6.0. These studies involve differential scanning calorimetry (DSC) measurements in the absence of urea and measurements of urea-induced denaturation curves monitored by observing changes in absorbance at 405, 530, and 695 nm and circular dichroism (CD) at 222, 405, and 416 nm. DSC measurements showed that denaturation of the protein is a two-state process, for calorimetric and van’t Hoff enthalpy changes are, within experimental errors, identical. Normalization of urea-induced denaturation curves monitored by optical properties leads to noncoincident sigmoidal curves. Heat-induced transition of g-cyt-c in the presence of different urea concentrations was monitored by CD at 222 nm and absorption at 405 nm. It was observed that these two different structural probes gave not only identical values of T m (transition temperature), ΔH m (change in enthalpy at T m ) and ΔC p (constant-pressure heat capacity change), but these thermodynamic parameters in the absence of urea are also in agreement with those obtained from DSC measurements
Studying pressure denaturation of a protein by molecular dynamics simulations.
Sarupria, Sapna; Ghosh, Tuhin; García, Angel E; Garde, Shekhar
2010-05-15
Many globular proteins unfold when subjected to several kilobars of hydrostatic pressure. This "unfolding-up-on-squeezing" is counter-intuitive in that one expects mechanical compression of proteins with increasing pressure. Molecular simulations have the potential to provide fundamental understanding of pressure effects on proteins. However, the slow kinetics of unfolding, especially at high pressures, eliminates the possibility of its direct observation by molecular dynamics (MD) simulations. Motivated by experimental results-that pressure denatured states are water-swollen, and theoretical results-that water transfer into hydrophobic contacts becomes favorable with increasing pressure, we employ a water insertion method to generate unfolded states of the protein Staphylococcal Nuclease (Snase). Structural characteristics of these unfolded states-their water-swollen nature, retention of secondary structure, and overall compactness-mimic those observed in experiments. Using conformations of folded and unfolded states, we calculate their partial molar volumes in MD simulations and estimate the pressure-dependent free energy of unfolding. The volume of unfolding of Snase is negative (approximately -60 mL/mol at 1 bar) and is relatively insensitive to pressure, leading to its unfolding in the pressure range of 1500-2000 bars. Interestingly, once the protein is sufficiently water swollen, the partial molar volume of the protein appears to be insensitive to further conformational expansion or unfolding. Specifically, water-swollen structures with relatively low radii of gyration have partial molar volume that are similar to that of significantly more unfolded states. We find that the compressibility change on unfolding is negligible, consistent with experiments. We also analyze hydration shell fluctuations to comment on the hydration contributions to protein compressibility. Our study demonstrates the utility of molecular simulations in estimating volumetric properties
DEFF Research Database (Denmark)
Groenning, Minna; Campos, Raul I; Hirschberg, Daniel
2015-01-01
describe an unexpectedly dynamic TTR protofibril structure which exchanges protomers with highly unfolded monomers in solution. The protofibrils only grow to an approximate final size of 2,900 kDa and a length of 70 nm and a comparative HXMS analysis of native and aggregated samples revealed a much higher...... average solvent exposure of TTR upon fibrillation. With SAXS, we reveal the continuous presence of a considerably unfolded TTR monomer throughout the fibrillation process, and show that a considerable fraction of the fibrillating protein remains in solution even at a late maturation state. Together......, these data reveal that the fibrillar state interchanges with the solution state. Accordingly, we suggest that TTR fibrillation proceeds via addition of considerably unfolded monomers, and the continuous presence of amyloidogenic structures near the protofibril surface offers a plausible explanation...
Directory of Open Access Journals (Sweden)
Christoph Sonderegger
Full Text Available The cysteine-rich, cationic, antifungal protein PAF is abundantly secreted into the culture supernatant of the filamentous Ascomycete Penicillium chrysogenum. The five β-strands of PAF form a compact β-barrel that is stabilized by three disulphide bonds. The folding of PAF allows the formation of four surface-exposed loops and distinct charged motifs on the protein surface that might regulate the interaction of PAF with the sensitive target fungus. The growth inhibitory activity of this highly stable protein against opportunistic fungal pathogens provides great potential in antifungal drug research. To understand its mode of action, we started to investigate the surface-exposed loops of PAF and replaced one aspartic acid at position 19 in loop 2 that is potentially involved in PAF active or binding site, with a serine (Asp19 to Ser19. We analysed the overall effects, such as unfolding, electrostatic changes, sporadic conformers and antifungal activity when substituting this specific amino acid to the fairly indifferent amino acid serine. Structural analyses revealed that the overall 3D solution structure is virtually identical with that of PAF. However, PAFD19S showed slightly increased dynamics and significant differences in the surface charge distribution. Thermal unfolding identified PAFD19S to be rather a two-state folder in contrast to the three-state folder PAF. Functional comparison of PAFD19S and PAF revealed that the exchange at residue 19 caused a dramatic loss of antifungal activity: the binding and internalization of PAFD19S by target cells was reduced and the protein failed to trigger an intracellular Ca2+ response, all of which are closely linked to the antifungal toxicity of PAF. We conclude that the negatively charged residue Asp19 in loop 2 is essential for full function of the cationic protein PAF.
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 IRE1α-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. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
International Nuclear Information System (INIS)
Bhattacharya, Jaydeep; GhoshMoulick, Ranjita; Choudhuri, Utpal; Chakrabarty, Prantar; Bhattacharya, Pranab K.; Lahiri, Prabir; Chakraborti, Bikas; Dasgupta, Anjan Kr.
2004-01-01
The unfolding pattern of crystal human hemoglobin and variants of hemoglobin obtained from hemolysate were studied using transverse urea gradient gel electrophoresis (TUGGE). A smooth sigmoid like increase of electrophoretic mobility was observed with increasing urea concentrations. A decrease in electrophoretic mobility resulted, if the protein was unfolded with guanidium hydrochloride (GdnHCl). The anomaly was resolved after the Stoke's radii (obtained using the photon correlation spectroscopy) and zeta potential (measured using laser Doppler velocimetry) measurements were made at different denaturant concentrations. Addition of denaturant led to formation of extended structure, irrespective of the nature of the denaturant, as indicated by increase in Stoke's radii in both cases (urea and GdnHCl). The unexpected increase in electrophoretic mobility in case of urea could be explained in terms of a critical redistribution of negative charge at intermediate stages of the unfolding process. In case of GdnHCl, the higher ionic strength masked the charge effect. The mobility, being solely dependent on size, decreased at higher denaturant concentration. Incidentally, folding loci of other hemoglobin variants (e.g. HbE) or that of post-translationally modified hemoglobin (e.g. HbA1c) could be determined by studying the charge distribution and hydrodynamic radius at varying denaturing stress and in each case the gel migration profile could be approximately scaled by the ratio of charge and hydrodynamic diameter of the protein. While unfolding induced charge effect was most pronounced in HbA0 (and crystal ferrous hemoglobin), the unfolding induced aggregation (manifested by the increase in Stoke's radii) was predominantly observed in the variant forms HbE and HbA1c. Representing the proteins by a plot, in which charge and hydrodynamic diameter are on independent axes, may be a useful way of characterizing protein variants having similar migration profiles on native gels
Tischer, Alexander; Auton, Matthew
2013-01-01
We have analyzed the thermodynamic properties of the von Willebrand factor (VWF) A3 domain using urea-induced unfolding at variable temperature and thermal unfolding at variable urea concentrations to generate a phase diagram that quantitatively describes the equilibrium between native and denatured states. From this analysis, we were able to determine consistent thermodynamic parameters with various spectroscopic and calorimetric methods that define the urea–temperature parameter plane from cold denaturation to heat denaturation. Urea and thermal denaturation are experimentally reversible and independent of the thermal scan rate indicating that all transitions are at equilibrium and the van't Hoff and calorimetric enthalpies obtained from analysis of individual thermal transitions are equivalent demonstrating two-state character. Global analysis of the urea–temperature phase diagram results in a significantly higher enthalpy of unfolding than obtained from analysis of individual thermal transitions and significant cross correlations describing the urea dependence of and that define a complex temperature dependence of the m-value. Circular dichroism (CD) spectroscopy illustrates a large increase in secondary structure content of the urea-denatured state as temperature increases and a loss of secondary structure in the thermally denatured state upon addition of urea. These structural changes in the denatured ensemble make up ∼40% of the total ellipticity change indicating a highly compact thermally denatured state. The difference between the thermodynamic parameters obtained from phase diagram analysis and those obtained from analysis of individual thermal transitions illustrates that phase diagrams capture both contributions to unfolding and denatured state expansion and by comparison are able to decipher these contributions. PMID:23813497
Directory of Open Access Journals (Sweden)
Kritika Singh
Full Text Available Wheat α-amylase, a multi-domain protein with immense industrial applications, belongs to α+β 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 α-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 α-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, α-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.
International Nuclear Information System (INIS)
Srivastava, Ritesh K.; Li, Changzhao; Chaudhary, Sandeep C.; Ballestas, Mary E.; Elmets, Craig A.; Robbins, David J.; Matalon, Sadis; Deshane, Jessy S.; Afaq, Farrukh; Bickers, David R.; Athar, Mohammad
2013-01-01
Arsenic exposure is known to disrupt innate immune functions in humans and in experimental animals. In this study, we provide a mechanism by which arsenic trioxide (ATO) disrupts macrophage functions. ATO treatment of murine macrophage cells diminished internalization of FITC-labeled latex beads, impaired clearance of phagocytosed fluorescent bacteria and reduced secretion of pro-inflammatory cytokines. These impairments in macrophage functions are associated with ATO-induced unfolded protein response (UPR) signaling pathway characterized by the enhancement in proteins such as GRP78, p-PERK, p-eIF2α, ATF4 and CHOP. The expression of these proteins is altered both at transcriptional and translational levels. Pretreatment with chemical chaperon, 4-phenylbutyric acid (PBA) attenuated the ATO-induced activation in UPR signaling and afforded protection against ATO-induced disruption of macrophage functions. This treatment also reduced ATO-mediated reactive oxygen species (ROS) generation. Interestingly, treatment with antioxidant N-acetylcysteine (NAC) prior to ATO exposure, not only reduced ROS production and UPR signaling but also improved macrophage functions. These data demonstrate that UPR signaling and ROS generation are interdependent and are involved in the arsenic-induced pathobiology of macrophage. These data also provide a novel strategy to block the ATO-dependent impairment in innate immune responses. - Highlights: • Inorganic arsenic to humans and experimental animals disrupt innate immune responses. • The mechanism underlying arsenic impaired macrophage functions involves UPR signaling. • Chemical chaperon attenuates arsenic-mediated macrophage function impairment. • Antioxidant, NAC blocks impairment in arsenic-treated macrophage functions
Energy Technology Data Exchange (ETDEWEB)
Srivastava, Ritesh K.; Li, Changzhao; Chaudhary, Sandeep C. [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL (United States); Ballestas, Mary E. [Department of Pediatrics Infectious Disease, Children' s of Alabama, School of Medicine, University of Alabama at Birmingham, AL (United States); Elmets, Craig A. [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL (United States); Robbins, David J. [Department of Surgery, Molecular Oncology Program, Miller School of Medicine, University of Miami, Miami (United States); Matalon, Sadis [Department of Anesthesiology, University of Alabama at Birmingham, Birmingham, AL (United States); Deshane, Jessy S. [Department of Medicine, Division of Pulmonary, Allergy and Critical Care Medicine, University of Alabama at Birmingham, Birmingham, AL (United States); Afaq, Farrukh [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL (United States); Bickers, David R. [Department of Dermatology, Columbia University Medical Center, New York (United States); Athar, Mohammad, E-mail: mathar@uab.edu [Department of Dermatology and Skin Diseases Research Center, University of Alabama at Birmingham, Birmingham, AL (United States)
2013-11-01
Arsenic exposure is known to disrupt innate immune functions in humans and in experimental animals. In this study, we provide a mechanism by which arsenic trioxide (ATO) disrupts macrophage functions. ATO treatment of murine macrophage cells diminished internalization of FITC-labeled latex beads, impaired clearance of phagocytosed fluorescent bacteria and reduced secretion of pro-inflammatory cytokines. These impairments in macrophage functions are associated with ATO-induced unfolded protein response (UPR) signaling pathway characterized by the enhancement in proteins such as GRP78, p-PERK, p-eIF2α, ATF4 and CHOP. The expression of these proteins is altered both at transcriptional and translational levels. Pretreatment with chemical chaperon, 4-phenylbutyric acid (PBA) attenuated the ATO-induced activation in UPR signaling and afforded protection against ATO-induced disruption of macrophage functions. This treatment also reduced ATO-mediated reactive oxygen species (ROS) generation. Interestingly, treatment with antioxidant N-acetylcysteine (NAC) prior to ATO exposure, not only reduced ROS production and UPR signaling but also improved macrophage functions. These data demonstrate that UPR signaling and ROS generation are interdependent and are involved in the arsenic-induced pathobiology of macrophage. These data also provide a novel strategy to block the ATO-dependent impairment in innate immune responses. - Highlights: • Inorganic arsenic to humans and experimental animals disrupt innate immune responses. • The mechanism underlying arsenic impaired macrophage functions involves UPR signaling. • Chemical chaperon attenuates arsenic-mediated macrophage function impairment. • Antioxidant, NAC blocks impairment in arsenic-treated macrophage functions.
Energy Technology Data Exchange (ETDEWEB)
Zheng, Wenjun, E-mail: wjzheng@buffalo.edu; Glenn, Paul [Department of Physics, University at Buffalo, Buffalo, New York 14260 (United States)
2015-01-21
The Bacteriophage T4 Lysozyme (T4L) is a prototype modular protein comprised of an N-terminal and a C-domain domain, which was extensively studied to understand the folding/unfolding mechanism of modular proteins. To offer detailed structural and dynamic insights to the folded-state stability and the mechanical unfolding behaviors of T4L, we have performed extensive equilibrium and steered molecular dynamics simulations of both the wild-type (WT) and a circular permutation (CP) variant of T4L using all-atom and coarse-grained force fields. Our all-atom and coarse-grained simulations of the folded state have consistently found greater stability of the C-domain than the N-domain in isolation, which is in agreement with past thermostatic studies of T4L. While the all-atom simulation cannot fully explain the mechanical unfolding behaviors of the WT and the CP variant observed in an optical tweezers study, the coarse-grained simulations based on the Go model or a modified elastic network model (mENM) are in qualitative agreement with the experimental finding of greater unfolding cooperativity in the WT than the CP variant. Interestingly, the two coarse-grained models predict different structural mechanisms for the observed change in cooperativity between the WT and the CP variant—while the Go model predicts minor modification of the unfolding pathways by circular permutation (i.e., preserving the general order that the N-domain unfolds before the C-domain), the mENM predicts a dramatic change in unfolding pathways (e.g., different order of N/C-domain unfolding in the WT and the CP variant). Based on our simulations, we have analyzed the limitations of and the key differences between these models and offered testable predictions for future experiments to resolve the structural mechanism for cooperative folding/unfolding of T4L.
Tellier, Julie; Shi, Wei; Minnich, Martina; Liao, Yang; Crawford, Simon; Smyth, Gordon K; Kallies, Axel; Busslinger, Meinrad; Nutt, Stephen L
2015-01-01
Plasma cell differentiation requires silencing of B cell transcription, while establishing antibody-secretory function and long-term survival. The transcription factors Blimp-1 and IRF4 are essential for plasma cell generation, however their function in mature plasma cells has remained elusive. We have found that while IRF4 was essential for plasma cell survival, Blimp-1 was dispensable. Blimp-1-deficient plasma cells retained their transcriptional identity, but lost the ability to secrete antibody. Blimp-1 regulated many components of the unfolded protein response (UPR), including XBP-1 and ATF6. The overlap of Blimp-1 and XBP-1 function was restricted to the UPR, with Blimp-1 uniquely regulating mTOR activity and plasma cell size. Thus, Blimp-1 is required for the unique physiological capacity of plasma cells that enables the secretion of protective antibody. PMID:26779600
Isothermal chemical denaturation of large proteins: Path-dependence and irreversibility.
Wafer, Lucas; Kloczewiak, Marek; Polleck, Sharon M; Luo, Yin
2017-12-15
State functions (e.g., ΔG) are path independent and quantitatively describe the equilibrium states of a thermodynamic system. Isothermal chemical denaturation (ICD) is often used to extrapolate state function parameters for protein unfolding in native buffer conditions. The approach is prudent when the unfolding/refolding processes are path independent and reversible, but may lead to erroneous results if the processes are not reversible. The reversibility was demonstrated in several early studies for smaller proteins, but was assumed in some reports for large proteins with complex structures. In this work, the unfolding/refolding of several proteins were systematically studied using an automated ICD instrument. It is shown that: (i) the apparent unfolding mechanism and conformational stability of large proteins can be denaturant-dependent, (ii) equilibration times for large proteins are non-trivial and may introduce significant error into calculations of ΔG, (iii) fluorescence emission spectroscopy may not correspond to other methods, such as circular dichroism, when used to measure protein unfolding, and (iv) irreversible unfolding and hysteresis can occur in the absence of aggregation. These results suggest that thorough confirmation of the state functions by, for example, performing refolding experiments or using additional denaturants, is needed when quantitatively studying the thermodynamics of protein unfolding using ICD. Copyright © 2017 Elsevier Inc. All rights reserved.
Energy Technology Data Exchange (ETDEWEB)
Bhattacharya, Jaydeep; GhoshMoulick, Ranjita; Choudhuri, Utpal; Chakrabarty, Prantar; Bhattacharya, Pranab K.; Lahiri, Prabir; Chakraborti, Bikas; Dasgupta, Anjan Kr
2004-09-27
The unfolding pattern of crystal human hemoglobin and variants of hemoglobin obtained from hemolysate were studied using transverse urea gradient gel electrophoresis (TUGGE). A smooth sigmoid like increase of electrophoretic mobility was observed with increasing urea concentrations. A decrease in electrophoretic mobility resulted, if the protein was unfolded with guanidium hydrochloride (GdnHCl). The anomaly was resolved after the Stoke's radii (obtained using the photon correlation spectroscopy) and zeta potential (measured using laser Doppler velocimetry) measurements were made at different denaturant concentrations. Addition of denaturant led to formation of extended structure, irrespective of the nature of the denaturant, as indicated by increase in Stoke's radii in both cases (urea and GdnHCl). The unexpected increase in electrophoretic mobility in case of urea could be explained in terms of a critical redistribution of negative charge at intermediate stages of the unfolding process. In case of GdnHCl, the higher ionic strength masked the charge effect. The mobility, being solely dependent on size, decreased at higher denaturant concentration. Incidentally, folding loci of other hemoglobin variants (e.g. HbE) or that of post-translationally modified hemoglobin (e.g. HbA1c) could be determined by studying the charge distribution and hydrodynamic radius at varying denaturing stress and in each case the gel migration profile could be approximately scaled by the ratio of charge and hydrodynamic diameter of the protein. While unfolding induced charge effect was most pronounced in HbA0 (and crystal ferrous hemoglobin), the unfolding induced aggregation (manifested by the increase in Stoke's radii) was predominantly observed in the variant forms HbE and HbA1c. Representing the proteins by a plot, in which charge and hydrodynamic diameter are on independent axes, may be a useful way of characterizing protein variants having similar migration profiles on
Hopf-pitchfork bifurcation and periodic phenomena in nonlinear financial system with delay
International Nuclear Information System (INIS)
Ding Yuting; Jiang Weihua; Wang Hongbin
2012-01-01
Highlights: ► We derive the unfolding of a financial system with Hopf-pitchfork bifurcation. ► We show the coexistence of a pair of stable small amplitudes periodic solutions. ► At the same time, also there is a pair of stable large amplitudes periodic solutions. ► Chaos can appear by period-doubling bifurcation far away from Hopf-pitchfork value. ► The study will be useful for interpreting economics phenomena in theory. - Abstract: In this paper, we identify the critical point for a Hopf-pitchfork bifurcation in a nonlinear financial system with delay, and derive the normal form up to third order with their unfolding in original system parameters near the bifurcation point by normal form method and center manifold theory. Furthermore, we analyze its local dynamical behaviors, and show the coexistence of a pair of stable periodic solutions. We also show that there coexist a pair of stable small-amplitude periodic solutions and a pair of stable large-amplitude periodic solutions for different initial values. Finally, we give the bifurcation diagram with numerical illustration, showing that the pair of stable small-amplitude periodic solutions can also exist in a large region of unfolding parameters, and the financial system with delay can exhibit chaos via period-doubling bifurcations as the unfolding parameter values are far away from the critical point of the Hopf-pitchfork bifurcation.
Dasgupta, Amrita; Udgaonkar, Jayant B; Das, Payel
2014-06-19
The unfolding of the SH3 domain of the PI3 kinase in aqueous urea has been studied using a synergistic experiment-simulation approach. The experimental observation of a transient wet molten globule intermediate, IU, with an unusual non-native burial of the sole Trp residue, W53, provides the benchmark for the unfolding simulations performed (eight in total, each at least 0.5 μs long). The simulations reveal that the partially unfolded IU ensemble is preceded by an early native-like molten globule intermediate ensemble I*. In the very initial stage of unfolding, dry globule conformations with the protein core filled with urea instead of water are transiently observed within the I* ensemble. Water penetration into the urea-filled core of dry globule conformations is frequently accompanied by very transient burial of W53. Later during gradual unfolding, W53 is seen to again become transiently buried in the IU ensemble for a much longer time. In the structurally heterogeneous IU ensemble, conformational flexibility of the C-terminal β-strands enables W53 burial by the formation of non-native, tertiary contacts with hydrophobic residues, which could serve to protect the protein from aggregation during unfolding.
International Nuclear Information System (INIS)
Guleria, Ayushi; Chandna, Sudhir
2016-01-01
Endoplasmic Reticulum (ER) performs multiple cellular functions such as proper folding of newly synthesized proteins and calcium homeostasis. ER stress triggers unfolded protein response (UPR) that attempts to restore normal ER function and resists damage-induced cell death. Lepidopteran Sf9 insect cells (derived from Spodoptera frugiperda) display 100-200 times higher radioresistance than mammalian cells. We have earlier reported that gamma-radiation doses <1000 Gy fail to trigger increase in cytosolic calcium in Sf9 cells, indicating resilience to calcium/ ER disturbances
Mechanics of collective unfolding
Caruel, M.; Allain, J.-M.; Truskinovsky, L.
2015-03-01
Mechanically induced unfolding of passive crosslinkers is a fundamental biological phenomenon encountered across the scales from individual macro-molecules to cytoskeletal actin networks. In this paper we study a conceptual model of athermal load-induced unfolding and use a minimalistic setting allowing one to emphasize the role of long-range interactions while maintaining full analytical transparency. Our model can be viewed as a description of a parallel bundle of N bistable units confined between two shared rigid backbones that are loaded through a series spring. We show that the ground states in this model correspond to synchronized, single phase configurations where all individual units are either folded or unfolded. We then study the fine structure of the wiggly energy landscape along the reaction coordinate linking the two coherent states and describing the optimal mechanism of cooperative unfolding. Quite remarkably, our study shows the fundamental difference in the size and the structure of the folding-unfolding energy barriers in the hard (fixed displacements) and soft (fixed forces) loading devices which persists in the continuum limit. We argue that both, the synchronization and the non-equivalence of the mechanical responses in hard and soft devices, have their origin in the dominance of long-range interactions. We then apply our minimal model to skeletal muscles where the power-stroke in acto-myosin crossbridges can be interpreted as passive folding. A quantitative analysis of the muscle model shows that the relative rigidity of myosin backbone provides the long-range interaction mechanism allowing the system to effectively synchronize the power-stroke in individual crossbridges even in the presence of thermal fluctuations. In view of the prototypical nature of the proposed model, our general conclusions pertain to a variety of other biological systems where elastic interactions are mediated by effective backbones.
Local Order in the Unfolded State: Conformational Biases and Nearest Neighbor Interactions
Directory of Open Access Journals (Sweden)
Siobhan Toal
2014-07-01
Full Text Available The discovery of Intrinsically Disordered Proteins, which contain significant levels of disorder yet perform complex biologically functions, as well as unwanted aggregation, has motivated numerous experimental and theoretical studies aimed at describing residue-level conformational ensembles. Multiple lines of evidence gathered over the last 15 years strongly suggest that amino acids residues display unique and restricted conformational preferences in the unfolded state of peptides and proteins, contrary to one of the basic assumptions of the canonical random coil model. To fully understand residue level order/disorder, however, one has to gain a quantitative, experimentally based picture of conformational distributions and to determine the physical basis underlying residue-level conformational biases. Here, we review the experimental, computational and bioinformatic evidence for conformational preferences of amino acid residues in (mostly short peptides that can be utilized as suitable model systems for unfolded states of peptides and proteins. In this context particular attention is paid to the alleged high polyproline II preference of alanine. We discuss how these conformational propensities may be modulated by peptide solvent interactions and so called nearest-neighbor interactions. The relevance of conformational propensities for the protein folding problem and the understanding of IDPs is briefly discussed.
Lahlali, Thomas; Plissonnier, Marie-Laure; Romero-López, Cristina; Michelet, Maud; Ducarouge, Benjamin; Berzal-Herranz, Alfredo; Zoulim, Fabien; Mehlen, Patrick; Parent, Romain
2016-05-01
Netrin-1, a multifunctional secreted protein, is up-regulated in cancer and inflammation. Netrin-1 blocks apoptosis induced by the prototypical dependence receptors deleted in colorectal carcinoma and uncoordinated phenotype-5. Although the unfolded protein response (UPR) triggers apoptosis on exposure to stress, it first attempts to restore endoplasmic reticulum homeostasis to foster cell survival. Importantly, UPR is implicated in chronic liver conditions including hepatic oncogenesis. Netrin-1's implication in cell survival on UPR in this context is unknown. Isolation of translational complexes, determination of RNA secondary structures by selective 2'-hydroxyl acylation and primer extension/dimethyl sulfate, bicistronic constructs, as well as conventional cell biology and biochemistry approaches were used on in vitro-grown hepatocytic cells, wild-type, and netrin-1 transgenic mice. HepaRG cells constitute a bona fide model for UPR studies in vitro through adequate activation of the 3 sensors of the UPR (protein kinase RNA-like endoplasmic reticulum kinase (PERK)), inositol requiring enzyme 1α (IRE1α), and activated transcription factor 6 (ATF6). The netrin-1 messenger RNA 5'-end was shown to fold into a complex double pseudoknot and bear E-loop motifs, both of which are representative hallmarks of related internal ribosome entry site regions. Cap-independent translation of netrin 5' untranslated region-driven luciferase was observed on UPR in vitro. Unlike several structurally related oncogenic transcripts (l-myc, c-myc, c-myb), netrin-1 messenger RNA was selected for translation during UPR both in human hepatocytes and in mice livers. Depletion of netrin-1 during UPR induces apoptosis, leading to cell death through an uncoordinated phenotype-5A/C-mediated involvement of protein phosphatase 2A and death-associated protein kinase 1 in vitro and in netrin transgenic mice. UPR-resistant, internal ribosome entry site-driven netrin-1 translation leads to
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Sasnauskas Kęstutis
2011-05-01
Full Text Available Abstract Background The expression of human virus surface proteins, as well as other mammalian glycoproteins, is much more efficient in cells of higher eukaryotes rather than yeasts. The limitations to high-level expression of active viral surface glycoproteins in yeast are not well understood. To identify possible bottlenecks we performed a detailed study on overexpression of recombinant mumps hemagglutinin-neuraminidase (MuHN and measles hemagglutinin (MeH in yeast Saccharomyces cerevisiae, combining the analysis of recombinant proteins with a proteomic approach. Results Overexpressed recombinant MuHN and MeH proteins were present in large aggregates, were inactive and totally insoluble under native conditions. Moreover, the majority of recombinant protein was found in immature form of non-glycosylated precursors. Fractionation of yeast lysates revealed that the core of viral surface protein aggregates consists of MuHN or MeH disulfide-linked multimers involving eukaryotic translation elongation factor 1A (eEF1A and is closely associated with small heat shock proteins (sHsps that can be removed only under denaturing conditions. Complexes of large Hsps seem to be bound to aggregate core peripherally as they can be easily removed at high salt concentrations. Proteomic analysis revealed that the accumulation of unglycosylated viral protein precursors results in specific cytosolic unfolded protein response (UPR-Cyto in yeast cells, characterized by different action and regulation of small Hsps versus large chaperones of Hsp70, Hsp90 and Hsp110 families. In contrast to most environmental stresses, in the response to synthesis of recombinant MuHN and MeH, only the large Hsps were upregulated whereas sHsps were not. Interestingly, the amount of eEF1A was also increased during this stress response. Conclusions Inefficient translocation of MuHN and MeH precursors through ER membrane is a bottleneck for high-level expression in yeast. Overexpression of
Glutathione Peroxidase-1 Suppresses the Unfolded Protein Response upon Cigarette Smoke Exposure
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Patrick Geraghty
2016-01-01
Full Text Available Oxidative stress provokes endoplasmic reticulum (ER stress-induced unfolded protein response (UPR in the lungs of chronic obstructive pulmonary (COPD subjects. The antioxidant, glutathione peroxidase-1 (GPx-1, counters oxidative stress induced by cigarette smoke exposure. Here, we investigate whether GPx-1 expression deters the UPR following exposure to cigarette smoke. Expression of ER stress markers was investigated in fully differentiated normal human bronchial epithelial (NHBE cells isolated from nonsmoking, smoking, and COPD donors and redifferentiated at the air liquid interface. NHBE cells from COPD donors expressed heightened ATF4, XBP1, GRP78, GRP94, EDEM1, and CHOP compared to cells from nonsmoking donors. These changes coincided with reduced GPx-1 expression. Reintroduction of GPx-1 into NHBE cells isolated from COPD donors reduced the UPR. To determine whether the loss of GPx-1 expression has a direct impact on these ER stress markers during smoke exposure, Gpx-1−/− mice were exposed to cigarette smoke for 1 year. Loss of Gpx-1 expression enhanced cigarette smoke-induced ER stress and apoptosis. Equally, induction of ER stress with tunicamycin enhanced antioxidant expression in mouse precision-cut lung slices. Smoke inhalation also exacerbated the UPR response during respiratory syncytial virus infection. Therefore, ER stress may be an antioxidant-related pathophysiological event in COPD.
Defining a methodology for benchmarking spectrum unfolding codes
International Nuclear Information System (INIS)
Meyer, W.; Kirmser, P.G.; Miller, W.H.; Hu, K.K.
1976-01-01
It has long been recognized that different neutron spectrum unfolding codes will produce significantly different results when unfolding the same measured data. In reviewing the results of such analyses it has been difficult to determine which result if any is the best representation of what was measured by the spectrometer detector. A proposal to develop a benchmarking procedure for spectrum unfolding codes is presented. The objective of the procedure will be to begin to develop a methodology and a set of data with a well established and documented result that could be used to benchmark and standardize the various unfolding methods and codes. It is further recognized that development of such a benchmark must involve a consensus of the technical community interested in neutron spectrum unfolding
Finkelstein, A. V.; Galzitskaya, O. V.
2004-04-01
Protein physics is grounded on three fundamental experimental facts: protein, this long heteropolymer, has a well defined compact three-dimensional structure; this structure can spontaneously arise from the unfolded protein chain in appropriate environment; and this structure is separated from the unfolded state of the chain by the “all-or-none” phase transition, which ensures robustness of protein structure and therefore of its action. The aim of this review is to consider modern understanding of physical principles of self-organization of protein structures and to overview such important features of this process, as finding out the unique protein structure among zillions alternatives, nucleation of the folding process and metastable folding intermediates. Towards this end we will consider the main experimental facts and simple, mostly phenomenological theoretical models. We will concentrate on relatively small (single-domain) water-soluble globular proteins (whose structure and especially folding are much better studied and understood than those of large or membrane and fibrous proteins) and consider kinetic and structural aspects of transition of initially unfolded protein chains into their final solid (“native”) 3D structures.
Ethanol cellular defense induce unfolded protein response in yeast
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Elisabet eNavarro-Tapia
2016-02-01
Full Text Available Ethanol is a valuable industrial product and a common metabolite used by many cell types. However, this molecule produces high levels of cytotoxicity affecting cellular performance at several levels. In the presence of ethanol, cells must adjust some of their components, such as the membrane lipids to maintain homeostasis. In the case of microorganism as Saccharomyces cerevisiae, ethanol is one of the principal products of their metabolism and is the main stress factor during fermentation. Although many efforts have been made, mechanisms of ethanol tolerance are not fully understood and very little evidence is available to date for specific signaling by ethanol in the cell. This work studied two Saccharomyces cerevisiae strains, CECT10094 and Temohaya-MI26, isolated from flor wine and agave fermentation (a traditional fermentation from Mexico respectively, which differ in ethanol tolerance, in order to understand the molecular mechanisms underlying the ethanol stress response and the reasons for different ethanol tolerance. The transcriptome was analyzed after ethanol stress and, among others, an increased activation of genes related with the unfolded protein response (UPR and its transcription factor, Hac1p, was observed in the tolerant strain CECT10094. We observed that this strain also resist more UPR agents than Temohaya-MI26 and the UPR-ethanol stress correlation was corroborated observing growth of 15 more strains and discarding UPR correlation with other stresses as thermal or oxidative stress. Furthermore, higher activation of UPR pathway in the tolerant strain CECT10094 was observed using a UPR mCherry reporter. Finally, we observed UPR activation in response to ethanol stress in other S. cerevisiae ethanol tolerant strains as the wine strains T73 and EC1118. This work demonstrates that the UPR pathway is activated under ethanol stress occurring in a standard fermentation and links this response to an enhanced ethanol tolerance. Thus
Solvent-Exposed Salt Bridges Influence the Kinetics of α-Helix Folding and Unfolding.
Meuzelaar, Heleen; Tros, Martijn; Huerta-Viga, Adriana; van Dijk, Chris N; Vreede, Jocelyne; Woutersen, Sander
2014-03-06
Salt bridges are known to play an essential role in the thermodynamic stability of the folded conformation of many proteins, but their influence on the kinetics of folding remains largely unknown. Here, we investigate the effect of Glu-Arg salt bridges on the kinetics of α-helix folding using temperature-jump transient-infrared spectroscopy and steady-state UV circular dichroism. We find that geometrically optimized salt bridges (Glu - and Arg + are spaced four peptide units apart, and the Glu/Arg order is such that the side-chain rotameric preferences favor salt-bridge formation) significantly speed up folding and slow down unfolding, whereas salt bridges with unfavorable geometry slow down folding and slightly speed up unfolding. Our observations suggest a possible explanation for the surprising fact that many biologically active proteins contain salt bridges that do not stabilize the native conformation: these salt bridges might have a kinetic rather than a thermodynamic function.
Complex oscillatory behaviour in a delayed protein cross talk model with periodic forcing
International Nuclear Information System (INIS)
Nikolov, Svetoslav
2009-01-01
The purpose of this paper is to examine the effects of periodic forcing on the time delay protein cross talk model behaviour. We assume periodic variation for the plasma membrane permeability. The dynamic behaviour of the system is simulated and bifurcation diagrams are obtained for different parameters. The results show that periodic forcing can very easily give rise to complex dynamics, including a period-doubling cascade, chaos, quasi-periodic oscillating, and periodic windows. Finally, we calculate the maximal Lyapunov exponent in the regions of the parameter space where chaotic motion of delayed protein cross talk model with periodic forcing exists.
Uncertainty analysis of dosimetry spectrum unfolding
International Nuclear Information System (INIS)
Perey, F.G.
1977-01-01
The propagation of uncertainties in the input data is analyzed for the usual dosimetry unfolding solution. A new formulation of the dosimetry unfolding problem is proposed in which the most likely value of the spectrum is obtained. The relationship of this solution to the usual one is discussed
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Jarom Heijmans
2013-04-01
Full Text Available Stem cells generate rapidly dividing transit-amplifying cells that have lost the capacity for self-renewal but cycle for a number of times until they exit the cell cycle and undergo terminal differentiation. We know very little of the type of signals that trigger the earliest steps of stem cell differentiation and mediate a stem cell to transit-amplifying cell transition. We show that in normal intestinal epithelium, endoplasmic reticulum (ER stress and activity of the unfolded protein response (UPR are induced at the transition from stem cell to transit-amplifying cell. Induction of ER stress causes loss of stemness in a Perk-eIF2α-dependent manner. Inhibition of Perk-eIF2α signaling results in stem cell accumulation in organoid culture of primary intestinal epithelium. Our findings show that the UPR plays an important role in the regulation of intestinal epithelial stem cell differentiation.
Activation of the unfolded protein response in sarcoma cells treated with rapamycin or temsirolimus.
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Joseph W Briggs
Full Text Available Activation of the unfolded protein response (UPR in eukaryotic cells represents an evolutionarily conserved response to physiological stress. Here, we report that the mTOR inhibitors rapamycin (sirolimus and structurally related temsirolimus are capable of inducing UPR in sarcoma cells. However, this effect appears to be distinct from the classical role for these drugs as mTOR inhibitors. Instead, we detected these compounds to be associated with ribosomes isolated from treated cells. Specifically, temsirolimus treatment resulted in protection from chemical modification of several rRNA residues previously shown to bind rapamycin in prokaryotic cells. As an application for these findings, we demonstrate maximum tumor cell growth inhibition occurring only at doses which induce UPR and which have been shown to be safely achieved in human patients. These results are significant because they challenge the paradigm for the use of these drugs as anticancer agents and reveal a connection to UPR, a conserved biological response that has been implicated in tumor growth and response to therapy. As a result, eIF2 alpha phosphorylation and Xbp-1 splicing may serve as useful biomarkers of treatment response in future clinical trials using rapamycin and rapalogs.
UNFOLDED REGULAR AND SEMI-REGULAR POLYHEDRA
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IONIŢĂ Elena
2015-06-01
Full Text Available This paper proposes a presentation unfolding regular and semi-regular polyhedra. Regular polyhedra are convex polyhedra whose faces are regular and equal polygons, with the same number of sides, and whose polyhedral angles are also regular and equal. Semi-regular polyhedra are convex polyhedra with regular polygon faces, several types and equal solid angles of the same type. A net of a polyhedron is a collection of edges in the plane which are the unfolded edges of the solid. Modeling and unfolding Platonic and Arhimediene polyhedra will be using 3dsMAX program. This paper is intended as an example of descriptive geometry applications.
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Céline Schaeffer
Full Text Available Uromodulin is the most abundant urinary protein in physiological conditions. It is exclusively produced by renal epithelial cells lining the thick ascending limb of Henle's loop (TAL and it plays key roles in kidney function and disease. Mutations in UMOD, the gene encoding uromodulin, cause autosomal dominant tubulointerstitial kidney disease uromodulin-related (ADTKD-UMOD, characterised by hyperuricemia, gout and progressive loss of renal function. While the primary effect of UMOD mutations, retention in the endoplasmic reticulum (ER, is well established, its downstream effects are still largely unknown. To gain insight into ADTKD-UMOD pathogenesis, we performed transcriptional profiling and biochemical characterisation of cellular models (immortalised mouse TAL cells of robust expression of wild type or mutant GFP-tagged uromodulin. In this model mutant uromodulin accumulation in the ER does not impact on cell viability and proliferation. Transcriptional profiling identified 109 genes that are differentially expressed in mutant cells relative to wild type ones. Up-regulated genes include several ER resident chaperones and protein disulphide isomerases. Consistently, pathway enrichment analysis indicates that mutant uromodulin expression affects ER function and protein homeostasis. Interestingly, mutant uromodulin expression induces the Unfolded Protein Response (UPR, and specifically the IRE1 branch, as shown by an increased splicing of XBP1. Consistent with UPR induction, we show increased interaction of mutant uromodulin with ER chaperones Bip, calnexin and PDI. Using metabolic labelling, we also demonstrate that while autophagy plays no role, mutant protein is partially degraded by the proteasome through ER-associated degradation. Our work demonstrates that ER stress could play a central role in ADTKD-UMOD pathogenesis. This sets the bases for future work to develop novel therapeutic strategies through modulation of ER homeostasis and
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Juhye Lena Kim
Full Text Available Recurrent hypoglycemia can occur as a major complication of insulin replacement therapy, limiting the long-term health benefits of intense glycemic control in type 1 and advanced type 2 diabetic patients. It impairs the normal counter-regulatory hormonal and behavioral responses to glucose deprivation, a phenomenon known as hypoglycemia associated autonomic failure (HAAF. The molecular mechanisms leading to defective counter-regulation are not completely understood. We hypothesized that both neuronal (excessive cholinergic signaling between the splanchnic nerve fibers and the adrenal medulla and humoral factors contribute to the impaired epinephrine production and release in HAAF. To gain further insight into the molecular mechanism(s mediating the blunted epinephrine responses following recurrent hypoglycemia, we utilized a global gene expression profiling approach. We characterized the transcriptomes during recurrent (defective counter-regulation model and acute hypoglycemia (normal counter-regulation group in the adrenal medulla of normal Sprague-Dawley rats. Based on comparison analysis of differentially expressed genes, a set of unique genes that are activated only at specific time points after recurrent hypoglycemia were revealed. A complementary bioinformatics analysis of the functional category, pathway, and integrated network indicated activation of the unfolded protein response. Furthermore, at least three additional pathways/interaction networks altered in the adrenal medulla following recurrent hypoglycemia were identified, which may contribute to the impaired epinephrine secretion in HAAF: greatly increased neuropeptide signaling (proenkephalin, neuropeptide Y, galanin; altered ion homeostasis (Na+, K+, Ca2+ and downregulation of genes involved in Ca2+-dependent exocytosis of secretory vesicles. Given the pleiotropic effects of the unfolded protein response in different organs, involved in maintaining glucose homeostasis, these
Study of chemically unfolded β-casein by means of small-angle neutron scattering
International Nuclear Information System (INIS)
Aschi, Adel; Gharbi, Abdelhafidh; Daoud, Mohamed; Douillard, Roger; Calmettes, Patrick
2007-01-01
β-casein is a flexible amphiphilic milk protein which forms an unfolded conformation in presence of very high denaturant concentrations. The structure of β-casein formed at the bulk was studied by small-angle neutron scattering (SANS). The value of the second virial coefficient of the protein solutions indicates that the interactions between the polypeptide chain and solvent are repulsive. The protein conformation is similar to an excluded volume chain. The corresponding values of the contour length, L, the statistical length, b and the apparent radius of the chain cross-section, R c are given
Experience with using unfolding procedures in ATLAS
Biondi, Silvia; The ATLAS collaboration
2016-01-01
In ATLAS, several unfolding methods are used to correct experimental measurements for detector effects, like acceptance and resolution. These methods use as input the raw experimental distributions, as well as Monte Carlo simulation for the description of the detector effects. The systematic uncertainties associated to the various unfolding methods are evaluated. The statistical and systematic uncertainties affecting the raw measurements and/or the simulation are propagated through the unfolding procedure. The resulting corrected measurements with their uncertainties can be directly compared with the corresponding theoretical predictions.
Applied multidimensional scaling and unfolding
Borg, Ingwer; Mair, Patrick
2018-01-01
This book introduces multidimensional scaling (MDS) and unfolding as data analysis techniques for applied researchers. MDS is used for the analysis of proximity data on a set of objects, representing the data as distances between points in a geometric space (usually of two dimensions). Unfolding is a related method that maps preference data (typically evaluative ratings of different persons on a set of objects) as distances between two sets of points (representing the persons and the objects, resp.). This second edition has been completely revised to reflect new developments and the coverage of unfolding has also been substantially expanded. Intended for applied researchers whose main interests are in using these methods as tools for building substantive theories, it discusses numerous applications (classical and recent), highlights practical issues (such as evaluating model fit), presents ways to enforce theoretical expectations for the scaling solutions, and addresses the typical mistakes that MDS/unfoldin...
Hempstead, Andrew D; Isberg, Ralph R
2015-12-08
Cells of the innate immune system recognize bacterial pathogens by detecting common microbial patterns as well as pathogen-specific activities. One system that responds to these stimuli is the IRE1 branch of the unfolded protein response (UPR), a sensor of endoplasmic reticulum (ER) stress. Activation of IRE1, in the context of Toll-like receptor (TLR) signaling, induces strong proinflammatory cytokine induction. We show here that Legionella pneumophila, an intravacuolar pathogen that replicates in an ER-associated compartment, blocks activation of the IRE1 pathway despite presenting pathogen products that stimulate this response. L. pneumophila TLR ligands induced the splicing of mRNA encoding XBP1s, the main target of IRE1 activity. L. pneumophila was able to inhibit both chemical and bacterial induction of XBP1 splicing via bacterial translocated proteins that interfere with host protein translation. A strain lacking five translocated translation elongation inhibitors was unable to block XBP1 splicing, but this could be rescued by expression of a single such inhibitor, consistent with limitation of the response by translation elongation inhibitors. Chemical inhibition of translation elongation blocked pattern recognition receptor-mediated XBP1 splicing, mimicking the effects of the bacterial translation inhibitors. In contrast, host cell-promoted inhibition of translation initiation in response to the pathogen was ineffective in blocking XBP1 splicing, demonstrating the need for the elongation inhibitors for protection from the UPR. The inhibition of host translation elongation may be a common strategy used by pathogens to limit the innate immune response by interfering with signaling via the UPR.
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Toru Hosoi
Full Text Available Stress signals cause abnormal proteins to accumulate in the endoplasmic reticulum (ER. Such stress is known as ER stress, which has been suggested to be involved in neurodegenerative diseases, diabetes, obesity and cancer. ER stress activates the unfolded protein response (UPR to reduce levels of abnormal proteins by inducing the production of chaperon proteins such as GRP78, and to attenuate translation through the phosphorylation of eIF2α. However, excessive stress leads to apoptosis by generating transcription factors such as CHOP. Casein kinase 2 (CK2 is a serine/threonine kinase involved in regulating neoplasia, cell survival and viral infections. In the present study, we investigated a possible linkage between CK2 and ER stress using mouse primary cultured glial cells. 4,5,6,7-tetrabromobenzotriazole (TBB, a CK2-specific inhibitor, attenuated ER stress-induced XBP-1 splicing and subsequent induction of GRP78 expression, but was ineffective against ER stress-induced eIF2α phosphorylation and CHOP expression. Similar results were obtained when endogenous CK2 expression was knocked-down by siRNA. Immunohistochemical analysis suggested that CK2 was present at the ER. These results indicate CK2 to be linked with UPR and to resist ER stress by activating the XBP-1-GRP78 arm of UPR.
International Nuclear Information System (INIS)
Lanza, Daniel Carlos Ferreira; Trindade, Daniel Maragno; Bressan, Gustavo Costa; Kobarg, Joerg
2008-01-01
The fasciculation and elongation protein Zeta1 (FEZ1) has been implicated in important functions in mammalian cells, ranging from molecular transport to transcriptional regulation. Theoretical predictions, circular dichroism spectroscopy and limiting proteolysis experiments all suggested that FEZ1 contains regions of low structural complexity and that it may belong to the growing family of natively unfolded proteins. We therefore performed Small Angle Scattering (SAXS) experiments which showed that FEZ1 is a dimer of elongated shape and that its conformation is mainly disordered. In parallel functional studies we observed that the overexpression of FEZ1 in human cells causes the so-called 'flower-like nuclei' phenotype, similar to what is observed in certain leukemic cells. Taken together, our results suggest that the FEZ1 dimer configuration may be critical to explain why its overexpression causes the formation of flower-like nuclei in human cells. (author)
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Nozomi Kawazoe
2017-06-01
Full Text Available Since acetic acid inhibits the growth and fermentation ability of Saccharomyces cerevisiae, it is one of the practical hindrances to the efficient production of bioethanol from a lignocellulosic biomass. Although extensive information is available on yeast response to acetic acid stress, the involvement of endoplasmic reticulum (ER and unfolded protein response (UPR has not been addressed. We herein demonstrated that acetic acid causes ER stress and induces the UPR. The accumulation of misfolded proteins in the ER and activation of Ire1p and Hac1p, an ER-stress sensor and ER stress-responsive transcription factor, respectively, were induced by a treatment with acetic acid stress (>0.2% v/v. Other monocarboxylic acids such as propionic acid and sorbic acid, but not lactic acid, also induced the UPR. Additionally, ire1Δ and hac1Δ cells were more sensitive to acetic acid than wild-type cells, indicating that activation of the Ire1p-Hac1p pathway is required for maximum tolerance to acetic acid. Furthermore, the combination of mild acetic acid stress (0.1% acetic acid and mild ethanol stress (5% ethanol induced the UPR, whereas neither mild ethanol stress nor mild acetic acid stress individually activated Ire1p, suggesting that ER stress is easily induced in yeast cells during the fermentation process of lignocellulosic hydrolysates. It was possible to avoid the induction of ER stress caused by acetic acid and the combined stress by adjusting extracellular pH.
Experience with using unfolding procedures in ATLAS
AUTHOR|(INSPIRE)INSPIRE-00407321; The ATLAS collaboration
2016-01-01
In the ATLAS experiment, several unfolding methods are used to correct experimental measurements for detector effects, like acceptance and resolution. These methods use as input the raw experimental distributions, as well as Monte Carlo simulation for the description of the detector effects. The systematic uncertainties associated to the various unfolding methods are evaluated. The statistical and systematic uncertainties affecting the raw measurements and/or the simulation are propagated through the unfolding procedure. The resulting corrected measurements with their uncertainties can be directly compared with the corresponding theoretical predictions.
Cheng, Tsing; Orlow, Seth J; Manga, Prashiela
2013-11-01
Accumulation of proteins in the endoplasmic reticulum (ER) typically induces stress and initiates the unfolded protein response (UPR) to facilitate recovery. If homeostasis is not restored, apoptosis is induced. However, adaptation to chronic UPR activation can increase resistance to subsequent acute ER stress. We therefore investigated adaptive mechanisms in Oculocutaneous albinism type 2 (Oca2)-null melanocytes where UPR signaling is arrested despite continued tyrosinase accumulation leading to resistance to the chemical ER stressor thapsigargin. Although thapsigargin triggers UPR activation, instead of Perk-mediated phosphorylation of eIF2α, in Oca2-null melanocytes, eIF2α was rapidly dephosphorylated upon treatment. Dephosphorylation was mediated by the Gadd34-PP1α phosphatase complex. Gadd34-complex inhibition blocked eIF2α dephosphorylation and significantly increased Oca2-null melanocyte sensitivity to thapsigargin. Thus, Oca2-null melanocytes adapt to acute ER stress by disruption of pro-apoptotic Perk signaling, which promotes cell survival. This is the first study to demonstrate rapid eIF2α dephosphorylation as an adaptive mechanism to ER stress. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.
Silva-Lucca, Rosemeire A; Andrade, Sheila S; Ferreira, Rodrigo Silva; Sampaio, Misako U; Oliva, Maria Luiza V
2013-12-24
Baupain belongs to the α+β class of proteins with a secondary structure-content of 44% α-helix, 16% β-sheet and 12% β-turn. The structural transition induced by pH was found to be noncooperative, with no important differences observed in the pH range from 3.0 to 10.5. At pH 2.0 the protein presented substantial non-native structure with strong ANS binding. Guanidine hydrochloride (GdnHCl)-induced unfolding did not change the protein structure significantly until 4.0 M, indicating the high rigidity of the molecule. The unfolding was cooperative, as seen by the sigmoidal transition curves with midpoints at 4.7±0.2 M and 5.0±0.2 M GdnHCl, as measured by CD and fluorescence spectroscopy. A red shift of 7 nm in intrinsic fluorescence was observed with 6.0 M GdnHCl. Temperature-induced unfolding of baupain was incomplete, and at least 35% of the native structure of the protein was retained, even at high temperature (90 °C). Baupain showed characteristics of a molten globule state, due to preferential ANS binding at pH 2.0 in comparison to the native form (pH 7.0) and completely unfolded (6.0 M GdnHCl) state. Combined with information about N-terminal sequence similarity, these results allow us to include baupain in the papain superfamily.
Neutron spectrum unfolding: Pt. 2
International Nuclear Information System (INIS)
Matiullah; Wiyaja, D.S.; Berzonis, M.A.; Bondars, H.; Lapenas, A.A.; Kudo, K.; Majeed, A.; Durrani, S.A.
1991-01-01
In Part I of this paper, we described the use of the computer code SAIPS in neutron spectrum unfolding. Here in Part II, we present our experimental work carried out to study the shape of the neutron spectrum in different experimental channels of a 5 MW light-water cooled and moderated research reactor. The spectral neutron flux was determined using various fission foils (placed in close contact with mica track detectors) and activation detectors. From the measured activities, the neutron spectrum was unfolded by SAIPS. (author)
Berg, Alexander K; Manokaran, Sumathra; Eiler, Daniel; Kooren, Joel; Mallik, Sanku; Srivastava, D K
2008-01-01
Peptide deformylase (PDF) catalyzes the removal of formyl group from the N-terminal methionine residues of nascent proteins in prokaryotes, and this enzyme is a high priority target for antibiotic design. In pursuit of delineating the structural-functional features of Escherichia coli PDF (EcPDF), we investigated the mechanistic pathway for the guanidinium chloride (GdmCl)-induced unfolding of the enzyme by monitoring the secondary structural changes via CD spectroscopy. The experimental data revealed that EcPDF is a highly stable enzyme, and it undergoes slow denaturation in the presence of varying concentrations of GdmCl. The most interesting aspect of these studies has been the abrupt reversal of the unfolding pathway at low to moderate concentrations of the denaturant, but not at high concentration. An energetic rationale for such an unprecedented feature in protein chemistry is offered.
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Emanuel eHanski
2014-08-01
Full Text Available The connection between bacterial pathogens and unfolded protein response (UPR is poorly explored. In this review we highlight the evidence showing that group A streptococcus (GAS induces endoplasmic reticulum (ER stress and UPR through which it captures the amino acid asparagine (ASN from the host. GAS acts extracellularly and during adherence to host cells it delivers the hemolysin toxins; streptolysin O (SLO and streptolysin S (SLS. By poorly understood pathways, these toxins trigger UPR leading to the induction of the transcriptional regulator ATF4 and consequently to the upregulation of asparagine synthetase (ASNS transcription leading to production and release of ASN. GAS senses ASN and alters gene expression profile accordingly, and increases the rate of multiplication. We suggest that induction of UPR by GAS and by other bacterial pathogens represent means through which bacterial pathogens gain nutrients from the host, obviating the need to become internalized or inflict irreversible cell damage.
Study of chemically unfolded {beta}-casein by means of small-angle neutron scattering
Energy Technology Data Exchange (ETDEWEB)
Aschi, Adel [Laboratoire de Physique de la Matiere Molle, Faculte des Sciences de Tunis, Campus Universitaire, 1060, Tunis (Tunisia)]. E-mail: aschi13@yahoo.fr; Gharbi, Abdelhafidh [Laboratoire de Physique de la Matiere Molle, Faculte des Sciences de Tunis, Campus Universitaire, 1060, Tunis (Tunisia); Daoud, Mohamed [Service de Physique de l' Etat Condense. CEA Saclay. 91191 Gif-sur-Yvette cedex (France); Douillard, Roger [Equipe de Biochimie des Macromolecules Vegetales, Centre de Recherche Agronomique, 2Esplanade R. Garros, BP 224, 51686 Reims cedex 2 (France); Calmettes, Patrick [Laboratoire Leon Brillouin, CEA Saclay, 91191 Gif-sur-Yvette cedex (France)
2007-01-01
{beta}-casein is a flexible amphiphilic milk protein which forms an unfolded conformation in presence of very high denaturant concentrations. The structure of {beta}-casein formed at the bulk was studied by small-angle neutron scattering (SANS). The value of the second virial coefficient of the protein solutions indicates that the interactions between the polypeptide chain and solvent are repulsive. The protein conformation is similar to an excluded volume chain. The corresponding values of the contour length, L, the statistical length, b and the apparent radius of the chain cross-section, R{sub c} are given.
A genetic algorithm based method for neutron spectrum unfolding
International Nuclear Information System (INIS)
Suman, Vitisha; Sarkar, P.K.
2013-03-01
An approach to neutron spectrum unfolding based on a stochastic evolutionary search mechanism - Genetic Algorithm (GA) is presented. It is tested to unfold a set of simulated spectra, the unfolded spectra is compared to the output of a standard code FERDOR. The method was then applied to a set of measured pulse height spectrum of neutrons from the AmBe source as well as of emitted neutrons from Li(p,n) and Ag(C,n) nuclear reactions carried out in the accelerator environment. The unfolded spectra compared to the output of FERDOR show good agreement in the case of AmBe spectra and Li(p,n) spectra. In the case of Ag(C,n) spectra GA method results in some fluctuations. Necessity of carrying out smoothening of the obtained solution is also studied, which leads to approximation of the solution yielding an appropriate solution finally. Few smoothing techniques like second difference smoothing, Monte Carlo averaging, combination of both and gaussian based smoothing methods are also studied. Unfolded results obtained after inclusion of the smoothening criteria are in close agreement with the output obtained from the FERDOR code. The present method is also tested on a set of underdetermined problems, the outputs of which is compared to the unfolded spectra obtained from the FERDOR applied to a completely determined problem, shows a good match. The distribution of the unfolded spectra is also studied. Uncertainty propagation in the unfolded spectra due to the errors present in the measurement as well as the response function is also carried out. The method appears to be promising for unfolding the completely determined as well as underdetermined problems. It also has provisions to carry out the uncertainty analysis. (author)
Novel insights into the unfolded protein response using Pichia pastoris specific DNA microarrays
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Kreil David P
2008-08-01
Full Text Available Abstract 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
Iterative nonlinear unfolding code: TWOGO
International Nuclear Information System (INIS)
Hajnal, F.
1981-03-01
a new iterative unfolding code, TWOGO, was developed to analyze Bonner sphere neutron measurements. The code includes two different unfolding schemes which alternate on successive iterations. The iterative process can be terminated either when the ratio of the coefficient of variations in terms of the measured and calculated responses is unity, or when the percentage difference between the measured and evaluated sphere responses is less than the average measurement error. The code was extensively tested with various known spectra and real multisphere neutron measurements which were performed inside the containments of pressurized water reactors
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Jeffrey W Perry
Full Text Available 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.
Branches of Triangulated Origami Near the Unfolded State
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Bryan Gin-ge Chen
2018-02-01
Full Text Available Origami structures are characterized by a network of folds and vertices joining unbendable plates. For applications to mechanical design and self-folding structures, it is essential to understand the interplay between the set of folds in the unfolded origami and the possible 3D folded configurations. When deforming a structure that has been folded, one can often linearize the geometric constraints, but the degeneracy of the unfolded state makes a linear approach impossible there. We derive a theory for the second-order infinitesimal rigidity of an initially unfolded triangulated origami structure and use it to study the set of nearly unfolded configurations of origami with four boundary vertices. We find that locally, this set consists of a number of distinct “branches” which intersect at the unfolded state, and that the number of these branches is exponential in the number of vertices. We find numerical and analytical evidence that suggests that the branches are characterized by choosing each internal vertex to either “pop up” or “pop down.” The large number of pathways along which one can fold an initially unfolded origami structure strongly indicates that a generic structure is likely to become trapped in a “misfolded” state. Thus, new techniques for creating self-folding origami are likely necessary; controlling the popping state of the vertices may be one possibility.
Branches of Triangulated Origami Near the Unfolded State
Chen, Bryan Gin-ge; Santangelo, Christian D.
2018-01-01
Origami structures are characterized by a network of folds and vertices joining unbendable plates. For applications to mechanical design and self-folding structures, it is essential to understand the interplay between the set of folds in the unfolded origami and the possible 3D folded configurations. When deforming a structure that has been folded, one can often linearize the geometric constraints, but the degeneracy of the unfolded state makes a linear approach impossible there. We derive a theory for the second-order infinitesimal rigidity of an initially unfolded triangulated origami structure and use it to study the set of nearly unfolded configurations of origami with four boundary vertices. We find that locally, this set consists of a number of distinct "branches" which intersect at the unfolded state, and that the number of these branches is exponential in the number of vertices. We find numerical and analytical evidence that suggests that the branches are characterized by choosing each internal vertex to either "pop up" or "pop down." The large number of pathways along which one can fold an initially unfolded origami structure strongly indicates that a generic structure is likely to become trapped in a "misfolded" state. Thus, new techniques for creating self-folding origami are likely necessary; controlling the popping state of the vertices may be one possibility.
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
Genes of the unfolded protein response pathway harbor risk alleles for primary open angle glaucoma.
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Mary Anna Carbone
Full Text Available The statistical power of genome-wide association (GWA studies to detect risk alleles for human diseases is limited by the unfavorable ratio of SNPs to study subjects. This multiple testing problem can be surmounted with very large population sizes when common alleles of large effects give rise to disease status. However, GWA approaches fall short when many rare alleles may give rise to a common disease, or when the number of subjects that can be recruited is limited. Here, we demonstrate that this multiple testing problem can be overcome by a comparative genomics approach in which an initial genome-wide screen in a genetically amenable model organism is used to identify human orthologues that may harbor risk alleles for adult-onset primary open angle glaucoma (POAG. Glaucoma is a major cause of blindness, which affects over 60 million people worldwide. Several genes have been associated with juvenile onset glaucoma, but genetic factors that predispose to adult onset primary open angle glaucoma (POAG remain largely unknown. Previous genome-wide analysis in a Drosophila ocular hypertension model identified transcripts with altered regulation and showed induction of the unfolded protein response (UPR upon overexpression of transgenic human glaucoma-associated myocilin (MYOC. We selected 16 orthologous genes with 62 polymorphic markers and identified in two independent human populations two genes of the UPR that harbor POAG risk alleles, BIRC6 and PDIA5. Thus, effectiveness of the UPR in response to accumulation of misfolded or aggregated proteins may contribute to the pathogenesis of POAG and provide targets for early therapeutic intervention.
Genes of the unfolded protein response pathway harbor risk alleles for primary open angle glaucoma.
Carbone, Mary Anna; Chen, Yuhong; Hughes, Guy A; Weinreb, Robert N; Zabriskie, Norman A; Zhang, Kang; Anholt, Robert R H
2011-01-01
The statistical power of genome-wide association (GWA) studies to detect risk alleles for human diseases is limited by the unfavorable ratio of SNPs to study subjects. This multiple testing problem can be surmounted with very large population sizes when common alleles of large effects give rise to disease status. However, GWA approaches fall short when many rare alleles may give rise to a common disease, or when the number of subjects that can be recruited is limited. Here, we demonstrate that this multiple testing problem can be overcome by a comparative genomics approach in which an initial genome-wide screen in a genetically amenable model organism is used to identify human orthologues that may harbor risk alleles for adult-onset primary open angle glaucoma (POAG). Glaucoma is a major cause of blindness, which affects over 60 million people worldwide. Several genes have been associated with juvenile onset glaucoma, but genetic factors that predispose to adult onset primary open angle glaucoma (POAG) remain largely unknown. Previous genome-wide analysis in a Drosophila ocular hypertension model identified transcripts with altered regulation and showed induction of the unfolded protein response (UPR) upon overexpression of transgenic human glaucoma-associated myocilin (MYOC). We selected 16 orthologous genes with 62 polymorphic markers and identified in two independent human populations two genes of the UPR that harbor POAG risk alleles, BIRC6 and PDIA5. Thus, effectiveness of the UPR in response to accumulation of misfolded or aggregated proteins may contribute to the pathogenesis of POAG and provide targets for early therapeutic intervention.
Haywood, J; Yammani, R R
2016-05-01
Obesity is the major risk factor for the development of osteoarthritis (OA); however, the mechanisms involved are not clearly understood. Obesity is associated with increased production of adipokine and elevated levels of circulating free fatty acids (FFA). A recent study has shown that saturated fatty acid palmitate induced pro-inflammatory and pro-apoptotic pathways in chondrocytes. Meniscus has been shown to be more susceptible than articular cartilage to catabolic stimuli. Thus, the aim of this study was to determine the effect of FFA (specifically, palmitate) on meniscus cells. Cultured primary porcine meniscus cells were stimulated with 500 μM FFA (palmitate and oleate) for 24 h to induce endoplasmic reticulum (ER) stress. After treatment, cell lysates were prepared and immunoblotted for C/EBP homologous protein (CHOP). To determine the activation of unfolded protein response (UPR) signaling, cell lysates were probed for cJun n-terminal kinase (JNK), cleaved caspase -3 and Xbp-1s, an alternative mRNA splicing product generated due to Ire1α activation. Treatment of isolated primary meniscus cells with palmitate but not oleate induced expression of CHOP and Xbp-1s. Palmitate treatment of meniscus cells also activated JNK and increased expression of caspase-3, thus promoting apoptosis in meniscus cells. Palmitate induces ER stress and promotes apoptotic pathways in meniscus cells. This is the first study to establish ER stress as a key metabolic mechanistic link between obesity and OA, in addition to (or operating with) biomechanical factors. Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
Delayed Toxicity Associated with Soluble Anthrax Toxin Receptor Decoy-Ig Fusion Protein Treatment
Cote, Christopher; Welkos, Susan; Manchester, Marianne; Young, John A. T.
2012-01-01
Soluble receptor decoy inhibitors, including receptor-immunogloubulin (Ig) fusion proteins, have shown promise as candidate anthrax toxin therapeutics. These agents act by binding to the receptor-interaction site on the protective antigen (PA) toxin subunit, thereby blocking toxin binding to cell surface receptors. Here we have made the surprising observation that co-administration of receptor decoy-Ig fusion proteins significantly delayed, but did not protect, rats challenged with anthrax lethal toxin. The delayed toxicity was associated with the in vivo assembly of a long-lived complex comprised of anthrax lethal toxin and the receptor decoy-Ig inhibitor. Intoxication in this system presumably results from the slow dissociation of the toxin complex from the inhibitor following their prolonged circulation. We conclude that while receptor decoy-Ig proteins represent promising candidates for the early treatment of B. anthracis infection, they may not be suitable for therapeutic use at later stages when fatal levels of toxin have already accumulated in the bloodstream. PMID:22511955
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Rosemeire A. Silva-Lucca
2013-12-01
Full Text Available Baupain belongs to the α+β class of proteins with a secondary structure-content of 44% α-helix, 16% β-sheet and 12% β-turn. The structural transition induced by pH was found to be noncooperative, with no important differences observed in the pH range from 3.0 to 10.5. At pH 2.0 the protein presented substantial non-native structure with strong ANS binding. Guanidine hydrochloride (GdnHCl-induced unfolding did not change the protein structure significantly until 4.0 M, indicating the high rigidity of the molecule. The unfolding was cooperative, as seen by the sigmoidal transition curves with midpoints at 4.7 ± 0.2 M and 5.0 ± 0.2 M GdnHCl, as measured by CD and fluorescence spectroscopy. A red shift of 7 nm in intrinsic fluorescence was observed with 6.0 M GdnHCl. Temperature-induced unfolding of baupain was incomplete, and at least 35% of the native structure of the protein was retained, even at high temperature (90 °C. Baupain showed characteristics of a molten globule state, due to preferential ANS binding at pH 2.0 in comparison to the native form (pH 7.0 and completely unfolded (6.0 M GdnHCl state. Combined with information about N-terminal sequence similarity, these results allow us to include baupain in the papain superfamily.
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Thomas Lahlali
2016-05-01
Full Text Available Background & Aims: Netrin-1, a multifunctional secreted protein, is up-regulated in cancer and inflammation. Netrin-1 blocks apoptosis induced by the prototypical dependence receptors deleted in colorectal carcinoma and uncoordinated phenotype-5. Although the unfolded protein response (UPR triggers apoptosis on exposure to stress, it first attempts to restore endoplasmic reticulum homeostasis to foster cell survival. Importantly, UPR is implicated in chronic liver conditions including hepatic oncogenesis. Netrin-1's implication in cell survival on UPR in this context is unknown. Methods: Isolation of translational complexes, determination of RNA secondary structures by selective 2â-hydroxyl acylation and primer extension/dimethyl sulfate, bicistronic constructs, as well as conventional cell biology and biochemistry approaches were used on in vitroâgrown hepatocytic cells, wild-type, and netrin-1 transgenic mice. Results: HepaRG cells constitute a bona fide model for UPR studies in vitro through adequate activation of the 3 sensors of the UPR (protein kinase RNAâlike endoplasmic reticulum kinase (PERK, inositol requiring enzyme 1α (IRE1α, and activated transcription factor 6 (ATF6. The netrin-1 messenger RNA 5'-end was shown to fold into a complex double pseudoknot and bear E-loop motifs, both of which are representative hallmarks of related internal ribosome entry site regions. Cap-independent translation of netrin 5' untranslated regionâdriven luciferase was observed on UPR in vitro. Unlike several structurally related oncogenic transcripts (l-myc, c-myc, c-myb, netrin-1 messenger RNA was selected for translation during UPR both in human hepatocytes and in mice livers. Depletion of netrin-1 during UPR induces apoptosis, leading to cell death through an uncoordinated phenotype-5A/Câmediated involvement of protein phosphatase 2A and death-associated protein kinase 1 in vitro and in netrin
Seo, Eunyoung; Yeom, Seon-In; Jo, Sunghwan; Jeong, Heejin; Kang, Byoung-Cheorl; Choi, Doil
2012-04-01
Secreted proteins are known to have multiple roles in plant development, metabolism, and stress response. In a previous study to understand the roles of secreted proteins, Capsicum annuum secreted proteins (CaS) were isolated by yeast secretion trap. Among the secreted proteins, we further characterized Capsicum annuum senescence-delaying 1 (CaSD1), a gene encoding a novel secreted protein that is present only in the genus Capsicum. The deduced CaSD1 contains multiple repeats of the amino acid sequence KPPIHNHKPTDYDRS. Interestingly, the number of repeats varied among cultivars and species in the Capsicum genus. CaSD1 is constitutively expressed in roots, and Agrobacterium-mediated transient overexpression of CaSD1 in Nicotiana benthamiana leaves resulted in delayed senescence with a dramatically increased number of trichomes and enlarged epidermal cells. Furthermore, senescence- and cell division-related genes were differentially regulated by CaSD1-overexpressing plants. These observations imply that the pepper-specific cell wall protein CaSD1 plays roles in plant growth and development by regulating cell division and differentiation.
Haze, K; Okada, T; Yoshida, H; Yanagi, H; Yura, T; Negishi, M; Mori, K
2001-04-01
Eukaryotic cells control the levels of molecular chaperones and folding enzymes in the endoplasmic reticulum (ER) by a transcriptional induction process termed the unfolded protein response (UPR). The mammalian UPR is mediated by the cis-acting ER stress response element consisting of 19 nt (CCAATN(9)CCACG), the CCACG part of which is considered to provide specificity. We recently identified the basic leucine zipper (bZIP) protein ATF6 as a mammalian UPR-specific transcription factor; ATF6 is activated by ER stress-induced proteolysis and binds directly to CCACG. Here we report that eukaryotic cells express another bZIP protein closely related to ATF6 in both structure and function. This protein encoded by the G13 (cAMP response element binding protein-related protein) gene is constitutively synthesized as a type II transmembrane glycoprotein anchored in the ER membrane and processed into a soluble form upon ER stress as occurs with ATF6. The proteolytic processing of ATF6 and the G13 gene product is accompanied by their relocation from the ER to the nucleus; their basic regions seem to function as a nuclear localization signal. Overexpression of the soluble form of the G13 product constitutively activates the UPR, whereas overexpression of a mutant lacking the activation domain exhibits a strong dominant-negative effect. Furthermore, the soluble forms of ATF6 and the G13 gene product are unable to bind to several point mutants of the cis-acting ER stress response element in vitro that hardly respond to ER stress in vivo. We thus concluded that the two related bZIP proteins are crucial transcriptional regulators of the mammalian UPR, and propose calling the ATF6 gene product ATF6alpha and the G13 gene product ATF6beta.
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Shvetank Sharma
Full Text Available Nonalcoholic fatty liver disease (NAFLD is a known outcome of hepatosteatosis. Free fatty acids (FFA induce the unfolded protein response (UPR or endoplasmic reticulum (ER stress that may induce apoptosis. Recent data indicate ER stress to be a major player in the progression of fatty liver to more aggressive lesions. Autophagy on the other hand has been demonstrated to be protective against ER stress-induced cell death. We hypothesized that exendin-4 (GLP-1 analog treatment of fat loaded hepatocytes can reduce steatosis by autophagy which leads to reduced ER stress-related hepatocyte apoptosis.Primary human hepatocytes were loaded with saturated, cis- and trans-unsaturated fatty acids (palmitic, oleic and elaidic acid respectively. Steatosis, induced with all three fatty acids, was significantly resolved after exendin-4 treatment. Exendin-4 sustained levels of GRP78 expression in fat-loaded cells when compared to untreated fat-loaded cells alone. In contrast, CHOP (C/EBP homologous protein; the penultimate protein that leads to ER stress-related cell death was significantly decreased by exendin-4 in hepatocytes loaded with fatty acids. Finally, exendin-4 in fat loaded hepatocytes clearly promoted gene products associated with macroautophagy as measured by enhanced production of both Beclin-1 and LC3B-II, markers for autophagy; and visualized by transmission electron microscopy (TEM. Similar observations were made in mouse liver lysates after mice were fed with high fat high fructose diet and treated with a long acting GLP-1 receptor agonist, liraglutide.GLP-1 proteins appear to protect hepatocytes from fatty acid-related death by prohibition of a dysfunctional ER stress response; and reduce fatty acid accumulation, by activation of both macro-and chaperone-mediated autophagy. These findings provide a novel role for GLP-1 proteins in halting the progression of more aggressive lesions from underlying steatosis in humans afflicted with NAFLD.
Jennings, Patricia
Entanglement and knots are naturally occurring, where, in the microscopic world, knots in DNA and homopolymers are well characterized. The most complex knots are observed in proteins which are harder to investigate, as proteins are heteropolymers composed of a combination of 20 different amino acids with different individual biophysical properties. As new-knotted topologies and new proteins containing knots continue to be discovered and characterized, the investigation of knots in proteins has gained intense interest. Thus far, the principle focus has been on the evolutionary origin of tying a knot, with questions of how a protein chain `self-ties' into a knot, what the mechanism(s) are that contribute to threading, and the biological relevance and functional implication of a knotted topology in vivo gaining the most insight. Efforts to study the fully untied and unfolded chain indicate that the knot is highly stable, remaining intact in the unfolded state orders of magnitude longer than first anticipated. The persistence of ``stable'' knots in the unfolded state, together with the challenge of defining an unfolded and untied chain from an unfolded and knotted chain, complicates the study of fully untied protein in vitro. Our discovery of a new class of knotted proteins, the Pierced Lassos (PL) loop topology, simplifies the knotting approach. While PLs are not easily recognizable by the naked eye, they have now been identified in many proteins in the PDB through the use of computation tools. PL topologies are diverse proteins found in all kingdoms of life, performing a large variety of biological responses such as cell signaling, immune responses, transporters and inhibitors (http://lassoprot.cent.uw.edu.pl/). Many of these PL topologies are secreted proteins, extracellular proteins, as well as, redox sensors, enzymes and metal and co-factor binding proteins; all of which provide a favorable environment for the formation of the disulphide bridge. In the PL
Redox Thermodynamics of Cytochromes c Subjected to Urea Induced Unfolding
Monari, S.; Ranieri, A.; Di Rocco, G.; van der Zwan, G.; Peressini, S.; Tavagnacco, C.; Millo, D.; Borsari, M.
2009-01-01
The thermodynamics of the electron transfer (ET) process for beef heart and yeast cytochromes c and the Lys72Ala/Lys73Ala/Lys79Ala mutant of the latter species subjected to progressive urea-induced unfolding was determined electrochemically. The results indicate the presence of at least three protein forms which were assigned to a low-temperature and a high-temperature His-Met intermediate species and a bis-histidinate form (although the presence of a His-Lys form cannot be excluded). The muc...
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Liu, Bao-Qin; Gao, Yan-Yan; Niu, Xiao-Fang [Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001 (China); Xie, Ji-Sheng [Youjiang Medical College for Nationalities, Guangxi 533000 (China); Meng, Xin; Guan, Yifu [Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001 (China); Wang, Hua-Qin, E-mail: wanghq_doctor@hotmail.com [Department of Biochemistry and Molecular Biology, China Medical University, Shenyang 110001 (China)
2010-01-01
Resveratrol (RES), a natural plant polyphenol, is an effective inducer of cell cycle arrest and apoptosis in a variety of carcinoma cell types. In addition, RES has been reported to inhibit tumorigenesis in several animal models suggesting that it functions as a chemopreventive and anti-tumor agent in vivo. The chemopreventive and chemotherapeutic properties associated with resveratrol offer promise for the design of new chemotherapeutic agents. However, the mechanisms by which RES mediates its effects are not yet fully understood. In this study, we showed that RES caused cell cycle arrest and proliferation inhibition via induction of unfolded protein response (UPR) in human leukemia K562 cell line. Treatment of K562 cells with RES induced a number of signature UPR markers, including transcriptional induction of GRP78 and CHOP, phosphorylation of eukaryotic initiation factor 2{alpha} (eIF2{alpha}), ER stress-specific XBP-1 splicing, suggesting the induction of UPR by RES. RES inhibited proliferation of K562 in a concentration-dependent manner. Flow cytometric analyses revealed that K562 cells were arrested in G1 phase upon RES treatment. Salubrinal, an eIF2{alpha} inhibitor, or overexpression of dominant negative mutants of PERK or eIF2{alpha}, effectively restored RES-induced cell cycle arrest, underscoring the important role of PERK/eIF2{alpha} branch of UPR in RES-induced inhibition of cell proliferation.
International Nuclear Information System (INIS)
Liu, Bao-Qin; Gao, Yan-Yan; Niu, Xiao-Fang; Xie, Ji-Sheng; Meng, Xin; Guan, Yifu; Wang, Hua-Qin
2010-01-01
Resveratrol (RES), a natural plant polyphenol, is an effective inducer of cell cycle arrest and apoptosis in a variety of carcinoma cell types. In addition, RES has been reported to inhibit tumorigenesis in several animal models suggesting that it functions as a chemopreventive and anti-tumor agent in vivo. The chemopreventive and chemotherapeutic properties associated with resveratrol offer promise for the design of new chemotherapeutic agents. However, the mechanisms by which RES mediates its effects are not yet fully understood. In this study, we showed that RES caused cell cycle arrest and proliferation inhibition via induction of unfolded protein response (UPR) in human leukemia K562 cell line. Treatment of K562 cells with RES induced a number of signature UPR markers, including transcriptional induction of GRP78 and CHOP, phosphorylation of eukaryotic initiation factor 2α (eIF2α), ER stress-specific XBP-1 splicing, suggesting the induction of UPR by RES. RES inhibited proliferation of K562 in a concentration-dependent manner. Flow cytometric analyses revealed that K562 cells were arrested in G1 phase upon RES treatment. Salubrinal, an eIF2α inhibitor, or overexpression of dominant negative mutants of PERK or eIF2α, effectively restored RES-induced cell cycle arrest, underscoring the important role of PERK/eIF2α branch of UPR in RES-induced inhibition of cell proliferation.
NEUPAC, Experimental Neutron Spectra Unfolding with Sensitivities
International Nuclear Information System (INIS)
Sasaki, Makoto; Nakazawa, Masaharu
1986-01-01
1 - Description of problem or function: The code is able to determine the integral quantities and their sensitivities, together with an estimate of the unfolded spectrum and integral quantities. The code also performs a chi-square test of the input/output data, and contains many options for the calculational routines. 2 - Method of solution: The code is based on the J1-type unfolding method, and the estimated neutron flux spectrum is obtained as its solution. 3 - Restrictions on the complexity of the problem: The maximum number of energy groups used for unfolding is 620. The maximum number of reaction rates and the window functions given as input is 20. The total storage requirement depends on the amount of input data
The effect of a DeltaK280 mutation on the unfolded state of a microtubule-binding repeat in Tau.
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Austin Huang
Full Text Available Tau is a natively unfolded protein that forms intracellular aggregates in the brains of patients with Alzheimer's disease. To decipher the mechanism underlying the formation of tau aggregates, we developed a novel approach for constructing models of natively unfolded proteins. The method, energy-minima mapping and weighting (EMW, samples local energy minima of subsequences within a natively unfolded protein and then constructs ensembles from these energetically favorable conformations that are consistent with a given set of experimental data. A unique feature of the method is that it does not strive to generate a single ensemble that represents the unfolded state. Instead we construct a number of candidate ensembles, each of which agrees with a given set of experimental constraints, and focus our analysis on local structural features that are present in all of the independently generated ensembles. Using EMW we generated ensembles that are consistent with chemical shift measurements obtained on tau constructs. Thirty models were constructed for the second microtubule binding repeat (MTBR2 in wild-type (WT tau and a DeltaK280 mutant, which is found in some forms of frontotemporal dementia. By focusing on structural features that are preserved across all ensembles, we find that the aggregation-initiating sequence, PHF6*, prefers an extended conformation in both the WT and DeltaK280 sequences. In addition, we find that residue K280 can adopt a loop/turn conformation in WT MTBR2 and that deletion of this residue, which can adopt nonextended states, leads to an increase in locally extended conformations near the C-terminus of PHF6*. As an increased preference for extended states near the C-terminus of PHF6* may facilitate the propagation of beta-structure downstream from PHF6*, these results explain how a deletion at position 280 can promote the formation of tau aggregates.
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Emily S. Riddle
2014-01-01
Full Text Available Intrauterine growth restriction (IUGR programs adult disease, including obesity and insulin resistance. Our group previously demonstrated that IUGR dysregulates adipose deposition in male, but not female, weanling rats. Dysregulated adipose deposition is often accompanied by the release of proinflammatory signaling molecules, such as tumor necrosis factor alpha (TNFα. TNFα contributes to adipocyte inflammation and impaired insulin signaling. TNFα has also been implicated in the activation of the unfolded protein response (UPR, which impairs insulin signaling. We hypothesized that, in male rat pups, IUGR would increase TNFα, TNFR1, and components of the UPR (Hspa5, ATF6, p-eIF2α, and Ddit3 prior to the onset of obesity. We further hypothesized that impaired glucose tolerance would occur after the onset of adipose dysfunction in male IUGR rats. To test this hypothesis, we used a well-characterized rat model of uteroplacental insufficiency-induced IUGR. Our primary findings are that, in male rats, IUGR (1 increased circulating and adipose TNFα, (2 increased mRNA levels of UPR components as well as p-eIF2a, and (3 impaired glucose tolerance after observed TNFα increased and after UPR activation. We speculate that programmed dysregulation of TNFα and UPR contributed to the development of glucose intolerance in male IUGR rats.
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Yan Ying
2010-08-01
Full Text Available Abstract Background Resveratrol (RES, a natural phytoalexin found at high levels in grapes and red wine, has been shown to induce anti-proliferation and apoptosis of human cancer cell lines. However, the underlying molecular mechanisms are at present only partially understood. Method The effects of RES on activation of unfolded protein responses (UPR were evaluated using Western blotting, semi-quantitative and real-time RT-PCR. Cell death was evaluated using Annexin V/PI staining and subsequent FACS. Results Similar as tunicamycin, treatment with RES lead to the activation of all 3 branches of the UPR, with early splicing of XBP-1 indicative of IRE1 activation, phosphorylation of eIF2α consistent with ER resident kinase (PERK activation, activating transcription factor 6 (ATF6 splicing, and increase in expression levels of the downstream molecules GRP78/BiP, GRP94 and CHOP/GADD153 in human Burkitt's lymphoma Raji and Daudi cell lines. RES was shown to induce cell death, which could be attenuated by thwarting upregulation of CHOP. Conclusions Our data suggest that activation of the apoptotic arm of the UPR and its downstream effector CHOP/GADD153 is involved, at least in part, in RES-induced apoptosis in Burkitt's lymphoma cells.
Unfolding methods in high-energy physics experiments
International Nuclear Information System (INIS)
Blobel, V.
1985-01-01
Distributions measured in high-energy physics experiments are often distorted or transformed by limited acceptance and finite resolution of the detectors. The unfolding of measured distributions is an important, but due to inherent instabilities a very difficult problem. Methods for unfolding, applicable for the analysis of high-energy physics experiments, and their properties are discussed. An introduction is given to the method of regularization. (orig.)
Unfolding methods in high-energy physics experiments
International Nuclear Information System (INIS)
Blobel, V.
1984-12-01
Distributions measured in high-energy physics experiments are often distorted or transformed by limited acceptance and finite resolution of the detectors. The unfolding of measured distributions is an important, but due to inherent instabilities a very difficult problem. Methods for unfolding, applicable for the analysis of high-energy physics experiments, and their properties are discussed. An introduction is given to the method of regularization. (orig.)
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.
Sengupta, Rituparna; Pantel, Adrian; Cheng, Xian; Shkel, Irina; Peran, Ivan; Stenzoski, Natalie; Raleigh, Daniel P; Record, M Thomas
2016-04-19
In vitro, replacing KCl with potassium glutamate (KGlu), the Escherichia coli cytoplasmic salt and osmolyte, stabilizes folded proteins and protein-nucleic acid complexes. To understand the chemical basis for these effects and rank Glu- in the Hofmeister anion series for protein unfolding, we quantify and interpret the strong stabilizing effect of KGlu on the ribosomal protein domain NTL9, relative to the effects of other stabilizers (KCl, KF, and K2SO4) and destabilizers (GuHCl and GuHSCN). GuHSCN titrations at 20 ° C, performed as a function of the concentration of KGlu or another salt and monitored by NTL9 fluorescence, are analyzed to obtain R-values quantifying the Hofmeister salt concentration (m3) dependence of the unfolding equilibrium constant K(obs) [r-value = −d ln K(obs)/dm3 = (1/RT) dΔG(obs) ° /dm3 = m-value/RT]. r-Values for both stabilizing K+ salts and destabilizing GuH+ salts are compared with predictions from model compound data. For two-salt mixtures, we find that contributions of stabilizing and destabilizing salts to observed r-values are additive and independent. At 20 ° C, we determine a KGlu r-value of 3.22 m(−1) and K2SO4, KF, KCl, GuHCl, and GuHSCN r-values of 5.38, 1.05, 0.64, −1.38, and −3.00 m(−1), respectively. The KGlu r-value represents a 25-fold (1.9 kcal) stabilization per molal KGlu added. KGlu is much more stabilizing than KF, and the stabilizing effect of KGlu is larger in magnitude than the destabilizing effect of GuHSCN. Interpretation of the data reveals good agreement between predicted and observed relative r-values and indicates the presence of significant residual structure in GuHSCN-unfolded NTL9 at 20 ° C.
Catalogue to select the initial guess spectrum during unfolding
Vega-Carrillo, H R
2002-01-01
A new method to select the initial guess spectrum is presented. Neutron spectra unfolded from Bonner sphere data are dependent on the initial guess spectrum used in the unfolding code. The method is based on a catalogue of detector count rates calculated from a set of reported neutron spectra. The spectra of three isotopic neutron sources sup 2 sup 5 sup 2 Cf, sup 2 sup 3 sup 9 PuBe and sup 2 sup 5 sup 2 Cf/D sub 2 O, were measured to test the method. The unfolding was carried out using the three initial guess options included in the BUNKIUT code. Neutron spectra were also calculated using MCNP code. Unfolded spectra were compared with those calculated; in all the cases our method gives the best results.
Kalman filter analysis of delayed neutron nondestructive assay measurements
International Nuclear Information System (INIS)
Aumeier, S. E.
1998-01-01
The ability to nondestructively determine the presence and quantity of fissile and fertile nuclei in various matrices is important in several nuclear applications including international and domestics safeguards, radioactive waste characterization and nuclear facility operations. Material irradiation followed by delayed neutron counting is a well known and useful nondestructive assay technique used to determine the fissile-effective content of assay samples. Previous studies have demonstrated the feasibility of using Kalman filters to unfold individual isotopic contributions to delayed neutron measurements resulting from the assay of mixes of uranium and plutonium isotopes. However, the studies in question used simulated measurement data and idealized parameters. We present the results of the Kalman filter analysis of several measurements of U/Pu mixes taken using Argonne National Laboratory's delayed neutron nondestructive assay device. The results demonstrate the use of Kalman filters as a signal processing tool to determine the fissile and fertile isotopic content of an assay sample from the aggregate delayed neutron response following neutron irradiation
Thermodynamics of protein destabilization in live cells.
Danielsson, Jens; Mu, Xin; Lang, Lisa; Wang, Huabing; Binolfi, Andres; Theillet, François-Xavier; Bekei, Beata; Logan, Derek T; Selenko, Philipp; Wennerström, Håkan; Oliveberg, Mikael
2015-10-06
Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a β-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 °C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein's interplay with the functionally optimized "interaction landscape" of the cellular interior.
OPERATOR NORM INEQUALITIES BETWEEN TENSOR UNFOLDINGS ON THE PARTITION LATTICE.
Wang, Miaoyan; Duc, Khanh Dao; Fischer, Jonathan; Song, Yun S
2017-05-01
Interest in higher-order tensors has recently surged in data-intensive fields, with a wide range of applications including image processing, blind source separation, community detection, and feature extraction. A common paradigm in tensor-related algorithms advocates unfolding (or flattening) the tensor into a matrix and applying classical methods developed for matrices. Despite the popularity of such techniques, how the functional properties of a tensor changes upon unfolding is currently not well understood. In contrast to the body of existing work which has focused almost exclusively on matricizations, we here consider all possible unfoldings of an order- k tensor, which are in one-to-one correspondence with the set of partitions of {1, …, k }. We derive general inequalities between the l p -norms of arbitrary unfoldings defined on the partition lattice. In particular, we demonstrate how the spectral norm ( p = 2) of a tensor is bounded by that of its unfoldings, and obtain an improved upper bound on the ratio of the Frobenius norm to the spectral norm of an arbitrary tensor. For specially-structured tensors satisfying a generalized definition of orthogonal decomposability, we prove that the spectral norm remains invariant under specific subsets of unfolding operations.
International Nuclear Information System (INIS)
Mujcic, Hilda; Rzymski, Tomasz; Rouschop, Kasper M.A.; Koritzinsky, Marianne; Milani, Manuela; Harris, Adrian L.; Wouters, Bradly G.
2009-01-01
Background and purpose: Tumour hypoxia contributes to failure of cancer treatment through its ability to protect against therapy and adversely influence tumour biology. In particular, several studies suggest that hypoxia promotes metastasis. Hypoxia-induced cellular changes are mediated by oxygen-sensitive signaling pathways that activate downstream transcription factors. We have investigated the induction and transcriptional regulation of a novel metastasis-associated gene, LAMP3 during hypoxia. Materials and methods: Microarray, quantitative PCR, Western blot analysis and immunohistochemistry were used to investigate hypoxic regulation of LAMP3. The mechanism for LAMP3 induction was investigated using transient RNAi and stable shRNA targeting components of the hypoxic response. Endoplasmic reticulum stress inducing agents, including proteasome inhibitors were assessed for their ability to regulate LAMP3. Results: LAMP3 is strongly induced by hypoxia at both the mRNA and protein levels in a large panel of human tumour cell lines. Induction of LAMP3 occurs as a consequence of the activation of the PERK/eIF2α/ATF4 arm of the unfolded protein response (UPR) and is independent of HIF-1α. LAMP3 is expressed heterogeneously within the microenvironment of tumours, overexpressed in breast cancer, and increases in tumours treated with avastin. Conclusions: These data identify LAMP3 as a novel hypoxia-inducible gene regulated by the UPR. LAMP3 is a new candidate biomarker of UPR activation by hypoxia in tumours and is a potential mediator of hypoxia-induced metastasis.
Pascal, Christine; Paté, Franck; Cheynier, Véronique; Delsuc, Marc-André
2009-09-01
Astringency is one of the major organoleptic properties of food and beverages that are made from plants, such as tea, chocolate, beer, or red wine. This sensation is thought to be due to interactions between tannins and salivary proline-rich proteins, which are natively unfolded proteins. A human salivary proline-rich protein, namely IB-5, was produced by the recombinant method. Its interactions with a model tannin, epigallocatechin gallate (EGCG), the major flavan-3-ol in green tea, were studied here. Circular dichroism experiments showed that IB-5 presents residual structures (PPII helices) when the ionic strength is close to that in saliva. In the presence of these residual structures, IB-5 undergoes an increase in structural content upon binding to EGCG. NMR data corroborated the presence of preformed structural elements within the protein prior to binding and a partial assignment was proposed, showing partial structuration. TOCSY experiments showed that amino acids that are involved in PPII helices are more likely to interact with EGCG than those in random coil regions, as if they were anchorage points for the ligand. The signal from IB-5 in the DOSY NMR spectrum revealed an increase in polydispersity upon addition of EGCG while the mean hydrodynamic radius remained unchanged. This strongly suggests the formation of IB-5/EGCG aggregates.
Review of unfolding methods for neutron flux dosimetry
International Nuclear Information System (INIS)
Stallmann, F.W.; Kam, F.B.K.
1975-01-01
The primary method in reactor dosimetry is the foil activation technique. To translate the activation measurements into neutron fluxes, a special data processing technique called unfolding is needed. Some general observations about the problems and the reliability of this approach to reactor dosimetry are presented. Current unfolding methods are reviewed. 12 references. (auth)
Deep Unfolding for Topic Models.
Chien, Jen-Tzung; Lee, Chao-Hsi
2018-02-01
Deep unfolding provides an approach to integrate the probabilistic generative models and the deterministic neural networks. Such an approach is benefited by deep representation, easy interpretation, flexible learning and stochastic modeling. This study develops the unsupervised and supervised learning of deep unfolded topic models for document representation and classification. Conventionally, the unsupervised and supervised topic models are inferred via the variational inference algorithm where the model parameters are estimated by maximizing the lower bound of logarithm of marginal likelihood using input documents without and with class labels, respectively. The representation capability or classification accuracy is constrained by the variational lower bound and the tied model parameters across inference procedure. This paper aims to relax these constraints by directly maximizing the end performance criterion and continuously untying the parameters in learning process via deep unfolding inference (DUI). The inference procedure is treated as the layer-wise learning in a deep neural network. The end performance is iteratively improved by using the estimated topic parameters according to the exponentiated updates. Deep learning of topic models is therefore implemented through a back-propagation procedure. Experimental results show the merits of DUI with increasing number of layers compared with variational inference in unsupervised as well as supervised topic models.
Toosi, Siavash; Orlow, Seth J.; Manga, Prashiela
2012-01-01
Vitiligo is characterized by depigmented skin patches due to loss of epidermal melanocytes. Oxidative stress may play 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 (4-TBP) and monobenzyl ether of hydroquinone (MBEH), known triggers of vitiligo. We show that expression of key UPR components, including the transcription factor X-box binding protein 1 (XBP1), are increased following exposure of melanocytes to phenols. XBP1 activation increases production of immune mediators interleukin-6 (IL6) and IL8. Co-treatment with XBP1 inhibitors reduced IL6 and IL8 production induced by phenols, while over-expression 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
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.
Houessinon, Aline; Gicquel, Albane; Bochereau, Flora; Louandre, Christophe; Nyga, Rémy; Godin, Corinne; Degonville, James; Fournier, Emma; Saidak, Zuzana; Drullion, Claire; Barbare, Jean-Claude; Chauffert, Bruno; François, Catherine; Pluquet, Olivier; Galmiche, Antoine
2016-01-28
Sorafenib is the treatment of reference for advanced hepatocellular carcinoma (HCC). A decrease in the serum levels of Alpha-fetoprotein (AFP) is reported to be the biological parameter that is best associated with disease control by sorafenib. In order to provide a biological rationale for the variations of AFP, we analyzed the various steps of AFP production in human HCC cell lines exposed to sorafenib. Sorafenib dramatically reduced the levels of AFP produced by HCC cells independently of its effect on cell viability. The mRNA levels of AFP decreased upon sorafenib treatment, while the AFP protein remained localized in the Golgi apparatus. Sorafenib activated the Regulated Inositol-Requiring Enzyme-1α (IRE-1α) and the PKR-like ER Kinase (PERK)-dependent arms of the Unfolded Protein Response (UPR). The inhibition of IRE-1α partially restored the mRNA levels of AFP upon treatment with sorafenib. The inhibition of both pathways partially prevented the drop in the production of AFP induced by sorafenib. The findings provide new insights on the regulation of AFP, and identify it as a biomarker suitable for the exploration of HCC cell proteostasis in the context of therapeutic targeting. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
Roussel, Benoit D; Newton, Timothy M; Malzer, Elke; Simecek, Nikol; Haq, Imran; Thomas, Sally E; Burr, Marian L; Lehner, Paul J; Crowther, Damian C; Marciniak, Stefan J; Lomas, David A
2013-11-15
Mutants of neuroserpin are retained as polymers within the endoplasmic reticulum (ER) of neurones to cause the autosomal dominant dementia familial encephalopathy with neuroserpin inclusion bodies or FENIB. The cellular consequences are unusual in that the ordered polymers activate the ER overload response (EOR) in the absence of the canonical unfolded protein response. We use both cell lines and Drosophila models to show that the G392E mutant of neuroserpin that forms polymers is degraded by UBE2j1 E2 ligase and Hrd1 E3 ligase while truncated neuroserpin, a protein that lacks 132 amino acids, is degraded by UBE2g2 (E2) and gp78 (E3) ligases. The degradation of G392E neuroserpin results from SREBP-dependent activation of the cholesterol biosynthetic pathway in cells that express polymers of neuroserpin (G392E). Inhibition of HMGCoA reductase, the limiting enzyme of the cholesterol biosynthetic pathway, reduced the ubiquitination of G392E neuroserpin in our cell lines and increased the retention of neuroserpin polymers in both HeLa cells and primary neurones. Our data reveal a reciprocal relationship between cholesterol biosynthesis and the clearance of mutant neuroserpin. This represents the first description of a link between sterol metabolism and modulation of the proteotoxicity mediated by the EOR.
The small angle x-ray scattering of globular proteins in solution during heat denaturation
Banuelos, Jose; Urquidi, Jacob
2008-10-01
The ability of proteins to change their conformation in response to changes in their environment has consequences in biological processes like metabolism, chemical regulation in cells, and is believed to play a role in the onset of several neurodegenerative diseases. Factors such as a change in temperature, pressure, and the introduction of ions into the aqueous environment of a protein can give rise to the folding/unfolding of a protein. As a protein unfolds, the ratio of nonpolar to polar groups exposed to water changes, affecting a protein's thermodynamic properties. Using small angle x-ray scattering (SAXS), we are currently studying the intermediate protein conformations that arise during the folding/unfolding process as a function of temperature for five globular proteins. Trends in the observed intermediate structures of these globular proteins, along with correlations with data on protein thermodynamics may help elucidate shared characteristics between all proteins in the folding/unfolding process. Experimental design considerations will be discussed and preliminary results for some of these systems will be presented.
Influence of cross-section structure on unfolded neutron spectra
International Nuclear Information System (INIS)
Ertek, C.; Vlasov, M.F.; Cross, B.; Smith, P.M.
1979-01-01
The influence of cross-section structure on neutron spectra unfolded by multiple foil activation technique, SAND-II case, has been studied. For three reactions with evident structure in neutron cross-section above threshold: 27Al(n,α)24Na, 31P(n,p)31Si and 32S(n,p)32P, two remarkably different sets of evaluated data were selected from the available evaluations; one set of data was ''smooth'', the structure having been averaged over by a smooth curve; the other set was ''sharp'' with structure given in detail. These data were used in unfolding procedure together with other reactions, the same in both cases (as well as input spectra and measured reaction rates). It was found that during unfolding calculations less iteration steps were needed to unfold the neutron flux spectrum with the set of ''sharp'' data. In case of ''smooth'' data it was difficult to obtain an agreement between measured and calculated activity values even by increasing the number of iteration steps. Contrary to expectations, considerable deformation of unfolded neutron flux spectrum has been observed in the case of the ''smooth'' data set. (author)
Optimization of expression and purification of human mortalin (Hsp70): Folding/unfolding analysis
Khan, Mohd Shahnawaz; Ahmed, Anwar; Tabrez, Shams; Islam, Badar ul; Rabbani, Nayyar; Malik, Ajamaluddin; Ismael, Mohamad A.; Alsenaidy, Mohammad A.; Alsenaidy, Abdulrahman M.
2017-12-01
Human mortalin is a Hsp70 mitochondrial protein that plays an essential role in the biogenesis of mitochondria. The deregulation of mortalin expression and its functions could lead to several age-associated disorders and some types of cancers. In the present study, we optimized the expression and purification of recombinant human mortalin by the use of two-step chromatography. Low temperature (18 °C) and 0.5 mM (IPTG) was required for optimum mortalin expression. Chaperone activity of mortalin was assessed by the citrate synthase and insulin protection assay, which suggested their protective role in mitochondria. Folding and unfolding assessments of mortalin were carried out in the presence of guanidine hydrochloride (GdnHCl) by intrinsic fluorescence measurement, ANS (8-analino 1-nephthlene sulfonic acid) binding and CD (circular dichroism) analysis. Under denaturing conditions, mortalin showed decrease in tryptophan fluorescence intensity along with a red shift of 11 nm. Moreover, ANS binding studies illustrated decrease in hydrophobicity. CD measurement of mortalin showed a predominant helical structure. However, the secondary structure was lost at low concentration of GdnHCl (1 M). We present a simple and robust method to produce soluble mortalin and warranted that chaperones are also susceptible to unfolding and futile to maintain protein homeostasis.
Zhou, Bin; Xie, Jingyi; Liu, Xiaokai; Wang, Bin; Pan, Li
2016-11-15
HacA is a conserved basic leucine zipper transcription factor that serves as the master transcriptional regulator in the unfolded protein response (UPR). To comprehensively evaluate the role of HacA in Aspergillus oryzae, a homokaryotic hacA disruption mutant (HacA-DE) and a strain that expressed a constitutively active form of HacA (HacA-CA) were successfully generated, and transcriptome analyses of these mutants were performed. Growth and phenotypic profiles demonstrated that hyphal growth and sporulation were impaired in the HacA-DE and HacA-CA strains that were grown on complete and minimal media, and the growth impairment was more pronounced for the HacA-CA strain. Compared with a wild-type (WT) strain, the transcriptome results indicated that differentially expressed genes in these mutants mainly fell into four categories: the protein secretory pathway, amino acid metabolism, lipid metabolism, and carbohydrate metabolism. Furthermore, we identified 80 and 36 genes of the secretory pathway whose expression significantly differed in the HacA-CA strain (compared with the WT and HacA-DE strains) and HacA-DE strain (compared with the WT strain), respectively, which mostly belonged to protein folding/UPR, glycosylation, and vesicle transport processes. Both the HacA-CA and HacA-DE strains exhibited reduced expression of extracellular enzymes, especially amylolytic enzymes, which resulted from the activation of the repression under secretion stress mechanism in response to endoplasmic reticulum stress. Collectively, our results suggest that the function of HacA is important not only for UPR induction, but also for growth and fungal physiology, as it serves to reduce secretion stress in A. oryzae. Copyright © 2016 Elsevier B.V. All rights reserved.
Surfactant protein D delays Fas- and TRAIL-mediated extrinsic pathway of apoptosis in T cells.
Djiadeu, Pascal; Kotra, Lakshmi P; Sweezey, Neil; Palaniyar, Nades
2017-05-01
Only a few extracellular soluble proteins are known to modulate apoptosis. We considered that surfactant-associated protein D (SP-D), an innate immune collectin present on many mucosal surfaces, could regulate apoptosis. Although SP-D is known to be important for immune cell homeostasis, whether SP-D affects apoptosis is unknown. In this study we aimed to determine the effects of SP-D on Jurkat T cells and human T cells dying by apoptosis. Here we show that SP-D binds to Jurkat T cells and delays the progression of Fas (CD95)-Fas ligand and TRAIL-TRAIL receptor induced, but not TNF-TNF receptor-mediated apoptosis. SP-D exerts its effects by reducing the activation of initiator caspase-8 and executioner caspase-3. SP-D also delays the surface exposure of phosphatidylserine. The effect of SP-D was ablated by the presence of caspase-8 inhibitor, but not by intrinsic pathway inhibitors. The binding ability of SP-D to dying cells decreases during the early stages of apoptosis, suggesting the release of apoptotic cell surface targets during apoptosis. SP-D also delays FasL-induced death of primary human T cells. SP-D delaying the progression of the extrinsic pathway of apoptosis could have important implications in regulating immune cell homeostasis at mucosal surfaces.
Sanders, Jarred; Scholz, Monika; Merutka, Ilaria; Biron, David
2017-08-28
Disrupting sleep during development leads to lasting deficits in chordates and arthropods. To address lasting impacts of sleep deprivation in Caenorhabditis elegans, we established a nonlethal deprivation protocol. Deprivation triggered protective insulin-like signaling and two unfolded protein responses (UPRs): the mitochondrial (UPR mt ) and the endoplasmic reticulum (UPR ER ) responses. While the latter is known to be triggered by sleep deprivation in rodent and insect brains, the former was not strongly associated with sleep deprivation previously. We show that deprivation results in a feeding defect when the UPR mt is deficient and in UPR ER -dependent germ cell apoptosis. In addition, when the UPR ER is deficient, deprivation causes excess twitching in vulval muscles, mirroring a trend caused by loss of egg-laying command neurons. These data show that nonlethal deprivation of C. elegans sleep causes proteotoxic stress. Unless mitigated, distinct types of deprivation-induced proteotoxicity can lead to anatomically and genetically separable lasting defects. The relative importance of different UPRs post-deprivation likely reflects functional, developmental, and genetic differences between the respective tissues and circuits.
Activation of the cellular unfolded protein response by recombinant adeno-associated virus vectors.
Directory of Open Access Journals (Sweden)
Balaji Balakrishnan
Full Text Available The unfolded protein response (UPR is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER. In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the three major UPR axes, namely, endoribonuclease inositol-requiring enzyme-1 (IRE1α, activating transcription factor 6 (ATF6 and PKR-like ER kinase (PERK in AAV infected cells. Total RNA from mock or AAV infected HeLa cells were used to determine the levels of 8 different ER-stress responsive transcripts from these pathways. We observed a significant up-regulation of IRE1α (up to 11 fold and PERK (up to 8 fold genes 12-48 hours after infection with self-complementary (scAAV2 but less prominent with single-stranded (ssAAV2 vectors. Further studies demonstrated that scAAV1 and scAAV6 also induce cellular UPR in vitro, with AAV1 vectors activating the PERK pathway (3 fold while AAV6 vectors induced a significant increase on all the three major UPR pathways [6-16 fold]. These data suggest that the type and strength of UPR activation is dependent on the viral capsid. We then examined if transient inhibition of UPR pathways by RNA interference has an effect on AAV transduction. siRNA mediated silencing of PERK and IRE1α had a modest effect on AAV2 and AAV6 mediated gene expression (∼1.5-2 fold in vitro. Furthermore, hepatic gene transfer of scAAV2 vectors in vivo, strongly elevated IRE1α and PERK pathways (2 and 3.5 fold, respectively. However, when animals were pre-treated with a pharmacological UPR inhibitor (metformin during scAAV2 gene transfer, the UPR signalling and its subsequent inflammatory response was attenuated concomitant to a modest 2.8 fold increase in transgene expression. Collectively, these data suggest that AAV vectors activate the cellular UPR pathways and their selective inhibition may be beneficial during AAV mediated gene transfer.
[Partially unfolded state of lysozyme with a developed secondary structure in dimethylsulfoxide].
Timchenko, A A; Kirkitadze, M D; Prokhorov, D A; Potekhin, S A; Serdiuk, I N
1996-06-01
The conformation of a chicken egg lysozyme molecule (dimensions, stoichiometry of its associates, and the degree of helicity) in DMSO was studied by small-angle neutron scattering, dynamic light scattering, and optical rotatory dispersion in the visible region of the spectrum. At high DMSO concentrations (70%), the protein was shown to exist as a dimer. The monomer molecules in the dimer adopt a partially unfolded conformation, with dimensions substantially greater than those in the native state and a high content of secondary structure (the degree of helicity is close to that of native lysozyme). This approach provides a unique possibility to assess the compactness of molecules in associates, which may be very useful in studying protein self-organization.
Directory of Open Access Journals (Sweden)
Gábor J. Szebeni
2017-10-01
Full Text Available Achiral Mannich-type curcumin analogs have been synthetized and assayed for their cytotoxic activity. The anti-proliferative and cytotoxic activity of curcuminoids has been tested on human non-small-cell lung carcinoma (A549, hepatocellular carcinoma (HepG2 and pancreatic cancer cell line (PANC-1. Based on the highest anti-proliferative activity nine drug candidates were further tested and proved to cause phosphatidylserine exposure as an early sign of apoptosis. Curcumin analogs with the highest apoptotic activity were selected for mechanistic studies in the most sensitive PANC-1 cells. Cytotoxic activity was accompanied by cytostatic effect since curcumin and analogs treatment led to G0/G1 cell cycle arrest. Moreover, cytotoxic effect could be also detected via the accumulation of curcuminoids in the endoplasmic reticulum (ER and the up-regulation of ER stress-related unfolded protein response (UPR genes: HSPA5, ATF4, XBP1, and DDIT3. The activated UPR induced mitochondrial membrane depolarization, caspase-3 activation and subsequent DNA breakdown in PANC-1 cells. Achiral curcumin analogs, C509, C521 and C524 possessed superior, 40-times more potent cytotoxic activity compared to natural dihydroxy-dimetoxycurcumin in PANC-1 cells.
Szebeni, Gábor J; Balázs, Árpád; Madarász, Ildikó; Pócz, Gábor; Ayaydin, Ferhan; Kanizsai, Iván; Fajka-Boja, Roberta; Alföldi, Róbert; Hackler, László; Puskás, László G
2017-10-07
Achiral Mannich-type curcumin analogs have been synthetized and assayed for their cytotoxic activity. The anti-proliferative and cytotoxic activity of curcuminoids has been tested on human non-small-cell lung carcinoma (A549), hepatocellular carcinoma (HepG2) and pancreatic cancer cell line (PANC-1). Based on the highest anti-proliferative activity nine drug candidates were further tested and proved to cause phosphatidylserine exposure as an early sign of apoptosis. Curcumin analogs with the highest apoptotic activity were selected for mechanistic studies in the most sensitive PANC-1 cells. Cytotoxic activity was accompanied by cytostatic effect since curcumin and analogs treatment led to G₀/G₁ cell cycle arrest. Moreover, cytotoxic effect could be also detected via the accumulation of curcuminoids in the endoplasmic reticulum (ER) and the up-regulation of ER stress-related unfolded protein response (UPR) genes: HSPA5 , ATF4, XBP1 , and DDIT3 . The activated UPR induced mitochondrial membrane depolarization, caspase-3 activation and subsequent DNA breakdown in PANC-1 cells. Achiral curcumin analogs, C509, C521 and C524 possessed superior, 40-times more potent cytotoxic activity compared to natural dihydroxy-dimetoxycurcumin in PANC-1 cells.
Zhang, Rui; Thamm, Douglas H; Misra, Vikram
2015-02-07
We had previously shown that the bLZip domain-containing transcription factor, Zhangfei/CREBZF inhibits the growth and the unfolded protein response (UPR) in cells of the D-17 canine osteosarcoma (OS) line and that the effects of Zhangfei are mediated by it stabilizing the tumour suppressor protein p53. To determine if our observations with D-17 cells applied more universally to canine OS, we examined three other independently isolated canine OS cell lines--Abrams, McKinley and Gracie. Like D-17, the three cell lines expressed p53 proteins that were capable of activating promoters with p53 response elements on their own, and synergistically with Zhangfei. Furthermore, as with D-17 cells, Zhangfei suppressed the growth and UPR-related transcripts in the OS cell lines. Zhangfei also induced the activation of osteocalcin expression, a marker of osteoblast differentiation and triggered programmed cell death. Osteosarcomas are common malignancies in large breeds of dogs. Although there has been dramatic progress in their treatment, these therapies often fail, leading to recurrence of the tumour and metastatic spread. Our results indicate that induction of the expression of Zhangfei in OS, where p53 is functional, may be an effective modality for the treatment of OS.
Conformational Entropy as Collective Variable for Proteins.
Palazzesi, Ferruccio; Valsson, Omar; Parrinello, Michele
2017-10-05
Many enhanced sampling methods rely on the identification of appropriate collective variables. For proteins, even small ones, finding appropriate descriptors has proven challenging. Here we suggest that the NMR S 2 order parameter can be used to this effect. We trace the validity of this statement to the suggested relation between S 2 and conformational entropy. Using the S 2 order parameter and a surrogate for the protein enthalpy in conjunction with metadynamics or variationally enhanced sampling, we are able to reversibly fold and unfold a small protein and draw its free energy at a fraction of the time that is needed in unbiased simulations. We also use S 2 in combination with the free energy flooding method to compute the unfolding rate of this peptide. We repeat this calculation at different temperatures to obtain the unfolding activation energy.
Thapliyal, Charu; Jain, Neha; Chaudhuri, Pratima
2015-01-01
A protein, differing in origin, may exhibit variable physicochemical behaviour, difference in sequence homology, fold and function. Thus studying structure-function relationship of proteins from altered sources is meaningful in the sense that it may give rise to comparative aspects of their sequence-structure-function relationship. Dihydrofolate reductase is an enzyme involved in cell cycle regulation. It is a significant enzyme as.a target for developing anticancer drugs. Hence, detailed understanding of structure-function relationships of wide variants of the enzyme dihydrofolate reductase would be important for developing an inhibitor or an antagonist against the enzyme involved in the cellular developmental processes. In this communication, we have reported the comparative structure-function relationship between E. coli and human dihydrofolate reductase. The differences in the unfolding behaviour of these two proteins have been investigated to understand various properties of these two proteins like relative' stability differences and variation in conformational changes under identical denaturing conditions. The equilibrium unfolding mechanism of dihydrofolate reductase proteins using guanidine hydrochloride as a denaturant in the presence of various types of osmolytes has been monitored using loss in enzymatic activity, intrinsic tryptophan fluorescence and an extrinsic fluorophore 8-anilino-1-naphthalene-sulfonic acid as probes. It has been observed that osmolytes, such as 1M sucrose, and 30% glycerol, provided enhanced stability to both variants of dihydrofolate reductase. Their level of stabilisation has been observed to be dependent on intrinsic protein stability. It was observed that 100 mM proline does not show any 'significant stabilisation to either of dihydrofolate reductases. In the present study, it has been observed that the human protein is relatively less stable than the E.coli counterpart.
Li, Shuli; Zhu, Guannan; Yang, Yuqi; Jian, Zhe; Guo, Sen; Dai, Wei; Shi, Qiong; Ge, Rui; Ma, Jingjing; Liu, Ling; Li, Kai; Luan, Qi; Wang, Gang; Gao, Tianwen; Li, Chunying
2017-07-01
In patients with vitiligo, an increased reactive oxygen species (ROS) level has been proved to be a key player during disease initiation and progression in melanocytes. Nevertheless, little is known about the effects of ROS on other cells involved in the aberrant microenvironment, such as keratinocytes and the following immune events. CXCL16 is constitutively expressed in keratinocytes and was recently found to mediate homing of CD8 + T cells in human skin. We sought to explicate the effect of oxidative stress on human keratinocytes and its capacity to drive CD8 + T-cell trafficking through CXCL16 regulation. We first detected putative T-cell skin-homing chemokines and ROS in serum and lesions of patients with vitiligo. The production of candidate chemokines was detected by using quantitative real-time PCR and ELISA in keratinocytes exposed to H 2 O 2 . Furthermore, the involved mediators were analyzed by using quantitative real-time PCR, Western blotting, ELISA, and immunofluorescence. Next, we tested the chemotactic migration of CD8 + T cells from patients with vitiligo mediated by the CXCL16-CXCR6 pair using the transwell assay. CXCL16 expression increased and showed a positive correlation with oxidative stress levels in serum and lesions of patients with vitiligo. The H 2 O 2 -induced CXCL16 expression was due to the activation of 2 unfolded protein response pathways: kinase RNA (PKR)-like ER kinase-eukaryotic initiation factor 2α and inositol-requiring enzyme 1α-X-box binding protein 1. CXCL16 produced by stressed keratinocytes induced migration of CXCR6 + CD8 + T cells derived from patients with vitiligo. CXCR6 + CD8 + T-cell skin infiltration is accompanied by melanocyte loss in lesions of patients with vitiligo. Our study demonstrated that CXCL16-CXCR6 mediates CD8 + T-cell skin trafficking under oxidative stress in patients with vitiligo. The CXCL16 expression in human keratinocytes induced by ROS is, at least in part, caused by unfolded protein response
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. © 2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
Pethica, Brian A
2010-07-21
Interpretations of data in the extensive literature on the unfolding of proteins in aqueous solution follow a variety of methods involving assumptions leading to estimates of thermodynamic quantities associated with the unfolding transition. Inconsistencies and thermodynamic errors in these methods are identified. Estimates of standard molar free energies and enthalpies of unfolding using incompletely defined equilibrium constants and the van't Hoff relation are unsound, and typically contradict model-free interpretation of the data. A widely used routine for estimating the change in heat capacity associated with unfolding based on changes in the unfolding temperature and enthalpy co-induced by addition of denaturant or protective additives is thermodynamically incorrect by neglect of the Phase Rule. Many models and simulations predicting thermodynamic measures of unfolding are presently making comparisons with insecure quantities derived by incorrect thermodynamic analyses of experimental data. Analysis of unfolding via the Gibbs-Duhem equation with the correct Phase Rule constraints avoids the assumptions associated with incomplete equilibrium constants and misuse of the van't Hoff relation, and applies equally to positive, negative, sitewise or diffuse solute binding to the protein. The method gives the necessary relations between the thermodynamic parameters for thermal and isothermal unfolding and is developed for the case of two-state unfolding. The differences in binding of denaturants or stabilizers to the folded and unfolded forms of the protein are identified as major determinants of the unfolding process. The Phase Rule requires the temperature and enthalpy of unfolding to depend generally on the protein concentration. The available evidence bears out this expectation for thermal unfolding, indicating that protein-protein interactions influence folding. A parallel dependence of the denaturant concentrations for isothermal unfolding on the protein
Directory of Open Access Journals (Sweden)
Yang Jin
2008-08-01
Full Text Available Abstract Background Although lung cancer is among the few malignancies for which we know the primary etiological agent (i.e., cigarette smoke, a precise understanding of the temporal sequence of events that drive tumor progression remains elusive. In addition to finding that cigarette smoke (CS impacts the functioning of key pathways with significant roles in redox homeostasis, xenobiotic detoxification, cell cycle control, and endoplasmic reticulum (ER functioning, our data highlighted a defensive role for the unfolded protein response (UPR program. The UPR promotes cell survival by reducing the accumulation of aberrantly folded proteins through translation arrest, production of chaperone proteins, and increased degradation. Importance of the UPR in maintaining tissue health is evidenced by the fact that a chronic increase in defective protein structures plays a pathogenic role in diabetes, cardiovascular disease, Alzheimer's and Parkinson's syndromes, and cancer. Methods Gene and protein expression changes in CS exposed human cell cultures were monitored by high-density microarrays and Western blot analysis. Tissue arrays containing samples from 110 lung cancers were probed with antibodies to proteins of interest using immunohistochemistry. Results We show that: 1 CS induces ER stress and activates components of the UPR; 2 reactive species in CS that promote oxidative stress are primarily responsible for UPR activation; 3 CS exposure results in increased expression of several genes with significant roles in attenuating oxidative stress; and 4 several major UPR regulators are increased either in expression (i.e., BiP and eIF2α or phosphorylation (i.e., phospho-eIF2α in a majority of human lung cancers. Conclusion These data indicate that chronic ER stress and recruitment of one or more UPR effector arms upon exposure to CS may play a pivotal role in the etiology or progression of lung cancers, and that phospho-eIF2α and BiP may have
Solving inverse problems with the unfolding program TRUEE: Examples in astroparticle physics
International Nuclear Information System (INIS)
Milke, N.; Doert, M.; Klepser, S.; Mazin, D.; Blobel, V.; Rhode, W.
2013-01-01
The unfolding program TRUEE is a software package for the numerical solution of inverse problems. The algorithm was first applied in the FORTRAN 77 program RUN. RUN is an event-based unfolding algorithm which makes use of the Tikhonov regularization. It has been tested and compared to different unfolding applications and stood out with notably stable results and reliable error estimation. TRUEE is a conversion of RUN to C++, which works within the powerful ROOT framework. The program has been extended for more user-friendliness and delivers unfolding results which are identical to RUN. Beside the simplicity of the installation of the software and the generation of graphics, there are new functions, which facilitate the choice of unfolding parameters and observables for the user. In this paper, we introduce the new unfolding program and present its performance by applying it to two exemplary data sets from astroparticle physics, taken with the MAGIC telescopes and the IceCube neutrino detector, respectively.
Interferences of Silica Nanoparticles in Green Fluorescent Protein Folding Processes.
Klein, Géraldine; Devineau, Stéphanie; Aude, Jean Christophe; Boulard, Yves; Pasquier, Hélène; Labarre, Jean; Pin, Serge; Renault, Jean Philippe
2016-01-12
We investigated the relationship between unfolded proteins, silica nanoparticles and chaperonin to determine whether unfolded proteins could stick to silica surfaces and how this process could impair heat shock protein activity. The HSP60 catalyzed green fluorescent protein (GFP) folding was used as a model system. The adsorption isotherms and adsorption kinetics of denatured GFP were measured, showing that denaturation increases GFP affinity for silica surfaces. This affinity is maintained even if the surfaces are covered by a protein corona and allows silica NPs to interfere directly with GFP folding by trapping it in its unstructured state. We determined also the adsorption isotherms of HSP60 and its chaperonin activity once adsorbed, showing that SiO2 NP can interfere also indirectly with protein folding through chaperonin trapping and inhibition. This inhibition is specifically efficient when NPs are covered first with a layer of unfolded proteins. These results highlight for the first time the antichaperonin activity of silica NPs and ask new questions about the toxicity of such misfolded proteins/nanoparticles assembly toward cells.
Can misfolded proteins be beneficial? The HAMLET case.
Pettersson-Kastberg, Jenny; Aits, Sonja; Gustafsson, Lotta; Mossberg, Anki; Storm, Petter; Trulsson, Maria; Persson, Filip; Mok, K Hun; Svanborg, Catharina
2009-01-01
By changing the three-dimensional structure, a protein can attain new functions, distinct from those of the native protein. Amyloid-forming proteins are one example, in which conformational change may lead to fibril formation and, in many cases, neurodegenerative disease. We have proposed that partial unfolding provides a mechanism to generate new and useful functional variants from a given polypeptide chain. Here we present HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) as an example where partial unfolding and the incorporation of cofactor create a complex with new, beneficial properties. Native alpha-lactalbumin functions as a substrate specifier in lactose synthesis, but when partially unfolded the protein binds oleic acid and forms the tumoricidal HAMLET complex. When the properties of HAMLET were first described they were surprising, as protein folding intermediates and especially amyloid-forming protein intermediates had been regarded as toxic conformations, but since then structural studies have supported functional diversity arising from a change in fold. The properties of HAMLET suggest a mechanism of structure-function variation, which might help the limited number of human protein genes to generate sufficient structural diversity to meet the diverse functional demands of complex organisms.
Heenan, Patrick R; Yu, Hao; Siewny, Matthew G W; Perkins, Thomas T
2018-03-28
Precisely quantifying the energetics that drive the folding of membrane proteins into a lipid bilayer remains challenging. More than 15 years ago, atomic force microscopy (AFM) emerged as a powerful tool to mechanically extract individual membrane proteins from a lipid bilayer. Concurrently, fluctuation theorems, such as the Jarzynski equality, were applied to deduce equilibrium free energies (ΔG 0 ) from non-equilibrium single-molecule force spectroscopy records. The combination of these two advances in single-molecule studies deduced the free-energy of the model membrane protein bacteriorhodopsin in its native lipid bilayer. To elucidate this free-energy landscape at a higher resolution, we applied two recent developments. First, as an input to the reconstruction, we used force-extension curves acquired with a 100-fold higher time resolution and 10-fold higher force precision than traditional AFM studies of membrane proteins. Next, by using an inverse Weierstrass transform and the Jarzynski equality, we removed the free energy associated with the force probe and determined the molecular free-energy landscape of the molecule under study, bacteriorhodopsin. The resulting landscape yielded an average unfolding free energy per amino acid (aa) of 1.0 ± 0.1 kcal/mol, in agreement with past single-molecule studies. Moreover, on a smaller spatial scale, this high-resolution landscape also agreed with an equilibrium measurement of a particular three-aa transition in bacteriorhodopsin that yielded 2.7 kcal/mol/aa, an unexpectedly high value. Hence, while average unfolding ΔG 0 per aa is a useful metric, the derived high-resolution landscape details significant local variation from the mean. More generally, we demonstrated that, as anticipated, the inverse Weierstrass transform is an efficient means to reconstruct free-energy landscapes from AFM data.
Thermodynamic effects of proline introduction on protein stability.
Prajapati, Ravindra Singh; Das, Mili; Sreeramulu, Sridhar; Sirajuddin, Minhajuddin; Srinivasan, Sankaranarayanan; Krishnamurthy, Vaishnavi; Ranjani, Ranganathan; Ramakrishnan, C; Varadarajan, Raghavan
2007-02-01
The amino acid Pro is more rigid than other naturally occurring amino acids and, in proteins, lacks an amide hydrogen. To understand the structural and thermodynamic effects of Pro substitutions, it was introduced at 13 different positions in four different proteins, leucine-isoleucine-valine binding protein, maltose binding protein, ribose binding protein, and thioredoxin. Three of the maltose binding protein mutants were characterized by X-ray crystallography to confirm that no structural changes had occurred upon mutation. In the remaining cases, fluorescence and CD spectroscopy were used to show the absence of structural change. Stabilities of wild type and mutant proteins were characterized by chemical denaturation at neutral pH and by differential scanning calorimetry as a function of pH. The mutants did not show enhanced stability with respect to chemical denaturation at room temperature. However, 6 of the 13 single mutants showed a small but significant increase in the free energy of thermal unfolding in the range of 0.3-2.4 kcal/mol, 2 mutants showed no change, and 5 were destabilized. In five of the six cases, the stabilization was because of reduced entropy of unfolding. However, the magnitude of the reduction in entropy of unfolding was typically several fold larger than the theoretical estimate of -4 cal K(-1) mol(-1) derived from the relative areas in the Ramachandran map accessible to Pro and Ala residues, respectively. Two double mutants were constructed. In both cases, the effects of the single mutations on the free energy of thermal unfolding were nonadditive. Copyright 2006 Wiley-Liss, Inc.
Park, Hyo-Jin; Park, Sun-Ji; Koo, Deog-Bon; Lee, Sang-Rae; Kong, Il-Keun; Ryoo, Jae-Woong; Park, Young-Il; Chang, Kyu-Tae; Lee, Dong-Seok
2014-09-15
We examined whether the three unfolded protein response (UPR) signaling pathways, which are activated in response to endoplasmic reticulum (ER)-stress, are involved in progesterone production in the luteal cells of the corpus luteum (CL) during the mouse estrous cycle. The luteal phase of C57BL/6 female mice (8 weeks old) was divided into two stages: the functional stage (16, 24, and 48 h) and the regression stage (72 and 96 h). Western blotting and reverse transcription (RT)-PCR were performed to analyze UPR protein/gene expression levels in each stage. We investigated whether ER stress affects the progesterone production by using Tm (0.5 μg/g BW) or TUDCA (0.5 μg/g BW) through intra-peritoneal injection. Our results indicate that expressions of Grp78/Bip, p-eIF2α/ATF4, p50ATF6, and p-IRE1/sXBP1 induced by UPR activation were predominantly maintained in functional and early regression stages of the CL. Furthermore, the expression of p-JNK, CHOP, and cleaved caspase3 as ER-stress mediated apoptotic factors increased during the regression stage. Cleaved caspase3 levels increased in the late-regression stage after p-JNK and CHOP expression in the early-regression stage. Additionally, although progesterone secretion and levels of steroidogenic enzymes decreased following intra-peritoneal injection of Tunicamycin, an ER stress inducer, the expression of Grp78/Bip, p50ATF6, and CHOP dramatically increased. These results suggest that the UPR signaling pathways activated in response to ER stress may play important roles in the regulation of the CL function. Furthermore, our findings enhance the understanding of the basic mechanisms affecting the CL life span. Copyright © 2014 Elsevier Inc. All rights reserved.
The effects of crowding agents Dextran-70k and PEG-8k on actin structure and unfolding reaction
Gagarskaia, Iuliia A.; Povarova, Olga I.; Uversky, Vladimir N.; Kuznetsova, Irina M.; Turoverov, Konstantin K.
2017-07-01
Recently, an increasing number of studies on proteins' structure, stability and folding are trying to bring the experimental conditions closer to those existing in a living cell, namely to the conditions of macromolecular crowding. In vitro such conditions are typically imitated by the ;inert; highly water-soluble polymers with different hydrodynamic dimensions. In this work, the effects of crowded milieu on the structure and conformational stability of actin, which is a key component of the muscle contraction system, was examined. The crowded milieu was simulated by high concentrations of PEG-8k or Dextran-70k. It was revealed that both crowding agents decelerated but not inhibited actin unfolding and made a compact state of inactivated actin thermodynamically more favorable in comparison with the unfolded state. At the same time, the high viscosity of the solution of crowding agents slowed down all processes and especially inactivated actin formation, since it involves the interaction of 14-16 partially unfolded actin molecules. The effects of crowding agent were larger when its hydrodynamic dimensions were closer to the size of globular actin.
AAA-ATPases in Protein Degradation
Directory of Open Access Journals (Sweden)
Ravikiran S. Yedidi
2017-06-01
Full Text Available Proteolytic machineries containing multisubunit protease complexes and AAA-ATPases play a key role in protein quality control and the regulation of protein homeostasis. In these protein degradation machineries, the proteolytically active sites are formed by either threonines or serines which are buried inside interior cavities of cylinder-shaped complexes. In eukaryotic cells, the proteasome is the most prominent protease complex harboring AAA-ATPases. To degrade protein substrates, the gates of the axial entry ports of the protease need to be open. Gate opening is accomplished by AAA-ATPases, which form a hexameric ring flanking the entry ports of the protease. Protein substrates with unstructured domains can loop into the entry ports without the assistance of AAA-ATPases. However, folded proteins require the action of AAA-ATPases to unveil an unstructured terminus or domain. Cycles of ATP binding/hydrolysis fuel the unfolding of protein substrates which are gripped by loops lining up the central pore of the AAA-ATPase ring. The AAA-ATPases pull on the unfolded polypeptide chain for translocation into the proteolytic cavity of the protease. Conformational changes within the AAA-ATPase ring and the adjacent protease chamber create a peristaltic movement for substrate degradation. The review focuses on new technologies toward the understanding of the function and structure of AAA-ATPases to achieve substrate recognition, unfolding and translocation into proteasomes in yeast and mammalian cells and into proteasome-equivalent proteases in bacteria and archaea.
AAA-ATPases in Protein Degradation.
Yedidi, Ravikiran S; Wendler, Petra; Enenkel, Cordula
2017-01-01
Proteolytic machineries containing multisubunit protease complexes and AAA-ATPases play a key role in protein quality control and the regulation of protein homeostasis. In these protein degradation machineries, the proteolytically active sites are formed by either threonines or serines which are buried inside interior cavities of cylinder-shaped complexes. In eukaryotic cells, the proteasome is the most prominent protease complex harboring AAA-ATPases. To degrade protein substrates, the gates of the axial entry ports of the protease need to be open. Gate opening is accomplished by AAA-ATPases, which form a hexameric ring flanking the entry ports of the protease. Protein substrates with unstructured domains can loop into the entry ports without the assistance of AAA-ATPases. However, folded proteins require the action of AAA-ATPases to unveil an unstructured terminus or domain. Cycles of ATP binding/hydrolysis fuel the unfolding of protein substrates which are gripped by loops lining up the central pore of the AAA-ATPase ring. The AAA-ATPases pull on the unfolded polypeptide chain for translocation into the proteolytic cavity of the protease. Conformational changes within the AAA-ATPase ring and the adjacent protease chamber create a peristaltic movement for substrate degradation. The review focuses on new technologies toward the understanding of the function and structure of AAA-ATPases to achieve substrate recognition, unfolding and translocation into proteasomes in yeast and mammalian cells and into proteasome-equivalent proteases in bacteria and archaea.
Gupta, Chinmaya; López, José Manuel; Azencott, Robert; Bennett, Matthew R; Josić, Krešimir; Ott, William
2014-05-28
Delay is an important and ubiquitous aspect of many biochemical processes. For example, delay plays a central role in the dynamics of genetic regulatory networks as it stems from the sequential assembly of first mRNA and then protein. Genetic regulatory networks are therefore frequently modeled as stochastic birth-death processes with delay. Here, we examine the relationship between delay birth-death processes and their appropriate approximating delay chemical Langevin equations. We prove a quantitative bound on the error between the pathwise realizations of these two processes. Our results hold for both fixed delay and distributed delay. Simulations demonstrate that the delay chemical Langevin approximation is accurate even at moderate system sizes. It captures dynamical features such as the oscillatory behavior in negative feedback circuits, cross-correlations between nodes in a network, and spatial and temporal information in two commonly studied motifs of metastability in biochemical systems. Overall, these results provide a foundation for using delay stochastic differential equations to approximate the dynamics of birth-death processes with delay.
Energy Technology Data Exchange (ETDEWEB)
Gupta, Chinmaya; López, José Manuel; Azencott, Robert; Ott, William [Department of Mathematics, University of Houston, Houston, Texas 77004 (United States); Bennett, Matthew R. [Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77204, USA and Institute of Biosciences and Bioengineering, Rice University, Houston, Texas 77005 (United States); Josić, Krešimir [Department of Mathematics, University of Houston, Houston, Texas 77004 (United States); Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204 (United States)
2014-05-28
Delay is an important and ubiquitous aspect of many biochemical processes. For example, delay plays a central role in the dynamics of genetic regulatory networks as it stems from the sequential assembly of first mRNA and then protein. Genetic regulatory networks are therefore frequently modeled as stochastic birth-death processes with delay. Here, we examine the relationship between delay birth-death processes and their appropriate approximating delay chemical Langevin equations. We prove a quantitative bound on the error between the pathwise realizations of these two processes. Our results hold for both fixed delay and distributed delay. Simulations demonstrate that the delay chemical Langevin approximation is accurate even at moderate system sizes. It captures dynamical features such as the oscillatory behavior in negative feedback circuits, cross-correlations between nodes in a network, and spatial and temporal information in two commonly studied motifs of metastability in biochemical systems. Overall, these results provide a foundation for using delay stochastic differential equations to approximate the dynamics of birth-death processes with delay.
International Nuclear Information System (INIS)
Gupta, Chinmaya; López, José Manuel; Azencott, Robert; Ott, William; Bennett, Matthew R.; Josić, Krešimir
2014-01-01
Delay is an important and ubiquitous aspect of many biochemical processes. For example, delay plays a central role in the dynamics of genetic regulatory networks as it stems from the sequential assembly of first mRNA and then protein. Genetic regulatory networks are therefore frequently modeled as stochastic birth-death processes with delay. Here, we examine the relationship between delay birth-death processes and their appropriate approximating delay chemical Langevin equations. We prove a quantitative bound on the error between the pathwise realizations of these two processes. Our results hold for both fixed delay and distributed delay. Simulations demonstrate that the delay chemical Langevin approximation is accurate even at moderate system sizes. It captures dynamical features such as the oscillatory behavior in negative feedback circuits, cross-correlations between nodes in a network, and spatial and temporal information in two commonly studied motifs of metastability in biochemical systems. Overall, these results provide a foundation for using delay stochastic differential equations to approximate the dynamics of birth-death processes with delay
Zero singularities of codimension two and three in delay differential equations
International Nuclear Information System (INIS)
Campbell, Sue Ann; Yuan Yuan
2008-01-01
We give conditions under which a general class of delay differential equations has a point of Bogdanov–Takens or a triple zero bifurcation. We show how a centre manifold projection of the delay equations reduces the dynamics to two- or three-dimensional systems of ordinary differential equations. We put these equations in normal form and determine how the coefficients of the normal forms depend on the original parameters in the model. Finally we apply our results to two neural models and compare the predictions of the theory with numerical bifurcation analysis of the full equations. One model involves a transcritical bifurcation, hence we derive and analyse the appropriate unfoldings for this case
[Unfolding item response model using best-worst scaling].
Ikehara, Kazuya
2015-02-01
In attitude measurement and sensory tests, the unfolding model is typically used. In this model, response probability is formulated by the distance between the person and the stimulus. In this study, we proposed an unfolding item response model using best-worst scaling (BWU model), in which a person chooses the best and worst stimulus among repeatedly presented subsets of stimuli. We also formulated an unfolding model using best scaling (BU model), and compared the accuracy of estimates between the BU and BWU models. A simulation experiment showed that the BWU modell performed much better than the BU model in terms of bias and root mean square errors of estimates. With reference to Usami (2011), the proposed models were apllied to actual data to measure attitudes toward tardiness. Results indicated high similarity between stimuli estimates generated with the proposed models and those of Usami (2011).
Nayar, Divya; Folberth, Angelina; van der Vegt, Nico F A
2017-07-19
Osmolytes affect hydrophobic collapse and protein folding equilibria. The underlying mechanisms are, however, not well understood. We report large-scale conformational sampling of two hydrophobic polymers with secondary and tertiary amide side chains using extensive molecular dynamics simulations. The calculated free energy of unfolding increases with urea for the secondary amide, yet decreases for the tertiary amide, in agreement with experiment. The underlying mechanism is rooted in opposing entropic driving forces: while urea screens the hydrophobic macromolecular interface and drives unfolding of the tertiary amide, urea's concomitant loss in configurational entropy drives collapse of the secondary amide. Only at sufficiently high urea concentrations bivalent urea hydrogen bonding interactions with the secondary amide lead to further stabilisation of its collapsed state. The observations provide a new angle on the interplay between side chain chemistry, urea hydrogen bonding, and the role of urea in attenuating or strengthening the hydrophobic effect.
Fundamentals of unfolding, refolding and aggregation of food proteins
Broersen, K.
2005-01-01
Protein functionality in food products strongly relies on the fact that proteins can undergo intermolecular interactions, called aggregation. It was found that very subtle dynamics inherent to the protein of interest can have consequences for the functional properties of proteins. The aim of this
Regularization and error assignment to unfolded distributions
Zech, Gunter
2011-01-01
The commonly used approach to present unfolded data only in graphical formwith the diagonal error depending on the regularization strength is unsatisfac-tory. It does not permit the adjustment of parameters of theories, the exclusionof theories that are admitted by the observed data and does not allow the com-bination of data from different experiments. We propose fixing the regulariza-tion strength by a p-value criterion, indicating the experimental uncertaintiesindependent of the regularization and publishing the unfolded data in additionwithout regularization. These considerations are illustrated with three differentunfolding and smoothing approaches applied to a toy example.
International Nuclear Information System (INIS)
Yan, Ying; Gao, Yan-Yan; Liu, Bao-Qin; Niu, Xiao-Fang; Zhuang, Ying; Wang, Hua-Qin
2010-01-01
Resveratrol (RES), a natural phytoalexin found at high levels in grapes and red wine, has been shown to induce anti-proliferation and apoptosis of human cancer cell lines. However, the underlying molecular mechanisms are at present only partially understood. The effects of RES on activation of unfolded protein responses (UPR) were evaluated using Western blotting, semi-quantitative and real-time RT-PCR. Cell death was evaluated using Annexin V/PI staining and subsequent FACS. Similar as tunicamycin, treatment with RES lead to the activation of all 3 branches of the UPR, with early splicing of XBP-1 indicative of IRE1 activation, phosphorylation of eIF2α consistent with ER resident kinase (PERK) activation, activating transcription factor 6 (ATF6) splicing, and increase in expression levels of the downstream molecules GRP78/BiP, GRP94 and CHOP/GADD153 in human Burkitt's lymphoma Raji and Daudi cell lines. RES was shown to induce cell death, which could be attenuated by thwarting upregulation of CHOP. Our data suggest that activation of the apoptotic arm of the UPR and its downstream effector CHOP/GADD153 is involved, at least in part, in RES-induced apoptosis in Burkitt's lymphoma cells
Directory of Open Access Journals (Sweden)
Yunjie Zhao
Full Text Available The zinc-binding Bbox1 domain in protein MID1, a member of the TRIM family of proteins, facilitates the ubiquitination of the catalytic subunit of protein phosphatase 2A and alpha4, a protein regulator of PP2A. The natural mutation of residue A130 to a valine or threonine disrupts substrate recognition and catalysis. While NMR data revealed the A130T mutant Bbox1 domain failed to coordinate both structurally essential zinc ions and resulted in an unfolded structure, the unfolding mechanism is unknown. Principle component analysis revealed that residue A130 served as a hinge point between the structured β-strand-turn-β-strand (β-turn-β and the lasso-like loop sub-structures that constitute loop1 of the ββα-RING fold that the Bbox1 domain adopts. Backbone RMSD data indicate significant flexibility and departure from the native structure within the first 5 ns of the molecular dynamics (MD simulation for the A130V mutant (>6 Å and after 30 ns for A130T mutant (>6 Å. Overall RMSF values were higher for the mutant structures and showed increased flexibility around residues 125 and 155, regions with zinc-coordinating residues. Simulated pKa values of the sulfhydryl group of C142 located near A130 suggested an increased in value to ~9.0, paralleling the increase in the apparent dielectric constants for the small cavity near residue A130. Protonation of the sulfhydryl group would disrupt zinc-coordination, directly contributing to unfolding of the Bbox1. Together, the increased motion of residues of loop 1, which contains four of the six zinc-binding cysteine residues, and the increased pKa of C142 could destabilize the structure of the zinc-coordinating residues and contribute to the unfolding.
Kinetic evidence for a two-stage mechanism of protein denaturation by guanidinium chloride.
Jha, Santosh Kumar; Marqusee, Susan
2014-04-01
Dry molten globular (DMG) intermediates, an expanded form of the native protein with a dry core, have been observed during denaturant-induced unfolding of many proteins. These observations are counterintuitive because traditional models of chemical denaturation rely on changes in solvent-accessible surface area, and there is no notable change in solvent-accessible surface area during the formation of the DMG. Here we show, using multisite fluorescence resonance energy transfer, far-UV CD, and kinetic thiol-labeling experiments, that the guanidinium chloride (GdmCl)-induced unfolding of RNase H also begins with the formation of the DMG. Population of the DMG occurs within the 5-ms dead time of our measurements. We observe that the size and/or population of the DMG is linearly dependent on [GdmCl], although not as strongly as the second and major step of unfolding, which is accompanied by core solvation and global unfolding. This rapid GdmCl-dependent population of the DMG indicates that GdmCl can interact with the protein before disrupting the hydrophobic core. These results imply that the effect of chemical denaturants cannot be interpreted solely as a disruption of the hydrophobic effect and strongly support recent computational studies, which hypothesize that chemical denaturants first interact directly with the protein surface before completely unfolding the protein in the second step (direct interaction mechanism).
Xiao, Yiming; Konermann, Lars
2015-08-01
Gas/water interfaces (such as air bubbles or foam) are detrimental to the stability of proteins, often causing aggregation. This represents a potential problem for industrial processes, for example, the production and handling of protein drugs. Proteins possess surfactant-like properties, resulting in a high affinity for gas/water interfaces. The tendency of previously buried nonpolar residues to maximize contact with the gas phase can cause significant structural distortion. Most earlier studies in this area employed spectroscopic tools that could only provide limited information. Here we use hydrogen/deuterium exchange (HDX) mass spectrometry (MS) for probing the conformational dynamics of the model protein myoglobin (Mb) in the presence of N(2) bubbles. HDX/MS relies on the principle that unfolded and/or highly dynamic regions undergo faster deuteration than tightly folded segments. In bubble-free solution Mb displays EX2 behavior, reflecting the occurrence of short-lived excursions to partially unfolded conformers. A dramatically different behavior is seen in the presence of N(2) bubbles; EX2 dynamics still take place, but in addition the protein shows EX1 behavior. The latter results from interconversion of the native state with conformers that are globally unfolded and long-lived. These unfolded species likely correspond to Mb that is adsorbed to the surface of gas bubbles. N(2) sparging also induces aggregation. To explain the observed behavior we propose a simple model, that is, "semi-unfolded" ↔ "native" ↔ "globally unfolded" → "aggregated". This model quantitatively reproduces the experimentally observed kinetics. To the best of our knowledge, the current study marks the first exploration of surface denaturation phenomena by HDX/MS. © 2015 The Protein Society.
Unfolding code for neutron spectrometry based on neural nets technology
International Nuclear Information System (INIS)
Ortiz R, J. M.; Vega C, H. R.
2012-10-01
The most delicate part of neutron spectrometry, is the unfolding process. The derivation of the spectral information is not simple because the unknown is not given directly as a result of the measurements. The drawbacks associated with traditional unfolding procedures have motivated the need of complementary approaches. Novel methods based on Artificial Neural Networks have been widely investigated. In this work, a neutron spectrum unfolding code based on neural nets technology is presented. This unfolding code called Neutron Spectrometry and Dosimetry by means of Artificial Neural Networks was designed in a graphical interface under LabVIEW programming environment. The core of the code is an embedded neural network architecture, previously optimized by the R obust Design of Artificial Neural Networks Methodology . The main features of the code are: is easy to use, friendly and intuitive to the user. This code was designed for a Bonner Sphere System based on a 6 Lil(Eu) neutron detector and a response matrix expressed in 60 energy bins taken from an International Atomic Energy Agency compilation. The main feature of the code is that as entrance data, only seven rate counts measurement with a Bonner spheres spectrometer are required for simultaneously unfold the 60 energy bins of the neutron spectrum and to calculate 15 dosimetric quantities, for radiation protection porpoises. This code generates a full report in html format with all relevant information. (Author)
A neutron spectrum unfolding computer code based on artificial neural networks
International Nuclear Information System (INIS)
Ortiz-Rodríguez, J.M.; Reyes Alfaro, A.; Reyes Haro, A.; Cervantes Viramontes, J.M.; Vega-Carrillo, H.R.
2014-01-01
The Bonner Spheres Spectrometer consists of a thermal neutron sensor placed at the center of a number of moderating polyethylene spheres of different diameters. From the measured readings, information can be derived about the spectrum of the neutron field where measurements were made. Disadvantages of the Bonner system are the weight associated with each sphere and the need to sequentially irradiate the spheres, requiring long exposure periods. Provided a well-established response matrix and adequate irradiation conditions, the most delicate part of neutron spectrometry, is the unfolding process. The derivation of the spectral information is not simple because the unknown is not given directly as a result of the measurements. The drawbacks associated with traditional unfolding procedures have motivated the need of complementary approaches. Novel methods based on Artificial Intelligence, mainly Artificial Neural Networks, have been widely investigated. In this work, a neutron spectrum unfolding code based on neural nets technology is presented. This code is called Neutron Spectrometry and Dosimetry with Artificial Neural networks unfolding code that was designed in a graphical interface. The core of the code is an embedded neural network architecture previously optimized using the robust design of artificial neural networks methodology. The main features of the code are: easy to use, friendly and intuitive to the user. This code was designed for a Bonner Sphere System based on a 6 LiI(Eu) neutron detector and a response matrix expressed in 60 energy bins taken from an International Atomic Energy Agency compilation. The main feature of the code is that as entrance data, for unfolding the neutron spectrum, only seven rate counts measured with seven Bonner spheres are required; simultaneously the code calculates 15 dosimetric quantities as well as the total flux for radiation protection purposes. This code generates a full report with all information of the unfolding
An FCS study of unfolding and refolding of CPM-labeled human serum albumin: role of ionic liquid.
Sasmal, Dibyendu Kumar; Mondal, Tridib; Sen Mojumdar, Supratik; Choudhury, Aparajita; Banerjee, Rajat; Bhattacharyya, Kankan
2011-11-10
The effect of a room temperature ionic liquid (RTIL) on the conformational dynamics of a protein, human serum albumin (HSA), is studied by fluorescence correlation spectroscopy (FCS). For this, the protein was covalently labeled by a fluorophore, 7-dimethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). On addition of a RTIL ([pmim][Br]) to the native protein, the diffusion coefficient (D(t)) decreases and the hydrodynamic radius (R(h)) increases. This suggests that the RTIL ([pmim][Br]) acts as a denaturant when the protein is in the native state. However, addition of [pmim][Br] to a protein denatured by GdnHCl causes an increases in D(t) and decrease in R(h). This suggests that in the presence of GdnHCl addition of RTIL helps the protein to refold. In the native state, the conformational dynamics of protein is described by three distinct time constants: ~3.6 ± 0.7, ~29 ± 4.5, and 133 ± 23 μs. The faster components (~3.6 ± 0.7 and ~29 ± 4.5 μs) are ascribed to chain dynamics of the protein, while the slowest component (133 μs) is responsible for interchain interaction or concerted motion. On addition of [pmim][Br], the conformational dynamics of HSA becomes slower (~5.1 ± 1, ~43.5 ± 2.8, and ~311 ± 2.3 μs in the presence of 1.5 M [pmim][Br]). The time constants for the protein denatured by 6 M GdnHCl are 3.2 ± 0.4, 34 ± 6, and 207 ± 38 μs. When 1.5 M [pmim][Br] is added to the denatured protein (in 6 M GdnHCl), the time constants become ~5 ± 1, ~41 ± 10, and ~230 ± 45 μs. The lifetime histogram shows that, on addition of GdnHCl to HSA, the contribution of the shorter lifetime component decreases and vanishes at 6 M GdnHCl. The shorter lifetime component immediately reappears after addition of RTIL to unfolded HSA. This suggests recoiling of the unfolded protein by RTIL.
Unfolding neutron spectra obtained from BS–TLD system using genetic algorithm
International Nuclear Information System (INIS)
Santos, J.A.L.; Silva, E.R.; Ferreira, T.A.E; Vilela, E.C.
2012-01-01
Due to the variability of neutron spectrum within the same environment, it is essential that the spectral distribution as a function of energy should be characterized. The precise information allows radiological quantities establishment related to that spectrum, but it is necessary that a spectrometric system covers a large interval of energy and an unfolding process is appropriate. This paper proposes use of a technique of Artificial Intelligence (AI) called genetic algorithm (GA), which uses bio-inspired mathematical models with the implementation of a specific matrix to unfolding data obtained from a combination of TLDs embedded in a BS system to characterize the neutron spectrum as a function of energy. The results obtained with this method were in accordance with reference spectra, thus enabling this technique to unfold neutron spectra with the BS–TLD system. - Highlights: ► The unfolding code used the artificial intelligence technique called genetic algorithms. ► A response matrix specific to the unfolding data obtained with the BS–TLD system is used by the AGLN. ► The observed results demonstrate the potential use of genetic algorithms in solving complex nuclear problems.
Unfolding in particle physics: A window on solving inverse problems
International Nuclear Information System (INIS)
Spano, F.
2013-01-01
Unfolding is the ensemble of techniques aimed at resolving inverse, ill-posed problems. A pedagogical introduction to the origin and main problems related to unfolding is presented and used as the the stepping stone towards the illustration of some of the most common techniques that are currently used in particle physics experiments. (authors)
There and back again: Two views on the protein folding puzzle.
Finkelstein, Alexei V; Badretdin, Azat J; Galzitskaya, Oxana V; Ivankov, Dmitry N; Bogatyreva, Natalya S; Garbuzynskiy, Sergiy O
2017-07-01
The ability of protein chains to spontaneously form their spatial structures is a long-standing puzzle in molecular biology. Experimentally measured folding times of single-domain globular proteins range from microseconds to hours: the difference (10-11 orders of magnitude) is the same as that between the life span of a mosquito and the age of the universe. This review describes physical theories of rates of overcoming the free-energy barrier separating the natively folded (N) and unfolded (U) states of protein chains in both directions: "U-to-N" and "N-to-U". In the theory of protein folding rates a special role is played by the point of thermodynamic (and kinetic) equilibrium between the native and unfolded state of the chain; here, the theory obtains the simplest form. Paradoxically, a theoretical estimate of the folding time is easier to get from consideration of protein unfolding (the "N-to-U" transition) rather than folding, because it is easier to outline a good unfolding pathway of any structure than a good folding pathway that leads to the stable fold, which is yet unknown to the folding protein chain. And since the rates of direct and reverse reactions are equal at the equilibrium point (as follows from the physical "detailed balance" principle), the estimated folding time can be derived from the estimated unfolding time. Theoretical analysis of the "N-to-U" transition outlines the range of protein folding rates in a good agreement with experiment. Theoretical analysis of folding (the "U-to-N" transition), performed at the level of formation and assembly of protein secondary structures, outlines the upper limit of protein folding times (i.e., of the time of search for the most stable fold). Both theories come to essentially the same results; this is not a surprise, because they describe overcoming one and the same free-energy barrier, although the way to the top of this barrier from the side of the unfolded state is very different from the way from the
2013-01-01
Background Two-dimensional gel electrophoresis (2DE) is one of the most popular methods in proteomics. Currently, most 2DE experiments are performed using immobilized pH gradient (IPG) in the first dimension; however, some laboratories still use carrier ampholytes-based isoelectric focusing technique. The aim of this study was to directly compare IPG-based and non-equilibrium pH gradient electrophoresis (NEPHGE)-based 2DE techniques by using the same samples and identical second dimension procedures. We have used commercially available Invitrogen ZOOM IPGRunner and WITAvision systems for IPG and NEPHGE, respectively. The effectiveness of IPG-based and NEPHGE-based 2DE methods was compared by analysing differential protein expression during cytosolic unfolded protein response (UPR-Cyto) in Saccharomyces cerevisiae. Results Protein loss during 2DE procedure was higher in IPG-based method, especially for basic (pI > 7) proteins. Overall reproducibility of spots was slightly better in NEPHGE-based method; however, there was a marked difference when evaluating basic and acidic protein spots. Using Coomassie staining, about half of detected basic protein spots were not reproducible by IPG-based 2DE, whereas NEPHGE-based method showed excellent reproducibility in the basic gel zone. The reproducibility of acidic proteins was similar in both methods. Absolute and relative volume variability of separate protein spots was comparable in both 2DE techniques. Regarding proteomic analysis of UPR-Cyto, the results exemplified parameters of general comparison of the methods. New highly basic protein Sis1p, overexpressed during UPR-Cyto stress, was identified by NEPHGE-based 2DE method, whereas IPG-based method showed unreliable results in the basic pI range and did not provide any new information on basic UPR-Cyto proteins. In the acidic range, the main UPR-Cyto proteins were detected and quantified by both methods. The drawback of NEPHGE-based 2DE method is its failure to
Declining global warming effects on the phenology of spring leaf unfolding.
Fu, Yongshuo H; Zhao, Hongfang; Piao, Shilong; Peaucelle, Marc; Peng, Shushi; Zhou, Guiyun; Ciais, Philippe; Huang, Mengtian; Menzel, Annette; Peñuelas, Josep; Song, Yang; Vitasse, Yann; Zeng, Zhenzhong; Janssens, Ivan A
2015-10-01
Earlier spring leaf unfolding is a frequently observed response of plants to climate warming. Many deciduous tree species require chilling for dormancy release, and warming-related reductions in chilling may counteract the advance of leaf unfolding in response to warming. Empirical evidence for this, however, is limited to saplings or twigs in climate-controlled chambers. Using long-term in situ observations of leaf unfolding for seven dominant European tree species at 1,245 sites, here we show that the apparent response of leaf unfolding to climate warming (ST, expressed in days advance of leaf unfolding per °C warming) has significantly decreased from 1980 to 2013 in all monitored tree species. Averaged across all species and sites, ST decreased by 40% from 4.0 ± 1.8 days °C(-1) during 1980-1994 to 2.3 ± 1.6 days °C(-1) during 1999-2013. The declining ST was also simulated by chilling-based phenology models, albeit with a weaker decline (24-30%) than observed in situ. The reduction in ST is likely to be partly attributable to reduced chilling. Nonetheless, other mechanisms may also have a role, such as 'photoperiod limitation' mechanisms that may become ultimately limiting when leaf unfolding dates occur too early in the season. Our results provide empirical evidence for a declining ST, but also suggest that the predicted strong winter warming in the future may further reduce ST and therefore result in a slowdown in the advance of tree spring phenology.
Unfolding code for neutron spectrometry based on neural nets technology
Energy Technology Data Exchange (ETDEWEB)
Ortiz R, J. M.; Vega C, H. R., E-mail: morvymm@yahoo.com.mx [Universidad Autonoma de Zacatecas, Unidad Academica de Ingenieria Electrica, Apdo. Postal 336, 98000 Zacatecas (Mexico)
2012-10-15
The most delicate part of neutron spectrometry, is the unfolding process. The derivation of the spectral information is not simple because the unknown is not given directly as a result of the measurements. The drawbacks associated with traditional unfolding procedures have motivated the need of complementary approaches. Novel methods based on Artificial Neural Networks have been widely investigated. In this work, a neutron spectrum unfolding code based on neural nets technology is presented. This unfolding code called Neutron Spectrometry and Dosimetry by means of Artificial Neural Networks was designed in a graphical interface under LabVIEW programming environment. The core of the code is an embedded neural network architecture, previously optimized by the {sup R}obust Design of Artificial Neural Networks Methodology{sup .} The main features of the code are: is easy to use, friendly and intuitive to the user. This code was designed for a Bonner Sphere System based on a {sup 6}Lil(Eu) neutron detector and a response matrix expressed in 60 energy bins taken from an International Atomic Energy Agency compilation. The main feature of the code is that as entrance data, only seven rate counts measurement with a Bonner spheres spectrometer are required for simultaneously unfold the 60 energy bins of the neutron spectrum and to calculate 15 dosimetric quantities, for radiation protection porpoises. This code generates a full report in html format with all relevant information. (Author)
UMG 3.3, Analysis of data measured with spectrometers using unfolding techniques
International Nuclear Information System (INIS)
Reginatto, Marcel; Wiegel, Burkhard; Zimbal, Andreas; Langner, Frank
2004-01-01
1 - Description of program or function: UMG (Unfolding with MAXED and GRAVEL) is a package of seven programs written for the analysis of data measured with spectrometers that require the use of unfolding techniques. The program MAXED applies the maximum entropy principle to the unfolding problem, and the program GRAVEL uses a modified SAND-II algorithm to do the unfolding. There are two versions of each: MXD F C33 and GRV F C33 for 'few-channel' unfolding (e.g., Bonner sphere spectrometers) and MXD M C33 and GRV M C33 for 'multi-channel' unfolding (e.g., NE-213). The program IQU can be used to calculate integral quantities for both MAXED and GRAVEL solution spectra and, in the case of MAXED solutions, it can also be used to calculate the uncertainty in these values as well as the uncertainty in the solution spectrum. The uncertainty calculation is handled in the following way: given a solution spectrum generated by MAXED, the program IQU considers variations in the measured data and in the default spectrum and uses standard Methods to do sensitivity analysis and uncertainty propagation. There are two versions: IQU F C33 for 'few-channel' unfolding and IQU M C33 for 'multi-channel' unfolding. The program UMGPlot can be used to display the results from the unfolding programs MAXED and GRAVEL in graphical form in a quick and easy way. 2 - Methods: MAXED is based on the maximum entropy principle. The solution to the unfolding problem is obtained by maximisation of the relative entropy (used here in the form due to Skilling, which is a generalisation of the usual expression to distributions that are not necessarily normalized) subject to constraints imposed by the measurements. This approach permits the inclusion of prior information in a well-defined and mathematically consistent way, and it leads to a solution spectrum that is a non-negative function which can be written in closed form. This last feature permits the use of standard Methods for sensitivity analysis and
One Peptide Reveals the Two Faces of α-Helix Unfolding-Folding Dynamics.
Jesus, Catarina S H; Cruz, Pedro F; Arnaut, Luis G; Brito, Rui M M; Serpa, Carlos
2018-04-12
The understanding of fast folding dynamics of single α-helices comes mostly from studies on rationally designed peptides displaying sequences with high helical propensity. The folding/unfolding dynamics and energetics of α-helix conformations in naturally occurring peptides remains largely unexplored. Here we report the study of a protein fragment analogue of the C-peptide from bovine pancreatic ribonuclease-A, RN80, a 13-amino acid residue peptide that adopts a highly populated helical conformation in aqueous solution. 1 H NMR and CD structural studies of RN80 showed that α-helix formation displays a pH-dependent bell-shaped curve, with a maximum near pH 5, and a large decrease in helical content in alkaline pH. The main forces stabilizing this short α-helix were identified as a salt bridge formed between Glu-2 and Arg-10 and the cation-π interaction involving Tyr-8 and His-12. Thus, deprotonation of Glu-2 or protonation of His-12 are essential for the RN80 α-helix stability. In the present study, RN80 folding and unfolding were triggered by laser-induced pH jumps and detected by time-resolved photoacoustic calorimetry (PAC). The photoacid proton release, amino acid residue protonation, and unfolding/folding events occur at different time scales and were clearly distinguished using time-resolved PAC. The partial unfolding of the RN80 α-helix, due to protonation of Glu-2 and consequent breaking of the stabilizing salt bridge between Glu-2 and Arg-10, is characterized by a concentration-independent volume expansion in the sub-microsecond time range (0.8 mL mol -1 , 369 ns). This small volume expansion reports the cost of peptide backbone rehydration upon disruption of a solvent-exposed salt bridge, as well as backbone intrinsic expansion. On the other hand, RN80 α-helix folding triggered by His-12 protonation and subsequent formation of a cation-π interaction leads to a microsecond volume contraction (-6.0 mL mol -1 , ∼1.7 μs). The essential role of two
Understanding curcumin-induced modulation of protein aggregation.
Ahmad, Basir; Borana, Mohanish S; Chaudhary, Ankur P
2017-07-01
Curcumin, a diarylheptanoid compound, found in spice turmeric is known to alter the aggregation of proteins and reduce the toxicity of the aggregates. This review looks at the molecular basis of modulating protein aggregation and toxicity of the aggregates. Foremost, we identify the interaction of curcumin and its derivatives with proteins/peptides and the effect of their interaction on the conformational stability and unfolding/folding pathway(s). The unfolding/folding processes generate partially folded/unfolded intermediate, which serve as aggregation precursor state. Secondly, we discuss the effect of curcumin binding on the kinetics parameters of the aggregation process, which give information about the mechanism of the aggregation inhibition. We describe, in addition, that curcumin can accelerate/promote fibril formation by binding to oligomeric intermediate(s) accumulated in the aggregation pathway. Finally, we discuss the correlation of curcumin-induced monomeric and/or oligomeric precursor states with aggregate structure and toxicity. On the basis of these discussions, we propose a model describing curcumin-induced inhibition/promotion of formation of amyloid-like fibrils. Copyright © 2016 Elsevier B.V. All rights reserved.
Romero-Fernandez, Wilber; Borroto-Escuela, Dasiel O; Alea, Mileidys Perez; Garcia-Mesa, Yoelvis; Garriga, Pere
2011-12-01
The human M(3) muscarinic acetylcholine receptor is present in both the central and peripheral nervous system, and it is involved in the pathophysiology of several neurodegenerative and autoimmune diseases. We suggested a possible N-glycosylation map for the M(3) muscarinic receptor expressed in COS-7 cells. Here, we examined the role that N-linked glycans play in the folding and in the cell surface trafficking of this receptor. The five potential asparagine-linked glycosylation sites in the muscarinic receptor were mutated and transiently expressed in COS-7 cells. The elimination of N-glycan attachment sites did not affect the cellular expression levels of the receptor. However, proper receptor localization to the plasma membrane was affected as suggested by reduced [(3)H]-N-methylscopolamine binding. Confocal microscopy confirmed this observation and showed that the nonglycosylated receptor was primarily localized in the intracellular compartments. The mutant variant showed an increase in phosphorylation of the α-subunit of eukaryote initiation factor 2, and other well-known endoplasmic reticulum stress markers of the unfolded protein response pathway, which further supports the proposal of the improper intracellular accumulation of the nonglycosylated receptor. The receptor devoid of glycans showed more susceptibility to events that culminate in apoptosis reducing cell viability. Our findings suggest up-regulation of pro-apoptotic Bax protein, down-regulation of anti-apoptotic Bcl-2, and cleavage of caspase-3 effectors. Collectively, our data provide experimental evidence of the critical role that N-glycan chains play in determining muscarinic receptor distribution, localization, as well as cell integrity. © The Author 2011. Published by Oxford University Press. All rights reserved.
Communication: Role of explicit water models in the helix folding/unfolding processes
Palazzesi, Ferruccio; Salvalaglio, Matteo; Barducci, Alessandro; Parrinello, Michele
2016-09-01
In the last years, it has become evident that computer simulations can assume a relevant role in modelling protein dynamical motions for their ability to provide a full atomistic image of the processes under investigation. The ability of the current protein force-fields in reproducing the correct thermodynamics and kinetics systems behaviour is thus an essential ingredient to improve our understanding of many relevant biological functionalities. In this work, employing the last developments of the metadynamics framework, we compare the ability of state-of-the-art all-atom empirical functions and water models to consistently reproduce the folding and unfolding of a helix turn motif in a model peptide. This theoretical study puts in evidence that the choice of the water models can influence the thermodynamic and the kinetics of the system under investigation, and for this reason cannot be considered trivial.
Unfolded equations for massive higher spin supermultiplets in AdS{sub 3}
Energy Technology Data Exchange (ETDEWEB)
Buchbinder, I.L. [Department of Theoretical Physics, Tomsk State Pedagogical University,60 Kievskaya Str., Tomsk, 634061 (Russian Federation); National Research Tomsk State University,36 Lenina Ave., Tomsk, 634050 (Russian Federation); Snegirev, T.V. [Department of Theoretical Physics, Tomsk State Pedagogical University,60 Kievskaya Str., Tomsk, 634061 (Russian Federation); Department of Higher Mathematics and Mathematical Physics,National Research Tomsk Polytechnic University, 30 Lenina Ave., Tomsk, 634050 (Russian Federation); Zinoviev, Yu.M. [Department of Theoretical Physics,Institute for High Energy Physics of National Research Center “Kurchatov Institute”, 1 Pobedy Str., Protvino, Moscow Region, 142280 (Russian Federation)
2016-08-10
In this paper we give an explicit construction of unfolded equations for massive higher spin supermultiplets of the minimal (1,0) supersymmetry in AdS{sub 3} space. For that purpose we use an unfolded formulation for massive bosonic and fermionic higher spins and find supertransformations leaving appropriate set of unfolded equations invariant. We provide two general supermultiplets (s,s+1/2) and (s,s−1/2) with arbitrary integer s, as well as a number of lower spin examples.
On unfolding counting-rate spectra of recoil-proton neutron detectors
International Nuclear Information System (INIS)
Yeivin, Yehuda
1983-01-01
This note proposes a possible scheme for unfolding recoil-proton neutron detector data, in which at first the undistorted proton source spectrum is derived. The main argument in favour of this scheme is that, compared with the conventional scheme, it necessitates somewhat weaker assumptions with respect to the unknown spectrum above the detector's upper energy cutoff, and would therefore be more reliable. We also demonstrate a simple, elementary proof of the wall effect correction for spherical detectors, and, in order to gain insight of the potential merits of the proposed unfolding scheme, illustrate our main argument by considering a hypothetic linear range-energy relation, in which case complete unfolding becomes possible with no assumptions at all on the proton spectrum above the cutoff energy. (author)
A neutron spectrum unfolding computer code based on artificial neural networks
Ortiz-Rodríguez, J. M.; Reyes Alfaro, A.; Reyes Haro, A.; Cervantes Viramontes, J. M.; Vega-Carrillo, H. R.
2014-02-01
The Bonner Spheres Spectrometer consists of a thermal neutron sensor placed at the center of a number of moderating polyethylene spheres of different diameters. From the measured readings, information can be derived about the spectrum of the neutron field where measurements were made. Disadvantages of the Bonner system are the weight associated with each sphere and the need to sequentially irradiate the spheres, requiring long exposure periods. Provided a well-established response matrix and adequate irradiation conditions, the most delicate part of neutron spectrometry, is the unfolding process. The derivation of the spectral information is not simple because the unknown is not given directly as a result of the measurements. The drawbacks associated with traditional unfolding procedures have motivated the need of complementary approaches. Novel methods based on Artificial Intelligence, mainly Artificial Neural Networks, have been widely investigated. In this work, a neutron spectrum unfolding code based on neural nets technology is presented. This code is called Neutron Spectrometry and Dosimetry with Artificial Neural networks unfolding code that was designed in a graphical interface. The core of the code is an embedded neural network architecture previously optimized using the robust design of artificial neural networks methodology. The main features of the code are: easy to use, friendly and intuitive to the user. This code was designed for a Bonner Sphere System based on a 6LiI(Eu) neutron detector and a response matrix expressed in 60 energy bins taken from an International Atomic Energy Agency compilation. The main feature of the code is that as entrance data, for unfolding the neutron spectrum, only seven rate counts measured with seven Bonner spheres are required; simultaneously the code calculates 15 dosimetric quantities as well as the total flux for radiation protection purposes. This code generates a full report with all information of the unfolding in
The construction of periodic unfolding operators on some compact Riemannian manifolds
DEFF Research Database (Denmark)
Dobberschütz, Sören; Böhm, Michael
2014-01-01
The notion of periodic unfolding has become a standard tool in the theory of periodic homogenization. However, all the results obtained so far are only applicable to the "flat" Euclidean space R n. In this paper, we present a generalization of the method of periodic unfolding applicable to struct...
NSDUAZ unfolding package for neutron spectrometry and dosimetry with Bonner spheres
Energy Technology Data Exchange (ETDEWEB)
Vega C, H. R.; Martinez B, M. R. [Universidad Autonoma de Zacatecas, Unidad Academica de Estudios Nucleares, Calle Cipres No. 10, Fracc. La Penuela, 98068 Zacatecas (Mexico); Ortiz R, J. M., E-mail: fermineutron@yahoo.com [Universidad Autonoma de Zacatecas, Unidad Academica de Ingenieria Electrica, Av. Ramon Lopez Velarde 801, Col. Centro, 98000 Zacatecas (Mexico)
2011-10-15
NSDUAZ (Neutron Spectrometry and Dosimetry for the Universidad Autonoma de Zacatecas) is a user friendly neutron unfolding package for Bonner sphere spectrometer with {sup 6}Lil(Eu) developed under Lab View environment. Unfolding is carried out using a recursive iterative procedure with the SPUNIT algorithm, where the starting spectrum is obtained from a library initial guess spectrum to start the iterations, the package include a statistical procedure based on the count rates relative to the count rate in the 8 inches-diameter sphere to select the initial spectrum. Neutron spectrum is unfolded in 32 energy groups ranging from 10{sup -8} up to 231.2 MeV. (Author)
Dynamics of a delayed intraguild predation model with harvesting
Collera, Juancho A.; Balilo, Aldrin T.
2018-03-01
In [1], a delayed three-species intraguild predation (IGP) model was considered. This particular tri-trophic community module includes a predator and its prey which share a common basal resource for their sustenance [3]. Here, it is assumed that in the absence of predation, the growth of the basal resource follows the delayed logistic equation. Without delay time, the IGP model in [1] reduces to the system considered in [7] where it was shown that IGP may induce chaos even if the functional responses are linear. Meanwhile, in [2] the delayed IGP model in [1] was generalized to include harvesting. Under the assumption that the basal resource has some economic value, a constant harvesting term on the basal resource was incorporated. However, both models in [1] and [2] use the delay time as the main parameter. In this research, we studied the delayed IGP model in [1] with the addition of linear harvesting term on each of the three species. The dynamical behavior of this system is examined using the harvesting rates as main parameter. In particular, we give conditions on the existence, stability, and bifurcations of equilibrium solutions of this system. This allows us to better understand the effects of harvesting in terms of the survival or extinction of one or more species in our system. Numerical simulations are carried out to illustrate our results. In fact, we show that the chaotic behavior in [7] unfolds when the harvesting rate parameter is varied.
Spectrum unfolding in X-ray spectrometry using the maximum entropy method
International Nuclear Information System (INIS)
Fernandez, Jorge E.; Scot, Viviana; Di Giulio, Eugenio
2014-01-01
The solution of the unfolding problem is an ever-present issue in X-ray spectrometry. The maximum entropy technique solves this problem by taking advantage of some known a priori physical information and by ensuring an outcome with only positive values. This method is implemented in MAXED (MAXimum Entropy Deconvolution), a software code contained in the package UMG (Unfolding with MAXED and GRAVEL) developed at PTB and distributed by NEA Data Bank. This package contains also the code GRAVEL (used to estimate the precision of the solution). This article introduces the new code UMESTRAT (Unfolding Maximum Entropy STRATegy) which applies a semi-automatic strategy to solve the unfolding problem by using a suitable combination of MAXED and GRAVEL for applications in X-ray spectrometry. Some examples of the use of UMESTRAT are shown, demonstrating its capability to remove detector artifacts from the measured spectrum consistently with the model used for the detector response function (DRF). - Highlights: ► A new strategy to solve the unfolding problem in X-ray spectrometry is presented. ► The presented strategy uses a suitable combination of the codes MAXED and GRAVEL. ► The applied strategy provides additional information on the Detector Response Function. ► The code UMESTRAT is developed to apply this new strategy in a semi-automatic mode
Genetic algorithms - A new technique for solving the neutron spectrum unfolding problem
International Nuclear Information System (INIS)
Freeman, David W.; Edwards, D. Ray; Bolon, Albert E.
1999-01-01
A new technique utilizing genetic algorithms has been applied to the Bonner sphere neutron spectrum unfolding problem. Genetic algorithms are part of a relatively new field of 'evolutionary' solution techniques that mimic living systems with computer-simulated 'chromosome' solutions. Solutions mate and mutate to create better solutions. Several benchmark problems, considered representative of radiation protection environments, have been evaluated using the newly developed UMRGA code which implements the genetic algorithm unfolding technique. The results are compared with results from other well-established unfolding codes. The genetic algorithm technique works remarkably well and produces solutions with relatively high spectral qualities. UMRGA appears to be a superior technique in the absence of a priori data - it does not rely on 'lucky' guesses of input spectra. Calculated personnel doses associated with the unfolded spectra match benchmark values within a few percent
Jethva, Prashant N; Udgaonkar, Jayant B
2017-09-07
Protein folding and unfolding reactions invariably appear to be highly cooperative reactions, but the structural and sequence determinants of cooperativity are poorly understood. Importantly, it is not known whether cooperative structural change occurs throughout the protein, or whether some parts change cooperatively and other parts change noncooperatively. In the current study, hydrogen exchange mass spectrometry has been used to show that the mechanism of unfolding of the PI3K SH3 domain is similar in the absence and presence of 5 M urea. The data are well described by a four state N ↔ I N ↔ I 2 ↔ U model, in which structural changes occur noncooperatively during the N ↔ I N and I N ↔ I 2 transitions, and occur cooperatively during the I 2 ↔ U transition. The nSrc-loop and RT-loop, as well as β strands 4 and 5 undergo noncooperative unfolding, while β strands 1, 2, and 3 unfold cooperatively in the absence of urea. However, in the presence of 5 M urea, the unfolding of β strand 4 switches to become cooperative, leading to an increase in the extent of cooperative structural change. The current study highlights the relationship between protein stability and cooperativity, by showing how the extent of cooperativity can be varied, using chemical denaturant to alter protein stability.
Sepulveda, Denisse; Rojas-Rivera, Diego; Rodríguez, Diego A; Groenendyk, Jody; Köhler, Andres; Lebeaupin, Cynthia; Ito, Shinya; Urra, Hery; Carreras-Sureda, Amado; Hazari, Younis; Vasseur-Cognet, Mireille; Ali, Maruf M U; Chevet, Eric; Campos, Gisela; Godoy, Patricio; Vaisar, Tomas; Bailly-Maitre, Béatrice; Nagata, Kazuhiro; Michalak, Marek; Sierralta, Jimena; Hetz, Claudio
2018-01-18
Maintenance of endoplasmic reticulum (ER) proteostasis is controlled by a dynamic signaling network known as the unfolded protein response (UPR). IRE1α is a major UPR transducer, determining cell fate under ER stress. We used an interactome screening to unveil several regulators of the UPR, highlighting the ER chaperone Hsp47 as the major hit. Cellular and biochemical analysis indicated that Hsp47 instigates IRE1α signaling through a physical interaction. Hsp47 directly binds to the ER luminal domain of IRE1α with high affinity, displacing the negative regulator BiP from the complex to facilitate IRE1α oligomerization. The regulation of IRE1α signaling by Hsp47 is evolutionarily conserved as validated using fly and mouse models of ER stress. Hsp47 deficiency sensitized cells and animals to experimental ER stress, revealing the significance of Hsp47 to global proteostasis maintenance. We conclude that Hsp47 adjusts IRE1α signaling by fine-tuning the threshold to engage an adaptive UPR. Copyright © 2018 Elsevier Inc. All rights reserved.
Mechanism-based strategies for protein thermostabilization.
Mozhaev, V V
1993-03-01
Strategies for stabilizing enzymes can be derived from a two-step model of irreversible inactivation that involves preliminary reversible unfolding, followed by an irreversible step. Reversible unfolding is best prevented by covalent immobilization, whereas methods such as covalent modification of amino acid residues or 'medium engineering' (by the addition of low-molecular-weight compounds) are effective against irreversible 'incorrect' refolding. Genetic modification of the protein sequence is the most effective approach for preventing chemical deterioration.
Svensson, Malin; Fast, Jonas; Mossberg, Ann-Kristin; Düringer, Caroline; Gustafsson, Lotta; Hallgren, Oskar; Brooks, Charles L; Berliner, Lawrence; Linse, Sara; Svanborg, Catharina
2003-12-01
HAMLET (human alpha-lactalbumin made lethal to tumor cells) is a complex of human alpha-lactalbumin and oleic acid (C18:1:9 cis) that kills tumor cells by an apoptosis-like mechanism. Previous studies have shown that a conformational change is required to form HAMLET from alpha-lactalbumin, and that a partially unfolded conformation is maintained in the HAMLET complex. This study examined if unfolding of alpha-lactalbumin is sufficient to induce cell death. We used the bovine alpha-lactalbumin Ca(2+) site mutant D87A, which is unable to bind Ca(2+), and thus remains partially unfolded regardless of solvent conditions. The D87A mutant protein was found to be inactive in the apoptosis assay, but could readily be converted to a HAMLET-like complex in the presence of oleic acid. BAMLET (bovine alpha-lactalbumin made lethal to tumor cells) and D87A-BAMLET complexes were both able to kill tumor cells. This activity was independent of the Ca(2+)site, as HAMLET maintained a high affinity for Ca(2+) but D87A-BAMLET was active with no Ca(2+) bound. We conclude that partial unfolding of alpha-lactalbumin is necessary but not sufficient to trigger cell death, and that the activity of HAMLET is defined both by the protein and the lipid cofactor. Furthermore, a functional Ca(2+)-binding site is not required for conversion of alpha-lactalbumin to the active complex or to cause cell death. This suggests that the lipid cofactor stabilizes the altered fold without interfering with the Ca(2+)site.
Directory of Open Access Journals (Sweden)
Ravi N Vellanki
Full Text Available OASIS is a transcription factor similar to ATF6 that is activated by endoplasmic reticulum stress. In this study we investigated the expression of OASIS in human glioma cell lines and the effect of OASIS knock-down on the ER stress response and cell migration. OASIS mRNA was detected in three distinct glioma cell lines (U373, A172 and U87 and expression levels were increased upon treatment with ER stress-inducing compounds in the U373 and U87 lines. OASIS protein, which is glycosylated on Asn-513, was detected in the U373 and U87 glioma lines at low levels in control cells and protein expression was induced by ER stress. Knock-down of OASIS in human glioma cell lines resulted in an attenuated unfolded protein response to ER stress (reduced GRP78/BiP and GRP94 induction and decreased expression of chondroitin sulfate proteoglycan extracellular matrix proteins, but induction of the collagen gene Col1a1 was unaffected. Cells in which OASIS was knocked-down exhibited altered cell morphology and reduced cell migration. These results suggest that OASIS is important for the ER stress response and maintenance of some extracellular matrix proteins in human glioma cells.
Brown, Marishka K; Strus, Ewa; Naidoo, Nirinjini
2017-07-01
Social isolation has a multitude of negative consequences on human health including the ability to endure challenges to the immune system, sleep amount and efficiency, and general morbidity and mortality. These adverse health outcomes are conserved in other social species. In the fruit fly Drosophila melanogaster, social isolation leads to increased aggression, impaired memory, and reduced amounts of daytime sleep. There is a correlation between molecules affected by social isolation and those implicated in sleep in Drosophila. We previously demonstrated that acute sleep loss in flies and mice induced the unfolded protein response (UPR), an adaptive signaling pathway. One mechanism indicating UPR upregulation is elevated levels of the endoplasmic reticular chaperone BiP/GRP78. We previously showed that BiP overexpression in Drosophila led to increased sleep rebound. Increased rebound sleep has also been demonstrated in socially isolated (SI) flies. D. melanogaster were used to study the effect of social isolation on cellular stress. SI flies displayed an increase in UPR markers; there were higher BiP levels, increased phosphorylation of the translation initiation factor eIF2α, and increased splicing of xbp1. These are all indicators of UPR activation. In addition, the effects of isolation on the UPR were reversible; pharmacologically and genetically altering sleep in the flies modulated the UPR. The reduction in sleep observed in SI flies is a cellular stressor that results in UPR induction. © Sleep Research Society 2017. Published by Oxford University Press [on behalf of the Sleep Research Society]. All rights reserved. For permissions, please email: journals.permissions@oup.com
Proving the correctness of unfold/fold program transformations using bisimulation
DEFF Research Database (Denmark)
Hamilton, Geoff W.; Jones, Neil
2011-01-01
by a labelled transition system whose bisimilarity relation is a congruence that coincides with contextual equivalence. Labelled transition systems are well-suited to represent global program behaviour. On the other hand, unfold/fold program transformations use generalization and folding, and neither is easy......This paper shows that a bisimulation approach can be used to prove the correctness of unfold/fold program transformation algorithms. As an illustration, we show how our approach can be use to prove the correctness of positive supercompilation (due to Sørensen et al). Traditional program equivalence...... to describe contextually, due to use of non-local information. We show that weak bisimulation on labelled transition systems gives an elegant framework to prove contextual equivalence of original and transformed programs. One reason is that folds can be seen in the context of corresponding unfolds....
DEFF Research Database (Denmark)
Lerche, A; Bisgaard, H; Christensen, J D
1988-01-01
allergic patients with nickel challenge in the chamber medium showed a time-dependent increase of mononuclear cells, eosinophils and basophils and a concomitant decrease of polymorphonuclear granulocytes, characteristic of a combined specific and unspecific inflammation. The morphology of the exudate...... in contact allergic patients exposed to nickel showed a dominance of polymorphonuclear granulocytes throughout the study period, while mononuclear cells, eosinophils and basophils were detected at a much lower quantity and with a considerable delay. Further, we studied the kinetics of the leucocyte granule...... proteins: lactoferrin, myeloperoxidase, lysozyme and eosinophil cationic protein in exudate fluid in a parallel test. A significant higher flux was found for all during the second day of allergen exposure compared to contact allergic patients without allergen challenge as well as normal volunteers...
Tamura, Yuki; Matsunaga, Yutaka; Kitaoka, Yu; Hatta, Hideo
2017-03-01
Mitochondrial and endoplasmic reticulum (ER) stress, and subsequently activated responses (mitochondrial/ER unfolded protein responses; UPRmt/UPRER), are involved in the pathogenesis of sarcopenia. To extend both basic and translational knowledge, we examined (i) whether age-induced mitochondrial and ER stress depend on skeletal muscle type in mice and (ii) whether heat stress treatment, a suggested strategy for sarcopenia, improves age-induced mitochondrial and ER stress. Aged (21-month-old) mice showed more severe mitochondrial stress and UPRmt than young (12-week-old) mice, based on increased oxidative stress, mitochondrial proteases, and mitochondrial E3 ubiquitin ligase. The aged mice also showed ER stress and UPRER, based on decreased ER enzymes and increased ER stress-related cell death. These changes were much more evident in soleus muscle than in gastrocnemius and plantaris muscles. After daily heat stress treatment (40 °C chamber for 30 minutes per day) for 4 weeks, mice showed remarkable improvements in age-related changes in soleus muscle. Heat stress had only minor effects in gastrocnemius and plantaris muscles. Based on these findings, age-associated mitochondrial stress, ER stress, and UPRmt/ER vary qualitatively with skeletal muscle type. Our results suggest a molecular basis for the beneficial effects of heat stress on muscle atrophy with age in soleus muscle. © The Author 2016. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.
Cysteine residue is not essential for CPM protein thermal-stability assay.
Wang, Zhaoshuai; Ye, Cui; Zhang, Xinyi; Wei, Yinan
2015-05-01
A popular thermal-stability assay developed especially for the study of membrane proteins uses a thiol-specific probe, 7-diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin (CPM). The fluorescence emission of CPM surges when it forms a covalent bond with the side chain of a free Cys, which becomes more readily accessible upon protein thermal denaturation. Interestingly, the melting temperatures of membrane proteins determined using the CPM assay in literature are closely clustered in the temperature range 45-55 °C. A thorough understanding of the mechanism behind the observed signal change is critical for the accurate interpretation of the protein unfolding. Here we used two α-helical membrane proteins, AqpZ and AcrB, as model systems to investigate the nature of the fluorescence surge in the CPM assay. We found that the transition temperatures measured using circular-dichroism (CD) spectroscopy and the CPM assay were significantly different. To eliminate potential artifact that might arise from the presence of detergent, we monitored the unfolding of two soluble proteins. We found that, contrary to current understanding, the presence of a sulfhydryl group was not a prerequisite for the CPM thermal-stability assay. The observed fluorescence increase is probably caused by binding of the fluorophore to hydrophobic patches exposed upon protein unfolding.
Non-leftmost Unfolding in Partial Evaluation of Logic Programs with Impure Predicates
DEFF Research Database (Denmark)
Albert, Elvira; Puebla, German; Gallagher, John Patrick
2006-01-01
-leftmost unfolding steps can result in incorrect results since the independence of the computation rule no longer holds in the presence of impure predicates. Existing proposals allow non-leftmost unfolding steps, but at the cost of accuracy: bindings and failure are not propagated backwards to predicates which...
Vincent, Laure-Anais; Attaoua, Chaker; Bellis, Michel; Rozkydalova, Lucie; Hadj-Kaddour, Kamel; Vian, Laurence; Cuq, Pierre
2015-04-01
On account of its strong ability to become chemoresistant after a primary response to drugs, malignant melanoma (MM) remains a therapeutic challenge. This study focuses on acquired resistance to vinca alkaloids (VAs) using VA-resistant MM cell lines (CAL1R-VCR, CAL1R-VDS, and CAL1R-VRB), established by long-term continuous exposure of parental CAL1-wt cells to vincristine (VCR), vindesine (VDS), or vinorelbine (VRB), respectively. Transcriptomic profiling using rma and rdam methods led to distinguish two cell groups: CAL1R-VCR and CAL1R-VDS, CAL1R-VRB, and CAL1-wt. mgsa of the specifically altered genes in the first group evidenced the GO terms 'lysosomal lumen' and 'vacuolar lumen' linked to underexpressed genes, and 'endoplasmic reticulum (ER) stress response' associated with overexpressed genes. A specific reduction of lysosomal enzymes, independent of acidic vacuole organelle (AVO) turnover, was observed (LTG probe) in CAL1R-VCR and CAL1R-VDS cells. It was associated with the specific lowering of cathepsin B and L, known to be involved in the lysosomal pathway of apoptosis. Confirming gene profiling, the same groups (CAL1R-VCR and CAL1R-VDS, CAL1-wt and CAL1R-VRB) could be distinguished regarding the VA-mediated changes on mean size areas and on acidic compartment volumes. These two parameters were reduced in CAL1R-VCR and CAL1R-VDS cells, suggesting a smaller AVO accumulation and thus a reduced sensitivity to lysosomal membrane permeabilization-mediated apoptosis. In addition, 'ER stress response' inhibition by tauroursodeoxycholic acid induced a higher VA sensitization of the first cell group. In conclusion, lysosomes and unfolded protein response could be key determinants of the differential resistance of MM to VAs. © 2015 Société Française de Pharmacologie et de Thérapeutique.
A novel neutron energy spectrum unfolding code using particle swarm optimization
International Nuclear Information System (INIS)
Shahabinejad, H.; Sohrabpour, M.
2017-01-01
A novel neutron Spectrum Deconvolution using Particle Swarm Optimization (SDPSO) code has been developed to unfold the neutron spectrum from a pulse height distribution and a response matrix. The Particle Swarm Optimization (PSO) imitates the bird flocks social behavior to solve complex optimization problems. The results of the SDPSO code have been compared with those of the standard spectra and recently published Two-steps Genetic Algorithm Spectrum Unfolding (TGASU) code. The TGASU code have been previously compared with the other codes such as MAXED, GRAVEL, FERDOR and GAMCD and shown to be more accurate than the previous codes. The results of the SDPSO code have been demonstrated to match well with those of the TGASU code for both under determined and over-determined problems. In addition the SDPSO has been shown to be nearly two times faster than the TGASU code. - Highlights: • Introducing a novel method for neutron spectrum unfolding. • Implementation of a particle swarm optimization code for neutron unfolding. • Comparing results of the PSO code with those of recently published TGASU code. • Match results of the PSO code with those of TGASU code. • Greater convergence rate of implemented PSO code than TGASU code.
Unfolding Semantics of the Untyped λ-Calculus with lectrec-Calculus with letrec
Rochel, J.
2016-01-01
We investigate the relationship between finite terms in lambda-letrec, the lambda calculus with letrec, and the infinite lambda terms they express. We say that a lambda-letrec term expresses a lambda term if the latter can be obtained as an infinite unfolding of the former. Unfolding is the process
Bosonic Fradkin-Tseytlin equations unfolded
Energy Technology Data Exchange (ETDEWEB)
Shaynkman, O.V. [I.E.Tamm Theory Department, Lebedev Physical Institute,Leninski prospect 53, 119991, Moscow (Russian Federation)
2016-12-22
We test infinite-dimensional extension of algebra su(k,k) proposed by Fradkin and Linetsky as the candidate for conformal higher spin algebra. Adjoint and twisted-adjoint representations of su(k,k) on the space of this algebra are carefully explored. For k=2 corresponding unfolded system is analyzed and it is shown to encode Fradkin-Tseytlin equations for the set of all integer spins 1,2,… with infinite multiplicity.
Kumar, Vijay; Prakash, Amresh; Pandey, Preeti; Lynn, Andrew M; Hassan, Md Imtaiyaz
2018-05-18
Misfolding and aggregation of Cu, Zn Superoxide dismutase (SOD1) is involved in the neurodegenerative disease, amyotrophic lateral sclerosis. Many studies have shown that metal-depleted, monomeric form of SOD1 displays substantial local unfolding dynamics and is the precursor for aggregation. Here, we have studied the structure and dynamics of different apo monomeric SOD1 variants associated with unfolding and aggregation in aqueous trifluoroethanol (TFE) through experiments and simulation. TFE induces partially unfolded β-sheet-rich extended conformations in these SOD1 variants, which subsequently develops aggregates with fibril-like characteristics. Fibrillation was achieved more easily in disulfide-reduced monomeric SOD1 when compared with wild-type and mutant monomeric SOD1. At higher concentrations of TFE, a native-like structure with the increase in α-helical content was observed. The molecular dynamics simulation results illustrate distinct structural dynamics for different regions of SOD1 variants and show uniform local unfolding of β-strands. The strands protected by the zinc-binding and electrostatic loops were found to unfold first in 20% (v/v) TFE, leading to a partial unfolding of β-strands 4, 5, and 6 which are prone to aggregation. Our results thus shed light on the role of local unfolding and conformational dynamics in SOD1 misfolding and aggregation. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.
Egg Yolk Protein Delays Recovery while Ovalbumin Is Useful in Recovery from Iron Deficiency Anemia
Directory of Open Access Journals (Sweden)
Yukiko Kobayashi
2015-06-01
Full Text Available Protein is a main nutrient involved in overall iron metabolism in vivo. In order to assess the prevention of iron deficiency anemia (IDA by diet, it is necessary to confirm the influence of dietary protein, which coexists with iron, on iron bioavailability. We investigated the usefulness of the egg structural protein in recovery from IDA. Thirty-one female Sprague-Dawley rats were divided into a control group (n = 6 fed a casein diet (4.0 mg Fe/100 g for 42 days and an IDA model group (n = 25 created by feeding a low-iron casein diet (LI, 0.4 mg Fe/100 g for 21 days and these IDA rats were fed normal iron diet with different proteins from eggs for another 21 days. The IDA rats were further divided into four subgroups depending on the proteins fed during the last 21 days, which were those with an egg white diet (LI-W, 4.0 mg Fe/100 g, n = 6, those with an ovalbumin diet (LI-A, 4.0 mg Fe/100 g, n = 7, those with an egg yolk-supplemented diet (LI-Y, 4.0 mg Fe/100 g, n = 6, and the rest with a casein diet (LI-C, 4.0 mg Fe/100 g, n = 6. In the LI-Y group, recovery of the hematocrit, hemoglobin, transferrin saturation level and the hepatic iron content were delayed compared to the other groups (p < 0.01, 0.01, 0.01, and 0.05, respectively, resulting in no recovery from IDA at the end of the experimental period. There were no significant differences in blood parameters in the LI-W and LI-A groups compared to the control group. The hepatic iron content of the LI-W and LI-A groups was higher than that of the LI-C group (p < 0.05. We found that egg white protein was useful for recovery from IDA and one of the efficacious components was ovalbumin, while egg yolk protein delayed recovery of IDA. This study demonstrates, therefore, that bioavailability of dietary iron varies depending on the source of dietary protein.
Comparison of neutron spectrum unfolding codes
International Nuclear Information System (INIS)
Zijp, W.
1979-02-01
This final report contains a set of four ECN-reports. The first is dealing with the comparison of the neutron spectrum unfolding codes CRYSTAL BALL, RFSP-JUL, SAND II and STAY'SL. The other three present the results of calculations about the influence of statistical weights in CRYSTAL BALL, SAND II and RFSP-JUL
Probabilistic analysis for identifying the driving force of protein folding
Tokunaga, Yoshihiko; Yamamori, Yu; Matubayasi, Nobuyuki
2018-03-01
Toward identifying the driving force of protein folding, energetics was analyzed in water for Trp-cage (20 residues), protein G (56 residues), and ubiquitin (76 residues) at their native (folded) and heat-denatured (unfolded) states. All-atom molecular dynamics simulation was conducted, and the hydration effect was quantified by the solvation free energy. The free-energy calculation was done by employing the solution theory in the energy representation, and it was seen that the sum of the protein intramolecular (structural) energy and the solvation free energy is more favorable for a folded structure than for an unfolded one generated by heat. Probabilistic arguments were then developed to determine which of the electrostatic, van der Waals, and excluded-volume components of the interactions in the protein-water system governs the relative stabilities between the folded and unfolded structures. It was found that the electrostatic interaction does not correspond to the preference order of the two structures. The van der Waals and excluded-volume components were shown, on the other hand, to provide the right order of preference at probabilities of almost unity, and it is argued that a useful modeling of protein folding is possible on the basis of the excluded-volume effect.
Energy Technology Data Exchange (ETDEWEB)
Harami, Taikan, E-mail: harami.taikan@jaea.go.jp [Japan Atomic Energy Agency (Japan); Kitao, Shinji; Kobayashi, Yasuhiro [Kyoto University, Research Reactor Institute (Japan); Mitsui, Takaya [Japan Atomic Energy Agency (Japan)
2008-01-15
The unfolded heme structure in myoglobin is controversial because of no chance of direct X-ray structure analyses. The unfolding of carbonmonoxy myoglobin (MbCO) by guanidine hydrochloride (GdnHCl) was studied by the Moessbauer spectroscopy. The spectra show the presence of a sort of spectrum in the unfolded MbCO, independent on the concentration of GdnHCl from 1 to 6 M and the increase of the fraction of unfolded MbCO, depending on the GdnHCl concentration. The isomer shift of the iron of heme in the unfolded MbCO was identified to be different from that of the native MbCO as the globin structure in Mb collapses under the unfolded conditions. This result and the existing related Moessbauer data proved that the heme in the unfolded MbCO may remain coordinated to the proximal histidine.
BONDI-97 A novel neutron energy spectrum unfolding tool using a genetic algorithm
Mukherjee, B
1999-01-01
The neutron spectrum unfolding procedure using the count rate data obtained from a set of Bonner sphere neutron detectors requires the solution of the Fredholm integral equation of the first kind by using complex mathematical methods. This paper reports a new approach for the unfolding of neutron spectra using the Genetic Algorithm tool BONDI-97 (BOnner sphere Neutron DIfferentiation). The BONDI-97 was used as the input for Genetic Algorithm engine EVOLVER to search for a globally optimised solution vector from a population of randomly generated solutions. This solution vector corresponds to the unfolded neutron energy spectrum. The Genetic Algorithm engine emulates the Darwinian 'Survival of the Fittest' strategy, the key ingredient of the 'Theory of Evolution'. The spectra of sup 2 sup 4 sup 1 Am/Be (alpha,n) and sup 2 sup 3 sup 9 Pu/Be (alpha,n) neutron sources were unfolded using the BONDI-97 tool. (author)
Delay-independent stability of genetic regulatory networks.
Wu, Fang-Xiang
2011-11-01
Genetic regulatory networks can be described by nonlinear differential equations with time delays. In this paper, we study both locally and globally delay-independent stability of genetic regulatory networks, taking messenger ribonucleic acid alternative splicing into consideration. Based on nonnegative matrix theory, we first develop necessary and sufficient conditions for locally delay-independent stability of genetic regulatory networks with multiple time delays. Compared to the previous results, these conditions are easy to verify. Then we develop sufficient conditions for global delay-independent stability for genetic regulatory networks. Compared to the previous results, this sufficient condition is less conservative. To illustrate theorems developed in this paper, we analyze delay-independent stability of two genetic regulatory networks: a real-life repressilatory network with three genes and three proteins, and a synthetic gene regulatory network with five genes and seven proteins. The simulation results show that the theorems developed in this paper can effectively determine the delay-independent stability of genetic regulatory networks.
International Nuclear Information System (INIS)
Mukhopadhyay, Somshuvra; Shah, Mehul; Patel, Kirit; Sehgal, Pravin B.
2006-01-01
The pyrrolizidine alkaloid monocrotaline (MCT) initiates pulmonary hypertension by inducing a 'megalocytosis' phenotype in target pulmonary arterial endothelial, smooth muscle and Type II alveolar epithelial cells. In cultured endothelial cells, a single exposure to the pyrrolic derivative of monocrotaline (MCTP) results in large cells with enlarged endoplasmic reticulum (ER) and Golgi and increased vacuoles. However, these cells fail to enter mitosis. Largely based upon data from endothelial cells, we proposed earlier that a disruption of the trafficking and mitosis-sensor functions of the Golgi (the 'Golgi blockade' hypothesis) may represent the subcellular mechanism leading to MCTP-induced megalocytosis. In the present study, we investigated the applicability of the Golgi blockade hypothesis to epithelial cells. MCTP induced marked megalocytosis in cultures of lung A549 and breast MCF-7 cells. This was associated with a change in the distribution of the cis-Golgi scaffolding protein GM130 from a discrete juxtanuclear localization to a circumnuclear distribution consistent with an anterograde block of GM130 trafficking to/through the Golgi. There was also a loss of plasma membrane caveolin-1 and E-cadherin, cortical actin together with a circumnuclear accumulation of clathrin heavy chain (CHC) and α-tubulin. Flotation analyses revealed losses/alterations in the association of caveolin-1, E-cadherin and CHC with raft microdomains. Moreover, megalocytosis was accompanied by an enhanced unfolded protein response (UPR) as evidenced by nuclear translocation of Ire1α and glucose regulated protein 58 (GRP58/ER-60/ERp57) and a circumnuclear accumulation of PERK kinase and protein disulfide isomerase (PDI). These data further support the hypothesis that an MCTP-induced Golgi blockade and enhanced UPR may represent the subcellular mechanism leading to enlargement of ER and Golgi and subsequent megalocytosis
International Nuclear Information System (INIS)
Zhu, Qingjun; Song, Fengquan; Ren, Jie; Chen, Xueyong; Zhou, Bin
2014-01-01
To further expand the application of an artificial neural network in the field of neutron spectrometry, the criteria for choosing between an artificial neural network and the maximum entropy method for the purpose of unfolding neutron spectra was presented. The counts of the Bonner spheres for IAEA neutron spectra were used as a database, and the artificial neural network and the maximum entropy method were used to unfold neutron spectra; the mean squares of the spectra were defined as the differences between the desired and unfolded spectra. After the information entropy of each spectrum was calculated using information entropy theory, the relationship between the mean squares of the spectra and the information entropy was acquired. Useful information from the information entropy guided the selection of unfolding methods. Due to the importance of the information entropy, the method for predicting the information entropy using the Bonner spheres' counts was established. The criteria based on the information entropy theory can be used to choose between the artificial neural network and the maximum entropy method unfolding methods. The application of an artificial neural network to unfold neutron spectra was expanded. - Highlights: • Two neutron spectra unfolding methods, ANN and MEM, were compared. • The spectrum's entropy offers useful information for selecting unfolding methods. • For the spectrum with low entropy, the ANN was generally better than MEM. • The spectrum's entropy was predicted based on the Bonner spheres' counts
FERD and FERDOR type unfolding codes
International Nuclear Information System (INIS)
Burrus, W.R.
1976-01-01
FERD and FERDO are unfolding codes which were developed at the Neutron Physics Division of Oak Ridge National Laboratory in 1965 and 1966. FERDO variants such as FERDOR and FORIST have been widely used, and many useful supplementary procedures have been developed for neutron and gamma-ray spectroscopy and other diverse applications. Algorithms for the codes are discussed
DEFF Research Database (Denmark)
Sehgal, Pankaj; Szalai, Paula; Olesen, Claus
2017-01-01
Calcium (Ca2+) is a fundamental regulator of cell signaling and function. Thapsigargin (Tg) blocks the sarco/endoplasmic reticulum (ER) Ca2+-ATPase (SERCA), disrupts Ca2+ homeostasis, and causes cell death. However, the exact mechanisms whereby SERCA-inhibition induces cell death are incompletely...... extensive drainage of the ER Ca2+ stores. This Ca2+ depletion was followed by markedly reduced cell proliferation rates and morphological changes that developed over 2–4 days and culminated in cell death. Interestingly, these changes were not accompanied by bulk increases in cytosolic Ca2+ levels. Moreover...... and their detrimental effects on cell viability. Furthermore, caspase activation and cell death were associated with a sustained unfolded protein response (UPR). We conclude that ER Ca2+ drainage and sustained UPR activation are key for initiation of apoptosis at low concentrations of Tg and Tg analogs, whereas high...
Shortening a loop can increase protein native state entropy.
Gavrilov, Yulian; Dagan, Shlomi; Levy, Yaakov
2015-12-01
Protein loops are essential structural elements that influence not only function but also protein stability and folding rates. It was recently reported that shortening a loop in the AcP protein may increase its native state conformational entropy. This effect on the entropy of the folded state can be much larger than the lower entropic penalty of ordering a shorter loop upon folding, and can therefore result in a more pronounced stabilization than predicted by polymer model for loop closure entropy. In this study, which aims at generalizing the effect of loop length shortening on native state dynamics, we use all-atom molecular dynamics simulations to study how gradual shortening a very long or solvent-exposed loop region in four different proteins can affect their stability. For two proteins, AcP and Ubc7, we show an increase in native state entropy in addition to the known effect of the loop length on the unfolded state entropy. However, for two permutants of SH3 domain, shortening a loop results only with the expected change in the entropy of the unfolded state, which nicely reproduces the observed experimental stabilization. Here, we show that an increase in the native state entropy following loop shortening is not unique to the AcP protein, yet nor is it a general rule that applies to all proteins following the truncation of any loop. This modification of the loop length on the folded state and on the unfolded state may result with a greater effect on protein stability. © 2015 Wiley Periodicals, Inc.
Paschek, Dietmar; Nymeyer, Hugh; García, Angel E
2007-03-01
We simulate the folding/unfolding equilibrium of the 20-residue miniprotein Trp-cage. We use replica exchange molecular dynamics simulations of the AMBER94 atomic detail model of the protein explicitly solvated by water, starting from a completely unfolded configuration. We employ a total of 40 replicas, covering the temperature range between 280 and 538 K. Individual simulation lengths of 100 ns sum up to a total simulation time of about 4 micros. Without any bias, we observe the folding of the protein into the native state with an unfolding-transition temperature of about 440 K. The native state is characterized by a distribution of root mean square distances (RMSD) from the NMR data that peaks at 1.8A, and is as low as 0.4A. We show that equilibration times of about 40 ns are required to yield convergence. A folded configuration in the entire extended ensemble is found to have a lifetime of about 31 ns. In a clamp-like motion, the Trp-cage opens up during thermal denaturation. In line with fluorescence quenching experiments, the Trp-residue sidechain gets hydrated when the protein opens up, roughly doubling the number of water molecules in the first solvation shell. We find the helical propensity of the helical domain of Trp-cage rather well preserved even at very high temperatures. In the folded state, we can identify states with one and two buried internal water molecules interconnecting parts of the Trp-cage molecule by hydrogen bonds. The loss of hydrogen bonds of these buried water molecules in the folded state with increasing temperature is likely to destabilize the folded state at elevated temperatures.
Energy Technology Data Exchange (ETDEWEB)
Marinkovic, P [Elektrotehnicki fakultet, Belgrade (Yugoslavia)
1988-07-01
Two unfolding procedures have been developed for obtaining fast neutron spectrum from proton-recoil spectrum assigned for spectrometry with organic scintillators. First is the method of differentiation of proton-recoil spectrum, and the second is the method based on solution of integral equation of Fredholm of first kind. (author)
Unfolding the phenomenon of inter-rater agreement
DEFF Research Database (Denmark)
Slaug, Bjørn; Schilling, Oliver; Helle, Tina
2011-01-01
Objective: The overall objective was to unfold the phenomenon of inter-rater agreement: to identify potential sources of variation in agreement data and to explore how they can be statistically accounted for. The ultimate aim was to propose recommendations for in-depth examination of agreement, i...
DEFF Research Database (Denmark)
Neess, Ditte; Bloksgaard, Maria; Sørensen, Signe Bek
2011-01-01
The acyl-CoA binding protein/diazepam binding inhibitor (ACBP/DBI) is an intracellular protein that binds C14-C22 acyl-CoA esters and is thought to act as an acyl-CoA transporter. In vitro analyses have indicated that ACBP can transport acyl-CoA esters between different enzymatic systems; however....... The delayed induction of SREBP target genes around weaning is caused by a compromised processing and decreased expression of SREBP precursors leading to reduced binding of SREBP to target sites in chromatin. In conclusion, lack of ACBP interferes with the normal metabolic adaptation to weaning and leads...
Sequence-dependent unfolding kinetics of DNA hairpins studied by nanopore force spectroscopy
International Nuclear Information System (INIS)
Renner, Stephan; Bessonov, Andrey; Simmel, Friedrich C; Gerland, Ulrich
2010-01-01
Nanopore force spectroscopy is used to study the unzipping kinetics of two DNA hairpin molecules with a 12 base pair long stem containing two contiguous stretches of six GC and six AT base pairs in interchanged order. Even though the thermodynamic stabilities of the two structures are nearly the same, they differ greatly in their unzipping kinetics. When the GC segment has to be broken before the AT segment, the unfolding rate is orders of magnitude smaller than in the opposite case. We also investigated hairpins with stem regions consisting only of AT or GC base pairs. The pure AT hairpins translocate much faster than the other hairpins, whereas the pure GC hairpins translocate on similar timescales to the hairpins with only an initial GC segment. For each hairpin, nanopore force spectroscopy is performed for different loading rates and the resulting unzipping distributions are mathematically transformed to a master curve that yields the unfolding rate as a function of applied voltage. This is compared with a stochastic model of the unfolding process for the two sequences for different voltages. The results can be rationalized in terms of the different natures of the free energy landscapes for the unfolding process.
Neutron spectra unfolding in Bonner spheres spectrometry using neural networks
International Nuclear Information System (INIS)
Kardan, M.R.; Setayeshi, S.; Koohi-Fayegh, R.; Ghiassi-Nejad, M.
2003-01-01
The neural network method has been used for the unfolding of neutron spectra in neutron spectrometry by Bonner spheres. A back propagation algorithm was used for training of neural networks 4mm x 4 mm bare LiI(Eu) and in a polyethylene sphere set: 2, 3, 4, 5, 6, 7, 8, 10, 12, 18 inch diameter have been used for unfolding of neutron spectra. Neural networks were trained by 199 sets of neutron spectra, which were subdivided into 6, 8, 10, 12, 15 and 20 energy bins and for each of them an appropriate neural network was designed and trained. The validation was performed by the 21 sets of neutron spectra. A neural network with 10 energy bins which had a mean value of error of 6% for dose equivalent estimation of spectra in the validation set showed the best results. The obtained results show that neural networks can be applied as an effective method for unfolding neutron spectra especially when the main target is neutron dosimetry. (author)
Kouvatsos, Nikolaos; Meldrum, Jill K; Searle, Mark S; Thomas, Neil R
2006-11-28
We have engineered a variant of the beta-clam shell protein ILBP which lacks the alpha-helical motif that caps the central binding cavity; the mutant protein is sufficiently destabilised that it is unfolded under physiological conditions, however, it unexpectedly binds its natural bile acid substrates with high affinity forming a native-like beta-sheet rich structure and demonstrating strong thermodynamic coupling between ligand binding and protein folding.
Lazar, Catalin; Uta, Mihaela; Petrescu, Stefana Maria; Branza-Nichita, Norica
2017-02-01
Cells replicating the human hepatitis B virus (HBV) express high levels of degradation-enhancing α-mannosidase-like proteins (EDEMs), a family of proteins involved in the endoplasmic reticulum associated degradation, one of the pathways activated during the unfolded protein response. Owing to their α-1,2 mannosidase activity, the EDEM1-3 proteins are able to process the N-linked glycans of misfolded or incompletely folded proteins, providing the recognition signal for their subsequent degradation. The HBV small (S), medium (M), and large (L) surface proteins bear an N-linked glycosylation site in the common S domain that is partially occupied in all proteins. The M protein contains an additional site in its preS2 domain, which is always functional. Here, we report that these oligosaccharides are processed by EDEMs, more efficiently by EDEM3, which induces degradation of L and S proteins, accompanied by a reduction of subviral particles production. In striking contrast, M not only is spared from degradation but its trafficking is also accelerated leading to an improved secretion. This unusual behavior of the M protein requires strictly the mannose trimming of the preS2 N-linked glycan. Furthermore, we show that HBV secretion is significantly inhibited under strong endoplasmic reticulum stress conditions when M expression is prevented by mutagenesis of the viral genome. These observations unfold unique properties of the M protein in the HBV life cycle during unfolded protein response and point to alternative mechanisms employed by EDEMs to alleviate this stress in case of necessity by promoting glycoprotein trafficking rather than degradation. © 2016 John Wiley & Sons Ltd.
Degradation of extracytoplasmic catalysts for protein folding in Bacillus subtilis
Krishnappa, Laxmi; Monteferrante, Carmine G; Neef, Jolanda; Dreisbach, Annette; van Dijl, Jan Maarten
The general protein secretion pathway of Bacillus subtilis has a high capacity for protein export from the cytoplasm, which is exploited in the biotechnological production of a wide range of enzymes. These exported proteins pass the membrane in an unfolded state, and accordingly, they have to fold
Inverse Problems for Nonlinear Delay Systems
2011-03-15
population dynamics. We consider the delay between birth and adulthood for neonate pea aphids and present a mathematical model that treats this delay as...which there is currently no known cure. For HIV, the core of the virus is composed of single-stranded viral RNA and protein components. As depicted in...at a CD4 receptor site and the viral core is injected into the cell. Once inside, the protein components enable transcription and integration of the
Directory of Open Access Journals (Sweden)
Alejandro Montenegro-Montero
Full Text Available 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.
An unfolding method for high energy physics experiments
International Nuclear Information System (INIS)
Blobel, V.
2002-06-01
Finite detector resolution and limited acceptance require one to apply unfolding methods in high energy physics experiments. Information on the detector resolution is usually given by a set of Monte Carlo events. Based on the experience with a widely used unfolding program (RUN) a modified method has been developed. The first step of the method is a maximum likelihood fit of the Monte Carlo distributions to the measured distribution in one, two or three dimensions; the finite statistics of the Monte Carlo events is taken into account by the use of Barlow's method with a new method of solution. A clustering method is used before combining bins in sparsely populated areas. In the second step a regularization is applied to the solution, which introduces only a small bias. The regularization parameter is determined from the data after a diagonalization and rotation procedure. (orig.)
FERDO/FERD, Unfolding of Pulse-Height Spectrometer Spectra
International Nuclear Information System (INIS)
Rust, B.W.; Ingersoll, D.T.; Burrus, W.R.
1985-01-01
1 - Description of problem or function: FERDO and FERD are unfolding codes which can be used to correct observed pulse-height distributions for the non-ideal response of a pulse-height spectrometer or to solve poorly conditioned linear equations. 2 - Method of solution: It is assumed that the response of the spectrometer is given by Ax = b, where A is the spectrometer response function matrix, x is the unknown spectrum, and b is the pulse-height distribution. FERDO does not resolve directly for x but instead solves for p = Wx, where W is a 'window function matrix'. Typically, W is the resolution function of an ideal spectrometer which has a single Gaussian response. The effective resolution of the unfolding solution may be varied by the choice of W. Confidence intervals are found for each element of the solution p
DEFF Research Database (Denmark)
Francis, C. J.; Lindorff-Larsen, Kresten; Best, R. B.
2006-01-01
dynamics simulations to characterise the residual structure of the 131 fragment of staphylococcal nuclease under physiological conditions. Our findings indicate that 131 under these conditions shows a tendency to form transiently hydrophobic clusters similar to those present in the native state of wild......The determination of the conformational preferences in unfolded states of proteins constitutes an important challenge in structural biology. We use inter-residue distances estimated from site-directed spin-labeling NMR experimental measurements as ensemble-averaged restraints in all-atom molecular...
Unfolding, aggregation, and seeded amyloid formation of lysine-58-cleaved beta(2)-microglobulin
DEFF Research Database (Denmark)
Heegaard, N.H.H.; Jørgensen, T.J.D.; Rozlosnik, N.
2005-01-01
. Using amide hydrogen/deuterium exchange monitored by mass spectrometry, we show that Delta K58-beta(2)m has increased unfolding rates compared to wt-beta(2)m and that unfolding is highly temperature dependent. The unfolding rate is I order of magnitude faster in Delta K58-beta(2)M than in wt-beta(2)m...... in the circulation of dialysis patients. This beta(2)M variant, Delta K58-beta(2)m, is a disulfide-linked two-chain molecule consisting of amino acid residues 1-57 and 59-99 of intact beta(2)m, and we here demonstrate and characterize its decreased conformational stability as compared to wild-type (wt) beta(2)M...
Directory of Open Access Journals (Sweden)
Atkinson Mark A
2011-02-01
Full Text Available Abstract Background Alpha-1 antitrypsin (AAT is a multi-functional protein that has anti-inflammatory and tissue protective properties. We previously reported that human AAT (hAAT gene therapy prevented autoimmune diabetes in non-obese diabetic (NOD mice and suppressed arthritis development in combination with doxycycline in mice. In the present study we investigated the feasibility of hAAT monotherapy for the treatment of chronic arthritis in collagen-induced arthritis (CIA, a mouse model of rheumatoid arthritis (RA. Methods DBA/1 mice were immunized with bovine type II collagen (bCII to induce arthritis. These mice were pretreated either with hAAT protein or with recombinant adeno-associated virus vector expressing hAAT (rAAV-hAAT. Control groups received saline injections. Arthritis development was evaluated by prevalence of arthritis and arthritic index. Serum levels of B-cell activating factor of the TNF-α family (BAFF, antibodies against both bovine (bCII and mouse collagen II (mCII were tested by ELISA. Results Human AAT protein therapy as well as recombinant adeno-associated virus (rAAV8-mediated hAAT gene therapy significantly delayed onset and ameliorated disease development of arthritis in CIA mouse model. Importantly, hAAT therapies significantly reduced serum levels of BAFF and autoantibodies against bCII and mCII, suggesting that the effects are mediated via B-cells, at least partially. Conclusion These results present a new drug for arthritis therapy. Human AAT protein and gene therapies are able to ameliorate and delay arthritis development and reduce autoimmunity, indicating promising potential of these therapies as a new treatment strategy for RA.
When fast is better: protein folding fundamentals and mechanisms from ultrafast approaches.
Muñoz, Victor; Cerminara, Michele
2016-09-01
Protein folding research stalled for decades because conventional experiments indicated that proteins fold slowly and in single strokes, whereas theory predicted a complex interplay between dynamics and energetics resulting in myriad microscopic pathways. Ultrafast kinetic methods turned the field upside down by providing the means to probe fundamental aspects of folding, test theoretical predictions and benchmark simulations. Accordingly, experimentalists could measure the timescales for all relevant folding motions, determine the folding speed limit and confirm that folding barriers are entropic bottlenecks. Moreover, a catalogue of proteins that fold extremely fast (microseconds) could be identified. Such fast-folding proteins cross shallow free energy barriers or fold downhill, and thus unfold with minimal co-operativity (gradually). A new generation of thermodynamic methods has exploited this property to map folding landscapes, interaction networks and mechanisms at nearly atomic resolution. In parallel, modern molecular dynamics simulations have finally reached the timescales required to watch fast-folding proteins fold and unfold in silico All of these findings have buttressed the fundamentals of protein folding predicted by theory, and are now offering the first glimpses at the underlying mechanisms. Fast folding appears to also have functional implications as recent results connect downhill folding with intrinsically disordered proteins, their complex binding modes and ability to moonlight. These connections suggest that the coupling between downhill (un)folding and binding enables such protein domains to operate analogically as conformational rheostats. © 2016 The Author(s).
DEFF Research Database (Denmark)
Larsen, Kristian Knus
2015-01-01
In recent years, many states have developed and implemented green solutions for defense. Building on these initiatives NATO formulated the NATO Green Defence Framework in 2014. The framework provides a broad basis for cooperation within the Alliance on green solutions for defense. This report aims...... to inform and support the further development of green solutions by unfolding how green technologies and green strategies have been developed and used to handle current security challenges. The report, initially, focuses on the security challenges that are being linked to green defense, namely fuel...... consumption in military operations, defense expenditure, energy security, and global climate change. The report then proceeds to introduce the NATO Green Defence Framework before exploring specific current uses of green technologies and green strategies for defense. The report concludes that a number...
An approach to unfold the response of a multi-element system using an artificial neural network
International Nuclear Information System (INIS)
Cordes, E.; Fehrenbacher, G.; Schuetz, R.; Sprunck, M.; Hahn, K.; Hofmann, R.; Wahl, W.
1998-01-01
An unfolding procedure is proposed which aims at obtaining spectral information of a neutron radiation field by the analysis of the response of a multi-element system consisting of converter type semiconductors. For the unfolding procedure an artificial neural network (feed forward network), trained by the back-propagation method, was used. The response functions of the single elements to neutron radiation were calculated by application of a computational model for an energy range from 10 -2 eV to 10 MeV. The training of the artificial neural network was based on the computation of responses of a six-element system for a set of 300 neutron spectra and the application of the back-propagation method. The validation was performed by the unfolding of 100 computed responses. Two unfolding examples were pointed out for the determination of the neutron spectra. The spectra resulting from the unfolding procedure agree well with the original spectra used for the response computation
Quantification of Drive-Response Relationships Between Residues During Protein Folding.
Qi, Yifei; Im, Wonpil
2013-08-13
Mutual correlation and cooperativity are commonly used to describe residue-residue interactions in protein folding/function. However, these metrics do not provide any information on the causality relationships between residues. Such drive-response relationships are poorly studied in protein folding/function and difficult to measure experimentally due to technical limitations. In this study, using the information theory transfer entropy (TE) that provides a direct measurement of causality between two times series, we have quantified the drive-response relationships between residues in the folding/unfolding processes of four small proteins generated by molecular dynamics simulations. Instead of using a time-averaged single TE value, the time-dependent TE is measured with the Q-scores based on residue-residue contacts and with the statistical significance analysis along the folding/unfolding processes. The TE analysis is able to identify the driving and responding residues that are different from the highly correlated residues revealed by the mutual information analysis. In general, the driving residues have more regular secondary structures, are more buried, and show greater effects on the protein stability as well as folding and unfolding rates. In addition, the dominant driving and responding residues from the TE analysis on the whole trajectory agree with those on a single folding event, demonstrating that the drive-response relationships are preserved in the non-equilibrium process. Our study provides detailed insights into the protein folding process and has potential applications in protein engineering and interpretation of time-dependent residue-based experimental observables for protein function.
PROTEINS IN VACUO . A MORE EFFICIENT MEANS OF ...
African Journals Online (AJOL)
With the aim of understanding solvent effects in protein folding, unfolding, stability and dynamic behavior, studies of protein ions in vacuo have become popular in recent years. One experimental descriptor which gives a general overview of ionic structure is the orientationally-averaged collision cross section , which is ...
Mechanisms of protein misfolding: Novel therapeutic approaches to protein-misfolding diseases
DEFF Research Database (Denmark)
Salahuddin, Parveen; Siddiqi, Mohammad Khursheed; Khan, Sanaullah
2016-01-01
’s disease (PD), Alzheimer’s disease (AD), Prion disease and Amylo lateral Sclerosis (ALS). Furthermore, tau protein shows intrinsically disorder conformation; therefore its interaction with microtubule is impaired and this protein undergoes aggregation. This is also underlying cause of Alzheimers and other......In protein misfolding, protein molecule acquires wrong tertiary structure, thereby induces protein misfolding diseases. Protein misfolding can occur through various mechanisms. For instance, changes in environmental conditions, oxidative stress, dominant negative mutations, error in post......-translational modifications, increase in degradation rate and trafficking error. All of these factors cause protein misfolding thereby leading to diseases conditions. Both in vitro and in vivo observations suggest that partially unfolded or misfolded intermediates are particularly prone to aggregation. These partially...
Joint mapping of genes and conditions via multidimensional unfolding analysis
Directory of Open Access Journals (Sweden)
Engelen Kristof
2007-06-01
Full Text Available Abstract Background Microarray compendia profile the expression of genes in a number of experimental conditions. Such data compendia are useful not only to group genes and conditions based on their similarity in overall expression over profiles but also to gain information on more subtle relations between genes and conditions. Getting a clear visual overview of all these patterns in a single easy-to-grasp representation is a useful preliminary analysis step: We propose to use for this purpose an advanced exploratory method, called multidimensional unfolding. Results We present a novel algorithm for multidimensional unfolding that overcomes both general problems and problems that are specific for the analysis of gene expression data sets. Applying the algorithm to two publicly available microarray compendia illustrates its power as a tool for exploratory data analysis: The unfolding analysis of a first data set resulted in a two-dimensional representation which clearly reveals temporal regulation patterns for the genes and a meaningful structure for the time points, while the analysis of a second data set showed the algorithm's ability to go beyond a mere identification of those genes that discriminate between different patient or tissue types. Conclusion Multidimensional unfolding offers a useful tool for preliminary explorations of microarray data: By relying on an easy-to-grasp low-dimensional geometric framework, relations among genes, among conditions and between genes and conditions are simultaneously represented in an accessible way which may reveal interesting patterns in the data. An additional advantage of the method is that it can be applied to the raw data without necessitating the choice of suitable genewise transformations of the data.
Dissecting Protein Configurational Entropy into Conformational and Vibrational Contributions.
Chong, Song-Ho; Ham, Sihyun
2015-10-01
Quantifying how the rugged nature of the underlying free-energy landscape determines the entropic cost a protein must incur upon folding and ligand binding is a challenging problem. Here, we present a novel computational approach that dissects the protein configurational entropy on the basis of the classification of protein dynamics on the landscape into two separate components: short-term vibrational dynamics related to individual free-energy wells and long-term conformational dynamics associated with transitions between wells. We apply this method to separate the configurational entropy of the protein villin headpiece subdomain into its conformational and vibrational components. We find that the change in configurational entropy upon folding is dominated by the conformational entropy despite the fact that the magnitude of the vibrational entropy is the significantly larger component in each of the folded and unfolded states, which is in accord with the previous empirical estimations. The straightforward applicability of our method to unfolded proteins promises a wide range of applications, including those related to intrinsically disordered proteins.
Directory of Open Access Journals (Sweden)
D. L. Fehl
2010-12-01
Full Text Available A five-channel, filtered-x-ray-detector (XRD array has been used to measure time-dependent, soft-x-ray flux emitted by z-pinch plasmas at the Z pulsed-power accelerator (Sandia National Laboratories, Albuquerque, New Mexico, USA. The preceding, companion paper [D. L. Fehl et al., Phys. Rev. ST Accel. Beams 13, 120402 (2010PRABFM1098-4402] describes an algorithm for spectral reconstructions (unfolds and spectrally integrated flux estimates from data obtained by this instrument. The unfolded spectrum S_{unfold}(E,t is based on (N=5 first-order B-splines (histograms in contiguous unfold bins j=1,…,N; the recovered x-ray flux F_{unfold}(t is estimated as ∫S_{unfold}(E,tdE, where E is x-ray energy and t is time. This paper adds two major improvements to the preceding unfold analysis: (a Error analysis.—Both data noise and response-function uncertainties are propagated into S_{unfold}(E,t and F_{unfold}(t. Noise factors ν are derived from simulations to quantify algorithm-induced changes in the noise-to-signal ratio (NSR for S_{unfold} in each unfold bin j and for F_{unfold} (ν≡NSR_{output}/NSR_{input}: for S_{unfold}, 1≲ν_{j}≲30, an outcome that is strongly spectrally dependent; for F_{unfold}, 0.6≲ν_{F}≲1, a result that is less spectrally sensitive and corroborated independently. For nominal z-pinch experiments, the combined uncertainty (noise and calibrations in F_{unfold}(t at peak is estimated to be ∼15%. (b Generalization of the unfold method.—Spectral sensitivities (called here passband functions are constructed for S_{unfold} and F_{unfold}. Predicting how the unfold algorithm reconstructs arbitrary spectra is thereby reduced to quadratures. These tools allow one to understand and quantitatively predict algorithmic distortions (including negative artifacts, to identify potentially troublesome spectra, and to design more useful response functions.
Directory of Open Access Journals (Sweden)
C.A. Royer
2005-08-01
Full Text Available A thorough understanding of protein structure and stability requires that we elucidate the molecular basis for the effects of both temperature and pressure on protein conformational transitions. While temperature effects are relatively well understood and the change in heat capacity upon unfolding has been reasonably well parameterized, the state of understanding of pressure effects is much less advanced. Ultimately, a quantitative parameterization of the volume changes (at the basis of pressure effects accompanying protein conformational transitions will be required. The present report introduces a qualitative hypothesis based on available model compound data for the molecular basis of volume change upon protein unfolding and its dependence on temperature.
Zeng, Xiaojun; Zhang, Liyun; Xiao, Xiuchan; Jiang, Yuanyuan; Guo, Yanzhi; Yu, Xinyan; Pu, Xuemei; Li, Menglong
2016-04-05
Thrombin-binding aptamer (TBA) with the sequence 5'GGTTGGTGTGGTTGG3' could fold into G-quadruplex, which correlates with functionally important genomic regionsis. However, unfolding mechanism involved in the structural stability of G-quadruplex has not been satisfactorily elucidated on experiments so far. Herein, we studied the unfolding pathway of TBA by a combination of molecular dynamics simulation (MD) and Markov State Model (MSM). Our results revealed that the unfolding of TBA is not a simple two-state process but proceeds along multiple pathways with multistate intermediates. One high flux confirms some observations from NMR experiment. Another high flux exhibits a different and simpler unfolding pathway with less intermediates. Two important intermediate states were identified. One is similar to the G-triplex reported in the folding of G-quadruplex, but lack of H-bonding between guanines in the upper plane. More importantly, another intermediate state acting as a connector to link the folding region and the unfolding one, was the first time identified, which exhibits higher population and stability than the G-triplex-like intermediate. These results will provide valuable information for extending our understanding the folding landscape of G-quadruplex formation.
Counting Unfolding Events in Stretched Helices with Induced Oscillation by Optical Tweezers
Bacabac, Rommel Gaud; Otadoy, Roland
Correlation measures based on embedded probe fluctuations, single or paired, are now widely used for characterizing the viscoelastic properties of biological samples. However, more robust applications using this technique are still lacking. Considering that the study of living matter routinely demonstrates new and complex phenomena, mathematical and experimental tools for analysis have to catch up in order to arrive at newer insights. Therefore, we derive ways of probing non-equilibrium events in helical biopolymers provided by stretching beyond thermal forces. We generalize, for the first time, calculations for winding turn probabilities to account for unfolding events in single fibrous biopolymers and globular proteins under tensile stretching using twin optical traps. The approach is based on approximating the ensuing probe fluctuations as originating from a damped harmonic oscillator under oscillatory forcing.
Lev, Avital; Lulla, Amriti R; Wagner, Jessica; Ralff, Marie D; Kiehl, Joshua B; Zhou, Yan; Benes, Cyril H; Prabhu, Varun V; Oster, Wolfgang; Astsaturov, Igor; Dicker, David T; El-Deiry, Wafik S
2017-10-10
Pancreatic cancer is chemo-resistant and metastasizes early with an overall five-year survival of ∼8.2%. First-in-class imipridone ONC201 is a small molecule in clinical trials with anti-cancer activity. ONC212, a fluorinated-ONC201 analogue, shows preclinical efficacy in melanoma and hepatocellular-cancer models. We investigated efficacy of ONC201 and ONC212 against pancreatic cancer cell lines ( N =16 including 9 PDX-cell lines). We demonstrate ONC212 efficacy in 4 in-vivo models including ONC201-resistant tumors. ONC212 is active in pancreatic cancer as single agent or in combination with 5-fluorouracil, irinotecan, oxaliplatin or RTK inhibitor crizotinib. Based on upregulation of pro-survival IGF1-R in some tumors, we found an active combination of ONC212 with inhibitor AG1024, including in vivo . We show a rationale for targeting pancreatic cancer using ONC212 combined with targeting the unfolded-protein response and ER chaperones such as GRP78/BIP. Our results lay the foundation to test imipridones, anti-cancer agents, in pancreatic cancer, that is refractory to most drugs.
Dante-unfolding code for energy spectra evaluation
International Nuclear Information System (INIS)
Petilli, M.
1979-01-01
The code DANTE, using the last square method in unfolding for dosimetry purpose, solves the neutron spectra evaluation problem starting by activity measurements. The code DANTE introduced for the first time the correlation between available data by mean of flux and activity variance-covariance matrices and the error propagation. In the present report the solution method is detailed described
Zhang, Ye; Zhou, Ning; Shi, Junfeng; Pochapsky, Susan Sondej; Pochapsky, Thomas C.; Zhang, Bei; Zhang, Xixiang; Xu, Bing
2015-01-01
While used extensively by nature to control the geometry of protein structures, and dynamics of proteins, such as self-organization, hydration forces and ionic interactions received less attention for controlling the behaviour of small molecules. Here we describe the synthesis and characterization of a novel zwitterionic metallopeptide consisting of a cationic core and three distal anionic groups linked by self-assembling peptide motifs. 2D NMR spectra, total correlated spectroscopy and nuclear Overhauser effect spectroscopy, show that the molecule exhibits a three-fold rotational symmetry and adopts a folded conformation in dimethyl sulfoxide due to Coulombic forces. When hydrated in water, the molecule unfolds to act as a self-assembling building block of supramolecular nanostructures. By combining ionic interactions with the unique geometry from metal complex and hydrophobic interactions from simple peptides, we demonstrate a new and effective way to design molecules for smart materials through mimicking a sophisticated biofunctional system using a conformational switch.
Zhang, Ye
2015-02-19
While used extensively by nature to control the geometry of protein structures, and dynamics of proteins, such as self-organization, hydration forces and ionic interactions received less attention for controlling the behaviour of small molecules. Here we describe the synthesis and characterization of a novel zwitterionic metallopeptide consisting of a cationic core and three distal anionic groups linked by self-assembling peptide motifs. 2D NMR spectra, total correlated spectroscopy and nuclear Overhauser effect spectroscopy, show that the molecule exhibits a three-fold rotational symmetry and adopts a folded conformation in dimethyl sulfoxide due to Coulombic forces. When hydrated in water, the molecule unfolds to act as a self-assembling building block of supramolecular nanostructures. By combining ionic interactions with the unique geometry from metal complex and hydrophobic interactions from simple peptides, we demonstrate a new and effective way to design molecules for smart materials through mimicking a sophisticated biofunctional system using a conformational switch.
A high-resolution neutron spectra unfolding method using the Genetic Algorithm technique
Mukherjee, B
2002-01-01
The Bonner sphere spectrometers (BSS) are commonly used to determine the neutron spectra within various nuclear facilities. Sophisticated mathematical tools are used to unfold the neutron energy distribution from the output data of the BSS. This paper highlights a novel high-resolution neutron spectra-unfolding method using the Genetic Algorithm (GA) technique. The GA imitates the biological evolution process prevailing in the nature to solve complex optimisation problems. The GA method was utilised to evaluate the neutron energy distribution, average energy, fluence and equivalent dose rates at important work places of a DIDO class research reactor and a high-energy superconducting heavy ion cyclotron. The spectrometer was calibrated with a sup 2 sup 4 sup 1 Am/Be (alpha,n) neutron standard source. The results of the GA method agreed satisfactorily with the results obtained by using the well-known BUNKI neutron spectra unfolding code.
Ahmad, Basir; Ahmed, Md Zulfazal; Haq, Soghra Khatun; Khan, Rizwan Hasan
2005-06-15
The effect of guanidine hydrochloride (GnHCl) on the global stability of human serum albumin (HSA) has been studied by fluorescence and circular dichroism spectroscopic measurements. The differential stability of native conformation of three HSA domains were explored by using domain-specific ligands, hemin (domain I), chloroform (domain II), bilirubin (at domain I/domain II interface) and diazepam (domain III). GnHCl induced unfolding transition curves as monitored by probes for secondary and tertiary structures were cooperative but noncoincidental. A strong ANS binding to the protein was observed around 1.8 M GnHCl, suggesting existence of intermediate states in the unfolding pathway of HSA. A gradual decrease (in the GnHCl concentration range 0.0-1.8 M) in the binding of diazepam indicates that domain III is the most labile to GnHCl denaturation. A significant increase in the binding of bilirubin up to 1.4 M GnHCl and decrease thereafter leading to complete abolishment of bilirubin binding at around 2.0 M GnHCl suggest favorable rearrangement and separation of domains I and II at 1.4 and 2.0 M GnHCl concentration, respectively. Above 1.6 M GnHCl, decrease of the binding of hemin, a ligand for domain I, chloroform, which binds in domain II and lone tryptophanyl fluorescence (Trp-214 located in domain II) indicate that at higher concentration of GnHCl domains I and II start unfolding simultaneously but the stability of domain I (7.4 Kcal/mol) is much more than domain II (4.3 Kcal/mol). A pictorial model for the unfolding of HSA domains, consistent with all these results, has been formulated, suggesting that domain III is the most labile followed by domain II while domain I is the most stable. A molten globule like state of domain III around 1.8 M GnHCl has also been identified and characterized.
Unfolding neutron spectra with BS-TLD system using genetic algorithms
Energy Technology Data Exchange (ETDEWEB)
Santos, Joelan A.L., E-mail: jasantos@cnen.gov.br [Universidade Federal de Pernambuco (UFPE), Recife, PE (Brazil). Dept. de Energia Nuclear; Centro Regional de Ciencias Nucleares do Nordeste (CRCN-NE/CNEN-PE), Recife, PE (Brazil); Silva, Everton R. [Universidade Federal de Pernambuco (UFPE), Recife, PE (Brazil). Centro de Informatica; Ferreira, Tiago A.E. [Universidade Federal Rural de Pernambuco (UFRPE), Recife, PE (Brazil). Dept. de Estatistica e Informatica; Fonseca, Evaldo S. [Instituto de Radioprotecao e Dosimetria (IRD/CNEN-RJ), Rio de Janeiro, RJ (Brazil); Vilela, Eudice C., E-mail: ecvilela@cnen.gov.br [Centro Regional de Ciencias Nucleares do Nordeste (CRCN-NE/CNEN-PE), Recife, PE (Brazil)
2011-07-01
Due to the variability of neutron spectrum within the same environment, it is essential that the spectral distribution as function of energy to be characterized. To perform this task, the neutron spectrometer has a primary role in determining the neutron flux ({Phi}{sub E}(E)). Precise information allows radiological quantities establishment related to that spectrum but it is necessary, however, a series of steps with a spectrometric system that can cover a large interval of energy and whose answer is isotropic. The most widely used for accomplishing this task is the spectrometric Bonner spheres system. One of the biggest problems related to neutron spectrometry is the process of data analysis, known as unfolding. Most of the work undertaken to implement new techniques of this process, using data obtained with the scintillator {sup 6}LiI(I). However, characteristics related to the dead time make it not be so effective when used in high flow neutron fields. An alternative to this problem is the use of thermoluminescent detectors (TLD), but the codes used do not provide a more specific response matrix to unfolding the information obtained through these materials, which makes the development of a specific response matrix important to adequately characterize the response obtained by them. This paper proposes using a technique of artificial intelligence called genetic algorithm, which uses bio-inspired mathematical models and through the implementation of a specific matrix to unfolding data obtained from a combination of TLDs embedded in a system of Bonner spheres, such as thermal neutron detectors, to characterize the neutron spectrum as a function of energy. The results obtained with this method were in accordance with reference spectra, thus enables of this technique to unfolding neutrons spectra with BS-TLD system. (author)
Unfolding neutron spectra with BS-TLD system using genetic algorithms
International Nuclear Information System (INIS)
Santos, Joelan A.L.; Silva, Everton R.; Vilela, Eudice C.
2011-01-01
Due to the variability of neutron spectrum within the same environment, it is essential that the spectral distribution as function of energy to be characterized. To perform this task, the neutron spectrometer has a primary role in determining the neutron flux (Φ E (E)). Precise information allows radiological quantities establishment related to that spectrum but it is necessary, however, a series of steps with a spectrometric system that can cover a large interval of energy and whose answer is isotropic. The most widely used for accomplishing this task is the spectrometric Bonner spheres system. One of the biggest problems related to neutron spectrometry is the process of data analysis, known as unfolding. Most of the work undertaken to implement new techniques of this process, using data obtained with the scintillator 6 LiI(I). However, characteristics related to the dead time make it not be so effective when used in high flow neutron fields. An alternative to this problem is the use of thermoluminescent detectors (TLD), but the codes used do not provide a more specific response matrix to unfolding the information obtained through these materials, which makes the development of a specific response matrix important to adequately characterize the response obtained by them. This paper proposes using a technique of artificial intelligence called genetic algorithm, which uses bio-inspired mathematical models and through the implementation of a specific matrix to unfolding data obtained from a combination of TLDs embedded in a system of Bonner spheres, such as thermal neutron detectors, to characterize the neutron spectrum as a function of energy. The results obtained with this method were in accordance with reference spectra, thus enables of this technique to unfolding neutrons spectra with BS-TLD system. (author)
International Nuclear Information System (INIS)
Junges, Leandro; Gallas, Jason A C
2015-01-01
The dynamics of two mutually delay-coupled semiconductor lasers has been frequently studied experimentally, numerically, and analytically either for weak or strong detuning between the lasers. Here, we present a systematic numerical investigation spanning all detuning ranges. We report high-resolution stability diagrams for wide ranges of the main control parameters of the laser, as described by the Lang–Kobayashi model. In particular, we detail the parameter influence on dynamical performance and map the distribution of chaotic pulsations and self-generated periodic spiking with arbitrary periodicity. Special attention is given to the unfolding of regular pulse packages for both symmetric and non-symmetric configurations with respect to detuning. The influence of the delay –time on the self-organization of periodic and chaotic laser phases as a function of the coupling and detuning is also described in detail. (paper)
Advanced path sampling of the kinetic network of small proteins
Du, W.
2014-01-01
This thesis is focused on developing advanced path sampling simulation methods to study protein folding and unfolding, and to build kinetic equilibrium networks describing these processes. In Chapter 1 the basic knowledge of protein structure and folding theories were introduced and a brief overview
UNFOLDINGS OF THE CYLINDRICA L SURFACES USED IN THE INDUSTRIAL INSTALLATIONS
Directory of Open Access Journals (Sweden)
VASILE GHEORGHITA
2013-02-01
Full Text Available The connections in the construction of the various industrial installations: pipes, boilers, joints elements and fittings have a cylindrical configuration, or similar cylindrical shape. The execution and their installation require knowledge of the unfolding and intersection curves, which compose them. The graphical solving of the problems of tech nical representation has enabled the formation of abstract geometric of the pieces forms and the ability to see into space. The paper proposes to establish the unfolding of a connection, used in the industrial equipments, by the classical method of the des criptive geometry and mathematics, using appropriate software
The AAA+ ATPase TRIP13 remodels HORMA domains through N-terminal engagement and unfolding
Energy Technology Data Exchange (ETDEWEB)
Ye, Qiaozhen; Kim, Dong Hyun; Dereli, Ihsan; Rosenberg, Scott C.; Hagemann, Goetz; Herzog, Franz; Tóth, Attila; Cleveland, Don W.; Corbett, Kevin D.
2017-06-28
Proteins of the conserved HORMA domain family, including the spindle assembly checkpoint protein MAD2 and the meiotic HORMADs, assemble into signaling complexes by binding short peptides termed “closure motifs”. The AAA+ ATPase TRIP13 regulates both MAD2 and meiotic HORMADs by disassembling these HORMA domain–closure motif complexes, but its mechanisms of substrate recognition and remodeling are unknown. Here, we combine X-ray crystallography and crosslinking mass spectrometry to outline how TRIP13 recognizes MAD2 with the help of the adapter protein p31comet. We show that p31comet binding to the TRIP13 N-terminal domain positions the disordered MAD2 N-terminus for engagement by the TRIP13 “pore loops”, which then unfold MAD2 in the presence of ATP. N-terminal truncation of MAD2 renders it refractory to TRIP13 action in vitro, and in cells causes spindle assembly checkpoint defects consistent with loss of TRIP13 function. Similar truncation of HORMAD1 in mouse spermatocytes compromises its TRIP13-mediated removal from meiotic chromosomes, highlighting a conserved mechanism for recognition and disassembly of HORMA domain–closure motif complexes by TRIP13.
International Nuclear Information System (INIS)
Fehl, D.L.; Chandler, G.A.; Biggs, F.; Dukart, R.J.; Moats, A.R.; Leeper, R.J.
1996-01-01
X-ray-producing hohlraums are being studied as indirect drives for Inertial Confinement Fusion targets. In a 1994 target series on the PBFAII accelerator, cylindrical hohlraum targets were heated by an intense Li + ion beam and viewed by an array of 13 time-resolved, filtered x-ray detectors (XRDs). The UFO unfold code and its suite of auxiliary functions were used extensively in obtaining time- resolved x-ray spectra and radiation temperatures from this diagnostic. UFO was also used to obtain fitted response functions from calibration data, to simulate data from blackbody x-ray spectra of interest, to determine the suitability of various unfolding parameters (e.g., energy domain, energy partition, smoothing conditions, and basis functions), to interpolate the XRD signal traces, and to unfold experimental data. The simulation capabilities of the code were useful in understanding an anomalous feature in the unfolded spectra at low photon energies (≤ 100 eV). Uncertainties in the differential and energy-integrated unfolded spectra were estimated from uncertainties in the data. The time-history of the radiation temperature agreed well with independent calculations of the wall temperature in the hohlraum
International Nuclear Information System (INIS)
Fehl, D.L.; Chandler, G.A.; Biggs, F.; Dukart, R.J.; Moats, A.R.; Leeper, R.J.
1997-01-01
X-ray-producing hohlraums are being studied as indirect drives for inertial confinement fusion targets. In a 1994 target series on the PBFAII accelerator, cylindrical hohlraum targets were heated by an intense Li + ion beam and viewed by an array of 13 time-resolved, filtered x-ray detectors (XRDs). The unfold operator (UFO) code and its suite of auxiliary functions were used extensively in obtaining time-resolved x-ray spectra and radiation temperatures from this diagnostic. The UFO was also used to obtain fitted response functions from calibration data, to simulate data from blackbody x-ray spectra of interest, to determine the suitability of various unfolding parameters (e.g., energy domain, energy partition, smoothing conditions, and basis functions), to interpolate the XRD signal traces, and to unfold experimental data. The simulation capabilities of the code were useful in understanding an anomalous feature in the unfolded spectra at low photon energies (≤100 eV). Uncertainties in the differential and energy-integrated unfolded spectra were estimated from uncertainties in the data. The time endash history of the radiation temperature agreed well with independent calculations of the wall temperature in the hohlraum. copyright 1997 American Institute of Physics
Adsorption of a small protein to a methyl-terminated hydrophobic surfaces
DEFF Research Database (Denmark)
Otzen, Daniel; Oliveberg, M.; Höök, F.
2003-01-01
We have studied the adsorption kinetics of a small monomeric protein S6 using the quartz crystal microbalance with dissipation monitoring (QCM-D) technique. Competitive adsorption from various proportions of native (Nat) and denatured (Den) protein in the bulk phase was carried out using a range...... of chemical denaturant concentrations. The ratio between Nat and Den in bulk has a profound affect on the adsorption behavior, most obvious from a significant (one order of magnitude) increase in the rate of a lag– and consolidation–adsorption phase when Nat is the major species present in bulk, signaling...... that these adsorption phases originates from the Den fraction of proteins in the bulk. To determine whether the kinetics of protein unfolding in the bulk phase are rate-limiting for adsorption of Nat, the adsorption kinetics of wildtype S6 with the mutant VA85 (whose unfolding kinetics are around 30 times more rapid...
Perspective: Structural fluctuation of protein and Anfinsen's thermodynamic hypothesis
Hirata, Fumio; Sugita, Masatake; Yoshida, Masasuke; Akasaka, Kazuyuki
2018-01-01
The thermodynamics hypothesis, casually referred to as "Anfinsen's dogma," is described theoretically in terms of a concept of the structural fluctuation of protein or the first moment (average structure) and the second moment (variance and covariance) of the structural distribution. The new theoretical concept views the unfolding and refolding processes of protein as a shift of the structural distribution induced by a thermodynamic perturbation, with the variance-covariance matrix varying. Based on the theoretical concept, a method to characterize the mechanism of folding (or unfolding) is proposed. The transition state, if any, between two stable states is interpreted as a gap in the distribution, which is created due to an extensive reorganization of hydrogen bonds among back-bone atoms of protein and with water molecules in the course of conformational change. Further perspective to applying the theory to the computer-aided drug design, and to the material science, is briefly discussed.
Deciphering hierarchical features in the energy landscape of adenylate kinase folding/unfolding
Taylor, J. Nicholas; Pirchi, Menahem; Haran, Gilad; Komatsuzaki, Tamiki
2018-03-01
Hierarchical features of the energy landscape of the folding/unfolding behavior of adenylate kinase, including its dependence on denaturant concentration, are elucidated in terms of single-molecule fluorescence resonance energy transfer (smFRET) measurements in which the proteins are encapsulated in a lipid vesicle. The core in constructing the energy landscape from single-molecule time-series across different denaturant concentrations is the application of rate-distortion theory (RDT), which naturally considers the effects of measurement noise and sampling error, in combination with change-point detection and the quantification of the FRET efficiency-dependent photobleaching behavior. Energy landscapes are constructed as a function of observation time scale, revealing multiple partially folded conformations at small time scales that are situated in a superbasin. As the time scale increases, these denatured states merge into a single basin, demonstrating the coarse-graining of the energy landscape as observation time increases. Because the photobleaching time scale is dependent on the conformational state of the protein, possible nonequilibrium features are discussed, and a statistical test for violation of the detailed balance condition is developed based on the state sequences arising from the RDT framework.
Identification of an Unfolding Intermediate for a DNA Lesion Bypass Polymerase
Sherrer, Shanen M.; Maxwell, Brian A.; Pack, Lindsey R.; Fiala, Kevin A.; Fowler, Jason D.; Zhang, Jun; Suo, Zucai
2012-01-01
Sulfolobus solfataricusDNA Polymerase IV (Dpo4), a prototype Y-family DNA polymerase, has been well characterized biochemically and biophysically at 37 °C or lower temperatures. However, the physiological temperature of the hyperthermophile S. solfataricus is approximately 80 °C. With such a large discrepancy in temperature, the in vivo relevance of these in vitro studies of Dpo4 has been questioned. Here, we employed circular dichroism spectroscopy and fluorescence-based thermal scanning to investigate the secondary structural changes of Dpo4 over a temperature range from 26 to 119 °C. Dpo4 was shown to display a high melting temperature characteristic of hyperthermophiles. Unexpectedly, the Little Finger domain of Dpo4, which is only found in the Y-family DNA polymerases, was shown to be more thermostable than the polymerase core. More interestingly, Dpo4 exhibited a three-state cooperative unfolding profile with an unfolding intermediate. The linker region between the Little Finger and Thumb domains of Dpo4 was found to be a source of structural instability. Through site-directed mutagenesis, the interactions between the residues in the linker region and the Palm domain were identified to play a critical role in the formation of the unfolding intermediate. Notably, the secondary structure of Dpo4 was not altered when the temperature was increased from 26 to 87.5 °C. Thus, in addition to providing structural insights into the thermal stability and an unfolding intermediate of Dpo4, our work also validated the relevance of the in vitro studies of Dpo4 performed at temperatures significantly lower than 80 °C. PMID:22667759
International Nuclear Information System (INIS)
Sitompul, Yos Panagaman; Shin, Hee-Sung; Park, Se-Hwan; Oh, Jong Myeong; Seo, Hee; Kim, Ho Dong
2013-01-01
An unfolding method has been developed to obtain a pin-wise source strength distribution of a 14 × 14 pressurized water reactor (PWR) spent fuel assembly. Sixteen measured gamma dose rates at 16 control rod guide tubes of an assembly are unfolded to 179 pin-wise source strengths of the assembly. The method calculates and optimizes five coefficients of the quadratic fitting function for X-Y source strength distribution, iteratively. The pin-wise source strengths are obtained at the sixth iteration, with a maximum difference between two sequential iterations of about 0.2%. The relative distribution of pin-wise source strength from the unfolding is checked using a comparison with the design code (Westinghouse APA code). The result shows that the relative distribution from the unfolding and design code is consistent within a 5% difference. The absolute value of the pin-wise source strength is also checked by reproducing the dose rates at the measurement points. The result shows that the pin-wise source strengths from the unfolding reproduce the dose rates within a 2% difference. (author)
Neutron response matrix for unfolding NE-213 measurements to 21 MeV
International Nuclear Information System (INIS)
Ingersoll, D.T.; Wehring, B.W.; Johnson, R.H.
1976-01-01
A neutron response matrix from measured neutron responses of NE-213 in the energy range of 0.2 to 22 MeV is presented. An interpolation scheme was used to construct an 81-column matrix from the data of Verbinski, Burrus, Love, Zobel, and Hill. As a test of the new response matrix, the Cf-252 neutron spectrum was measured and unfolded using the new response matrix and the FORIST unfolding code. The spectrum agrees well with previous measurements at lower energies, while providing new information above 8 MeV
Conserved salt-bridge competition triggered by phosphorylation regulates the protein interactome
Skinner, John J.
2017-12-05
Phosphorylation is a major regulator of protein interactions; however, the mechanisms by which regulation occurs are not well understood. Here we identify a salt-bridge competition or “theft” mechanism that enables a phospho-triggered swap of protein partners by Raf Kinase Inhibitory Protein (RKIP). RKIP transitions from inhibiting Raf-1 to inhibiting G-protein–coupled receptor kinase 2 upon phosphorylation, thereby bridging MAP kinase and G-Protein–Coupled Receptor signaling. NMR and crystallography indicate that a phosphoserine, but not a phosphomimetic, competes for a lysine from a preexisting salt bridge, initiating a partial unfolding event and promoting new protein interactions. Structural elements underlying the theft occurred early in evolution and are found in 10% of homo-oligomers and 30% of hetero-oligomers including Bax, Troponin C, and Early Endosome Antigen 1. In contrast to a direct recognition of phosphorylated residues by binding partners, the salt-bridge theft mechanism represents a facile strategy for promoting or disrupting protein interactions using solvent-accessible residues, and it can provide additional specificity at protein interfaces through local unfolding or conformational change.
Conserved salt-bridge competition triggered by phosphorylation regulates the protein interactome
Skinner, John J.; Wang, Sheng; Lee, Jiyoung; Ong, Colin; Sommese, Ruth; Sivaramakrishnan, Sivaraj; Koelmel, Wolfgang; Hirschbeck, Maria; Schindelin, Hermann; Kisker, Caroline; Lorenz, Kristina; Sosnick, Tobin R.; Rosner, Marsha Rich
2017-01-01
Phosphorylation is a major regulator of protein interactions; however, the mechanisms by which regulation occurs are not well understood. Here we identify a salt-bridge competition or “theft” mechanism that enables a phospho-triggered swap of protein partners by Raf Kinase Inhibitory Protein (RKIP). RKIP transitions from inhibiting Raf-1 to inhibiting G-protein–coupled receptor kinase 2 upon phosphorylation, thereby bridging MAP kinase and G-Protein–Coupled Receptor signaling. NMR and crystallography indicate that a phosphoserine, but not a phosphomimetic, competes for a lysine from a preexisting salt bridge, initiating a partial unfolding event and promoting new protein interactions. Structural elements underlying the theft occurred early in evolution and are found in 10% of homo-oligomers and 30% of hetero-oligomers including Bax, Troponin C, and Early Endosome Antigen 1. In contrast to a direct recognition of phosphorylated residues by binding partners, the salt-bridge theft mechanism represents a facile strategy for promoting or disrupting protein interactions using solvent-accessible residues, and it can provide additional specificity at protein interfaces through local unfolding or conformational change.
Mosoni, Laurent; Gatineau, Eva; Gatellier, Philippe; Migné, Carole; Savary-Auzeloux, Isabelle; Rémond, Didier; Rocher, Emilie; Dardevet, Dominique
2014-01-01
Our aim was to compare and combine 3 nutritional strategies to slow down the age-related loss of muscle mass in healthy old rats: 1) increase protein intake, which is likely to stimulate muscle protein anabolism; 2) use leucine rich, rapidly digested whey proteins as protein source (whey proteins are recognized as the most effective proteins to stimulate muscle protein anabolism). 3) Supplement animals with a mixture of chamomile extract, vitamin E, vitamin D (reducing inflammation and oxidative stress is also effective to improve muscle anabolism). Such comparisons and combinations were never tested before. Nutritional groups were: casein 12% protein, whey 12% protein, whey 18% protein and each of these groups were supplemented or not with polyphenols/antioxidants. During 6 months, we followed changes of weight, food intake, inflammation (plasma fibrinogen and alpha-2-macroglobulin) and body composition (DXA). After 6 months, we measured muscle mass, in vivo and ex-vivo fed and post-absorptive muscle protein synthesis, ex-vivo muscle proteolysis, and oxidative stress parameters (liver and muscle glutathione, SOD and total antioxidant activities, muscle carbonyls and TBARS). We showed that although micronutrient supplementation reduced inflammation and oxidative stress, the only factor that significantly reduced the loss of lean body mass was the increase in whey protein intake, with no detectable effect on muscle protein synthesis, and a tendency to reduce muscle proteolysis. We conclude that in healthy rats, increasing protein intake is an effective way to delay sarcopenia. PMID:25268515
Energy Technology Data Exchange (ETDEWEB)
Zeng, Fanxing; Tee, Catherine; Liu, Michelle [Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1 (Canada); Sherry, James P. [Aquatic Contaminants Research Division, Environment Canada, Burlington, Ontario L7R 4A6 (Canada); Dixon, Brian; Duncker, Bernard P. [Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1 (Canada); Bols, Niels C., E-mail: ncbols@uwaterloo.ca [Department of Biology, University of Waterloo, Waterloo, Ontario N2L 3G1 (Canada)
2014-01-15
Highlights: •2-Phenylethynesulfonamide (PES) is an inhibitor of p53 and HSP 70 in mammals. •In the fish epithelial cell line, RTgill-W1, PES enhanced ROS generation and was cytotoxic. •RTgill-W1 death was by apoptosis and blocked by the anti-oxidant N-acetylcysteine. •This is the first report linking PES-induced cell death to ROS. •With this background PES should be useful for studying fish cell survival pathways. -- Abstract: The effect of 2-phenylethynesulfonamide (PES), which is a p53 and HSP70 inhibitor in mammalian cells, was studied on the rainbow trout (Oncorhynchus mykiss) gill epithelial cell line, RTgill-W1, in order to evaluate PES as a tool for understanding the cellular survival pathways operating in fish. As judged by three viability assays, fish cells were killed by 24 h exposures to PES, but cell death was blocked by the anti-oxidant N-acetylcysteine (NAC). Cell death had several hallmarks of apoptosis: DNA laddering, nuclear fragmentation, Annexin V staining, mitochondrial membrane potential decline, and caspases activation. Reactive oxygen species (ROS) production peaked in several hours after the addition of PES and before cell death. HSP70 and BiP levels were higher in cultures treated with PES for 24 h, but this was blocked by NAC. As well, PES treatment caused HSP70, BiP and p53 to accumulate in the detergent-insoluble fraction, and this too was prevented by NAC. Of several possible scenarios to explain the results, the following one is the simplest. PES enhances the generation of ROS, possibly by inhibiting the anti-oxidant actions of p53 and HSP70. ER stress arises from the ROS and from PES inhibiting the chaperone activities of HSP70. The ER stress in turn initiates the unfolded protein response (UPR), but this fails to restore ER homeostasis so proteins aggregate and cells die. Despite these multiple actions, PES should be useful for studying fish cellular survival pathways.
Model based rib-cage unfolding for trauma CT
von Berg, Jens; Klinder, Tobias; Lorenz, Cristian
2018-03-01
A CT rib-cage unfolding method is proposed that does not require to determine rib centerlines but determines the visceral cavity surface by model base segmentation. Image intensities are sampled across this surface that is flattened using a model based 3D thin-plate-spline registration. An average rib centerline model projected onto this surface serves as a reference system for registration. The flattening registration is designed so that ribs similar to the centerline model are mapped onto parallel lines preserving their relative length. Ribs deviating from this model appear deviating from straight parallel ribs in the unfolded view, accordingly. As the mapping is continuous also the details in intercostal space and those adjacent to the ribs are rendered well. The most beneficial application area is Trauma CT where a fast detection of rib fractures is a crucial task. Specifically in trauma, automatic rib centerline detection may not be guaranteed due to fractures and dislocations. The application by visual assessment on the large public LIDC data base of lung CT proved general feasibility of this early work.
Contribution of buried aspartic acid to the stability of the PDZ2 protein
International Nuclear Information System (INIS)
Jayasimha, Pruthvi; Shanmuganathan, Aranganathan; Suladze, Saba; Makhatadze, George I.
2012-01-01
Highlights: ► Buried Asp residues on average form 2.5 to 3 hydrogen bonds and/or 0.8 salt bridges. ► Contribution of buried Asp to stability was estimated using model protein PDZ2. ► The energetic contribution of Asp56 to PDZ2 stability estimated to be 18 kJ · mol −1 . ► Findings are discussed in terms of contribution of Asp residues to protein stability. - Abstract: Statistical analysis of protein structures shows that buried aspartic acid residues on average form 2.5 to 3 hydrogen bonds and/or 0.8 potential ionic interactions with other protein groups. To estimate the energetic contribution of such buried groups to the Gibbs free energy of proteins, we measured the effects of amino acid substitutions of D56 in a model protein PDZ2 on its stability. We used temperature-induced unfolding monitored by DSC and denaturant-induced unfolding monitored by the changes in fluorescence intensity. We find that all substitutions of D56 lead to protein unfolding, thus suggesting that this buried hydrogen bonded aspartic acid has a significant contribution to the stability. To quantify the changes in the Gibbs free energy, one of the variants, D56N was stabilized by addition of the protective osmolyte TMAO. Comparison of the stability of the D56N variant with the wild-type PDZ2 in the presence and absence of TMAO allowed us to estimate the contribution of D56 to the protein stability to be 18 kJ · mol −1 . These findings are discussed in terms of contribution of buried ionizable groups to protein stability.
Interaction of sucralose with whey protein: Experimental and molecular modeling studies
Zhang, Hongmei; Sun, Shixin; Wang, Yanqing; Cao, Jian
2017-12-01
The objective of this research was to study the interactions of sucralose with whey protein isolate (WPI) by using the three-dimensional fluorescence spectroscopy, circular dichroism spectroscopy and molecular modeling. The results showed that the peptide strands structure of WPI had been changed by sucralose. Sucralose binding induced the secondary structural changes and increased content of aperiodic structure of WPI. Sucralose decreased the thermal stability of WPI and acted as a structure destabilizer during the thermal unfolding process of protein. In addition, the existence of sucralose decreased the reversibility of the unfolding of WPI. Nonetheless, sucralose-WPI complex was less stable than protein alone. The molecular modeling result showed that van der Waals and hydrogen bonding interactions contribute to the complexation free binding energy. There are more than one possible binding sites of WPI with sucralose by surface binding mode.
Yokota, Jun-Ichi; Shiro, Daisuke; Tanaka, Mizuki; Onozaki, Yasumichi; Mizutani, Osamu; Kakizono, Dararat; Ichinose, Sakurako; Shintani, Tomoko; Gomi, Katsuya; Shintani, Takahiro
2017-03-01
Filamentous fungi are often used as cell factories for recombinant protein production because of their ability to secrete large quantities of hydrolytic enzymes. However, even using strong transcriptional promoters, yields of nonfungal proteins are generally much lower than those of fungal proteins. Recent analyses revealed that expression of certain nonfungal secretory proteins induced the unfolded protein response (UPR), suggesting that they are recognized as proteins with folding defects in filamentous fungi. More recently, however, even highly expressed endogenous secretory proteins were found to evoke the UPR. These findings raise the question of whether the unfolded or misfolded state of proteins is selectively recognized by quality control mechanisms in filamentous fungi. In this study, a fungal secretory protein (1,2-α-D-mannosidase; MsdS) with a mutation that decreases its thermostability was expressed at different levels in Aspergillus oryzae. We found that, at moderate expression levels, wild-type MsdS was secreted to the medium, while the mutant was not. In the strain with a deletion for the hrdA gene, which is involved in the endoplasmic reticulum-associated degradation pathway, mutant MsdS had specifically increased levels in the intracellular fraction but was not secreted. When overexpressed, the mutant protein was secreted to the medium to a similar extent as the wild-type protein; however, the mutant underwent hyperglycosylation and induced the UPR. Deletion of α-amylase (the most abundant secretory protein in A. oryzae) alleviated the UPR induction by mutant MsdS overexpression. These findings suggest that misfolded MsdS and unfolded species of α-amylase might act synergistically for UPR induction.
Kesby, James P; Markou, Athina; Semenova, Svetlana
2016-10-01
Depression and psychostimulant abuse are common comorbidities among humans with immunodeficiency virus (HIV) disease. The HIV regulatory protein TAT is one of multiple HIV-related proteins associated with HIV-induced neurotoxicity. TAT-induced dysfunction of dopamine and serotonin systems in corticolimbic brain areas may result in impaired reward function, thus, contributing to depressive symptoms and psychostimulant abuse. Transgenic mice with doxycycline-induced TAT protein expression in the brain (TAT+, TAT- control) show neuropathology resembling brain abnormalities in HIV+ humans. We evaluated brain reward function in response to TAT expression, nicotine and methamphetamine administration in TAT+ and TAT- mice using the intracranial self-stimulation procedure. We evaluated the brain dopamine and serotonin systems with high-performance liquid chromatography. The effects of TAT expression on delay-dependent working memory in TAT+ and TAT- mice using the operant delayed nonmatch-to-position task were also assessed. During doxycycline administration, reward thresholds were elevated by 20% in TAT+ mice compared with TAT- mice. After the termination of doxycycline treatment, thresholds of TAT+ mice remained significantly higher than those of TAT- mice and this was associated with changes in mesolimbic serotonin and dopamine levels. TAT+ mice showed a greater methamphetamine-induced threshold lowering compared with TAT- mice. TAT expression did not alter delay-dependent working memory. These results indicate that TAT expression in mice leads to reward deficits, a core symptom of depression, and a greater sensitivity to methamphetamine-induced reward enhancement. Our findings suggest that the TAT protein may contribute to increased depressive-like symptoms and continued methamphetamine use in HIV-positive individuals. Copyright © 2016 Elsevier Ltd. All rights reserved.
Clinical evaluation of coronary territory map by using unfolded map of Tl-201 myocardial SPECT
International Nuclear Information System (INIS)
Uehara, Toshiisa; Nishimura, Tsunehiko; Katafuchi, Tetsuro; Yamagami, Hidetoshi; Kumita, Shinichirou; Hayashida, Kohei; Hayashi, Makoto
1990-01-01
Coronary territory map was developed on unfolded map of exercise Tl-201 myocardial SPECT. Each coronary territory was determined by summing the each unfolded map of 54 cases of single vessel disease respectively, and standardizing with normal pattern obtained from normal patients. The diagnostic accuracy of coronary territory map to identify the diseased coronary artery was analyzed in 104 clinical cases and was compared with that of planar and SPECT visual diagnosis, simple unfolded map (raw map) and extent and severity map. The results were as follows. (1) Territory map showed excellent diagnostic accuracy in single or double vessel disease, especially in diagnosis of left circumflex coronary artery lesion. (2) In triple vessel disease, the diagnostic accuracy of territory map or other unfolded maps was 30% at best, and was inferior to planar or SPECT visual analysis. The cause of this inferiority seemed that the quantitatively analyzed map had no information about the degree of Tl-uptake into lung or myocardium, which give useful information in visual diagnosis. (3) The diagnostic agreement ratio in two observers was the highest in territory map diagnosis, so that the territory map diagnosis seemed to be the most objective one. (4) The unfolded map diagnosis with apical display obtained from long-axis tomogram was useful to diagnose left anteior descending coronary (LAD) lesion, which improve not only the sensitivity of LAD but also specificity of right coronary artery single vessel disease. (author)
Origins of pressure-induced protein transitions.
Chalikian, Tigran V; Macgregor, Robert B
2009-12-18
The molecular mechanisms underlying pressure-induced protein denaturation can be analyzed based on the pressure-dependent differences in the apparent volume occupied by amino acids inside the protein and when they are exposed to water in an unfolded conformation. We present here an analysis for the peptide group and the 20 naturally occurring amino acid side chains based on volumetric parameters for the amino acids in the interior of the native state, the micelle-like interior of the pressure-induced denatured state, and the unfolded conformation modeled by N-acetyl amino acid amides. The transfer of peptide groups from the protein interior to water becomes increasingly favorable as pressure increases. Thus, solvation of peptide groups represents a major driving force in pressure-induced protein denaturation. Polar side chains do not appear to exhibit significant pressure-dependent changes in their preference for the protein interior or solvent. The transfer of nonpolar side chains from the protein interior to water becomes more unfavorable as pressure increases. We conclude that a sizeable population of nonpolar side chains remains buried inside a solvent-inaccessible core of the pressure-induced denatured state. At elevated pressures, this core may become packed almost as tightly as the interior of the native state. The presence and partial disappearance of large intraglobular voids is another driving force facilitating pressure-induced denaturation of individual proteins. Our data also have implications for the kinetics of protein folding and shed light on the nature of the folding transition state ensemble.
International Nuclear Information System (INIS)
Ortiz R, J. M.; Martinez B, M. R.; Vega C, H. R.; Gallego D, E.; Lorente F, A.; Mendez V, R.; Los Arcos M, J. M.; Guerrero A, J. E.
2011-01-01
With the Bonner spheres spectrometer neutron spectrum is obtained through an unfolding procedure. Monte Carlo methods, Regularization, Parametrization, Least-squares, and Maximum Entropy are some of the techniques utilized for unfolding. In the last decade methods based on Artificial Intelligence Technology have been used. Approaches based on Genetic Algorithms and Artificial Neural Networks (Ann) have been developed in order to overcome the drawbacks of previous techniques. Nevertheless the advantages of Ann still it has some drawbacks mainly in the design process of the network, vg the optimum selection of the architectural and learning Ann parameters. In recent years the use of hybrid technologies, combining Ann and genetic algorithms, has been utilized to. In this work, several Ann topologies were trained and tested using Ann and Genetically Evolved Artificial Neural Networks in the aim to unfold neutron spectra using the count rates of a Bonner sphere spectrometer. Here, a comparative study of both procedures has been carried out. (Author)
Characteristics of SiC neutron sensor spectrum unfolding process based on Bayesian inference
Energy Technology Data Exchange (ETDEWEB)
Cetnar, Jerzy; Krolikowski, Igor [Faculty of Energy and Fuels AGH - University of Science and Technology, Al. Mickiewicza 30, 30-059 Krakow (Poland); Ottaviani, L. [IM2NP, UMR CNRS 7334, Aix-Marseille University, Case 231 -13397 Marseille Cedex 20 (France); Lyoussi, A. [CEA, DEN, DER, Instrumentation Sensors and Dosimetry Laboratory, Cadarache, F-13108 St-Paul-Lez-Durance (France)
2015-07-01
This paper deals with SiC detector signal interpretation in neutron radiation measurements in mixed neutron gamma radiation fields, which is called the detector inverse problem or the spectrum unfolding, and it aims in finding a representation of the primary radiation, based on the measured detector signals. In our novel methodology we resort to Bayesian inference approach. In the developed procedure the resultant spectra is unfolded form detector channels reading, where the estimated neutron fluence in a group structure is obtained with its statistical characteristic comprising of standard deviation and correlation matrix. In the paper we present results of unfolding process for case of D-T neutron source in neutron moderating environment. Discussions of statistical properties of obtained results are presented as well as of the physical meaning of obtained correlation matrix of estimated group fluence. The presented works has been carried out within the I-SMART project, which is part of the KIC InnoEnergy R and D program. (authors)
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.
All-Atom Molecular Dynamics Simulation of Protein Translocation through an α-Hemolysin Nanopore
Di Marino, Daniele
2015-08-06
© 2015 American Chemical Society. Nanopore sensing is attracting the attention of a large and varied scientific community. One of the main issues in nanopore sensing is how to associate the measured current signals to specific features of the molecule under investigation. This is particularly relevant when the translocating molecule is a protein and the pore is sufficiently narrow to necessarily involve unfolding of the translocating protein. Recent experimental results characterized the cotranslocational unfolding of Thioredoxin (Trx) passing through an α-hemolisin pore, providing evidence for the existence of a multistep process. In this study we report the results of all-atom molecular dynamics simulations of the same system. Our data indicate that Trx translocation involves two main barriers. The first one is an unfolding barrier associated with a translocation intermediate where the N-terminal region of Trx is stuck at the pore entrance in a conformation that strongly resembles the native one. After the abrupt unfolding of the N-terminal region, the Trx enters the α-hemolisin vestibule. During this stage, the constriction is occupied not only by the translocating residue but also by a hairpin-like structure forming a tangle in the constriction. The second barrier is associated with the disentangling of this region.
All-Atom Molecular Dynamics Simulation of Protein Translocation through an α-Hemolysin Nanopore
Di Marino, Daniele; Bonome, Emma Letizia; Tramontano, Anna; Chinappi, Mauro
2015-01-01
© 2015 American Chemical Society. Nanopore sensing is attracting the attention of a large and varied scientific community. One of the main issues in nanopore sensing is how to associate the measured current signals to specific features of the molecule under investigation. This is particularly relevant when the translocating molecule is a protein and the pore is sufficiently narrow to necessarily involve unfolding of the translocating protein. Recent experimental results characterized the cotranslocational unfolding of Thioredoxin (Trx) passing through an α-hemolisin pore, providing evidence for the existence of a multistep process. In this study we report the results of all-atom molecular dynamics simulations of the same system. Our data indicate that Trx translocation involves two main barriers. The first one is an unfolding barrier associated with a translocation intermediate where the N-terminal region of Trx is stuck at the pore entrance in a conformation that strongly resembles the native one. After the abrupt unfolding of the N-terminal region, the Trx enters the α-hemolisin vestibule. During this stage, the constriction is occupied not only by the translocating residue but also by a hairpin-like structure forming a tangle in the constriction. The second barrier is associated with the disentangling of this region.
RDANN a new methodology to solve the neutron spectra unfolding problem
Energy Technology Data Exchange (ETDEWEB)
Ortiz R, J.M.; Martinez B, M.R.; Vega C, H.R. [UAZ, Av. Ramon Lopez Velarde No. 801, 98000 Zacatecas (Mexico)
2006-07-01
The optimization processes known as Taguchi method and DOE methodology are applied to the design, training and testing of Artificial Neural Networks in the neutron spectrometry field, which offer potential benefits in the evaluation of the behavior of the net as well as the ability to examine the interaction of the weights and neurons inside the same one. In this work, the Robust Design of Artificial Neural Networks methodology is used to solve the neutron spectra unfolding problem, designing, training and testing an ANN using a set of 187 neutron spectra compiled by the International Atomic Energy Agency, to obtain the better neutron spectra unfolded from the Bonner spheres spectrometer's count rates. (Author)
RDANN a new methodology to solve the neutron spectra unfolding problem
International Nuclear Information System (INIS)
Ortiz R, J.M.; Martinez B, M.R.; Vega C, H.R.
2006-01-01
The optimization processes known as Taguchi method and DOE methodology are applied to the design, training and testing of Artificial Neural Networks in the neutron spectrometry field, which offer potential benefits in the evaluation of the behavior of the net as well as the ability to examine the interaction of the weights and neurons inside the same one. In this work, the Robust Design of Artificial Neural Networks methodology is used to solve the neutron spectra unfolding problem, designing, training and testing an ANN using a set of 187 neutron spectra compiled by the International Atomic Energy Agency, to obtain the better neutron spectra unfolded from the Bonner spheres spectrometer's count rates. (Author)
Li, Xianchan; Zheng, Wei; Zhang, Limin; Yu, Ping; Lin, Yuqing; Su, Lei; Mao, Lanqun
2009-10-15
This study demonstrates a facile and effective electrochemical method for investigation of hemoglobin (Hb) unfolding based on the electrochemical redox property of heme groups in Hb at bare glassy carbon (GC) electrodes. In the native state, the heme groups are deeply buried in the hydrophobic pockets of Hb with a five-coordinate high-spin complex and thus show a poor electrochemical property at bare GC electrodes. Upon the unfolding of Hb induced by the denaturant of guanidine hydrochloride (GdnHCl), the fifth coordinative bond between the heme groups and the residue of the polypeptides (His-F8) is broken, and as a result, the heme groups initially buried deeply in the hydrophobic pockets dissociate from the polypeptide chains and are reduced electrochemically at GC electrodes, which can be used to probe the unfolding of Hb. The results on the GdnHCl-induced Hb unfolding obtained with the electrochemical method described here well coincide with those studied with other methods, such as UV-vis spectroscopy, fluorescence, and circular dichroism. The application of the as-established electrochemical method is illustrated to study the kinetics of GdnHCl-induced Hb unfolding, the GdnHCl-induced unfolding of another kind of hemoprotein, catalase, and the pH-induced Hb unfolding/refolding.
Directory of Open Access Journals (Sweden)
Jose J. Virgen-Ortíz
2017-01-01
Full Text Available Lipases from Candida antarctica (isoform B and Rhizomucor miehei (CALB and RML have been immobilized on octyl-agarose (OC and further coated with polyethylenimine (PEI and dextran sulfate (DS. The enzymes just immobilized on OC supports could be easily released from the support using 2% SDS at pH 7, both intact or after thermal inactivation (in fact, after inactivation most enzyme molecules were already desorbed. The coating with PEI and DS greatly reduced the enzyme release during thermal inactivation and improved enzyme stability. However, using OC-CALB/RML-PEI-DS, the full release of the immobilized enzyme to reuse the support required more drastic conditions: a pH value of 3, a buffer concentration over 2 M, and temperatures above 45 °C. However, even these conditions were not able to fully release the thermally inactivated enzyme molecules from the support, being necessary to increase the buffer concentration to 4 M sodium phosphate and decrease the pH to 2.5. The formation of unfolded protein/polymers composites seems to be responsible for this strong interaction between the octyl and some anionic groups of OC supports. The support could be reused five cycles using these conditions with similar loading capacity of the support and stability of the immobilized enzyme.
Singh, Tanoj K; Øiseth, Sofia K; Lundin, Leif; Day, Li
2014-11-01
Protein intake is essential for growth and repair of body cells, the normal functioning of muscles, and health related immune functions. Most food proteins are consumed after undergoing various degrees of processing. Changes in protein structure and assembly as a result of processing impact the digestibility of proteins. Research in understanding to what extent the protein structure impacts the rate of proteolysis under human physiological conditions has gained considerable interest. In this work, four whey protein gels were prepared using heat processing at two different pH values, 6.8 and 4.6, with and without applied shear. The gels showed different protein network microstructures due to heat induced unfolding (at pH 6.8) or lack of unfolding, thus resulting in fine stranded protein networks. When shear was applied during heating, particulate protein networks were formed. The differences in the gel microstructures resulted in considerable differences in their rheological properties. An in vitro gastric and intestinal model was used to investigate the resulting effects of these different gel structures on whey protein digestion. In addition, the rate of digestion was monitored by taking samples at various time points throughout the in vitro digestion process. The peptides in the digesta were profiled using SDS-polyacrylamide gel electrophoresis, reversed-phase-HPLC and LC-MS. Under simulated gastric conditions, whey proteins in structured gels were hydrolysed faster than native proteins in solution. The rate of peptides released during in vitro digestion differed depending on the structure of the gels and extent of protein aggregation. The outcomes of this work highlighted that changes in the network structure of the protein can influence the rate and pattern of its proteolysis under gastrointestinal conditions. Such knowledge could assist the food industry in designing novel food formulations to control the digestion kinetics and the release of biologically
Equilibrium amide hydrogen exchange and protein folding kinetics
International Nuclear Information System (INIS)
Bai Yawen
1999-01-01
The classical Linderstrom-Lang hydrogen exchange (HX) model is extended to describe the relationship between the HX behaviors (EX1 and EX2) and protein folding kinetics for the amide protons that can only exchange by global unfolding in a three-state system including native (N), intermediate (I), and unfolded (U) states. For these slowly exchanging amide protons, it is shown that the existence of an intermediate (I) has no effect on the HX behavior in an off-pathway three-state system (I↔U↔N). On the other hand, in an on-pathway three-state system (U↔I↔N), the existence of a stable folding intermediate has profound effect on the HX behavior. It is shown that fast refolding from the unfolded state to the stable intermediate state alone does not guarantee EX2 behavior. The rate of refolding from the intermediate state to the native state also plays a crucial role in determining whether EX1 or EX2 behavior should occur. This is mainly due to the fact that only amide protons in the native state are observed in the hydrogen exchange experiment. These new concepts suggest that caution needs to be taken if one tries to derive the kinetic events of protein folding from equilibrium hydrogen exchange experiments
Unfolding intermediates of the mutant His-107-Tyr of human ...
Indian Academy of Sciences (India)
Srabani Taraphder
We present in this article a detailed analysis of representative structures and proton transfer activity of .... cal molecular dynamics simulations to identify potential unfolding ... clustering parameters to carry out K-means cluster- ing of different ...
Kim, Ki Hyung; Yun, Soi; Mok, K H; Lee, E K
2016-09-01
A fluorescent reporter, 8-anilino-1-naphthalene sulfonic acid (ANS), can serve as a reference molecule for conformational transition of a protein because its aromatic carbons have strong affinity with hydrophobic cores of partially unfolded molten globules. Using a typical calcium-binding protein, bovine α-lactalbumin (BLA), as a model protein, we compared the ANS binding thermodynamics to the decalcified (10 mM EDTA treated) apo-BLA at two representative temperatures: 20 and 40 °C. This is because the authentic molten globule is known to form more heavily at an elevated temperature such as 40 °C. Isothermal titration calorimetry experiments revealed that the BLA-ANS interactions at both temperatures were entropy-driven, and the dissociation constants were similar on the order of 10(-4) M, but there was a dramatic changeover in the binding thermodynamics from endothermic at 20 °C to exothermic at 40 °C. We believe that the higher subpopulation of authentic molten globules at 40 °C than 20 °C would be responsible for the results, which also indicate that weak binding is sufficient to alter the ANS binding mechanisms. We expect that the thermodynamic properties obtained from this study would serve as a useful reference for investigating the binding of other hydrophobic ligands such as oleic acid to apo-BLA, because oleic acid is known to have tumor-selective cytotoxicity when complexed with partially unfolded α-lactalbumin. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.
Neutron spectrum unfolding using genetic algorithm in a Monte Carlo simulation
Energy Technology Data Exchange (ETDEWEB)
Suman, Vitisha [Health Physics Division, Bhabha Atomic Research Centre, Mumbai 400085 (India); Sarkar, P.K., E-mail: pksarkar02@gmail.com [Manipal Centre for Natural Sciences, Manipal University, Manipal 576104 (India)
2014-02-11
A spectrum unfolding technique GAMCD (Genetic Algorithm and Monte Carlo based spectrum Deconvolution) has been developed using the genetic algorithm methodology within the framework of Monte Carlo simulations. Each Monte Carlo history starts with initial solution vectors (population) as randomly generated points in the hyper dimensional solution space that are related to the measured data by the response matrix of the detection system. The transition of the solution points in the solution space from one generation to another are governed by the genetic algorithm methodology using the techniques of cross-over (mating) and mutation in a probabilistic manner adding new solution points to the population. The population size is kept constant by discarding solutions having lesser fitness values (larger differences between measured and calculated results). Solutions having the highest fitness value at the end of each Monte Carlo history are averaged over all histories to obtain the final spectral solution. The present method shows promising results in neutron spectrum unfolding for both under-determined and over-determined problems with simulated test data as well as measured data when compared with some existing unfolding codes. An attractive advantage of the present method is the independence of the final spectra from the initial guess spectra.
Neutron spectrum unfolding using genetic algorithm in a Monte Carlo simulation
International Nuclear Information System (INIS)
Suman, Vitisha; Sarkar, P.K.
2014-01-01
A spectrum unfolding technique GAMCD (Genetic Algorithm and Monte Carlo based spectrum Deconvolution) has been developed using the genetic algorithm methodology within the framework of Monte Carlo simulations. Each Monte Carlo history starts with initial solution vectors (population) as randomly generated points in the hyper dimensional solution space that are related to the measured data by the response matrix of the detection system. The transition of the solution points in the solution space from one generation to another are governed by the genetic algorithm methodology using the techniques of cross-over (mating) and mutation in a probabilistic manner adding new solution points to the population. The population size is kept constant by discarding solutions having lesser fitness values (larger differences between measured and calculated results). Solutions having the highest fitness value at the end of each Monte Carlo history are averaged over all histories to obtain the final spectral solution. The present method shows promising results in neutron spectrum unfolding for both under-determined and over-determined problems with simulated test data as well as measured data when compared with some existing unfolding codes. An attractive advantage of the present method is the independence of the final spectra from the initial guess spectra
A method for unfolding high-energy scintillation gamma-ray spectra up to 8 MeV
International Nuclear Information System (INIS)
Dymke, N.; Hofmann, B.
1982-01-01
In unfolding a high-energy scintillation gamma-ray spectrum up to 8 MeV with the help of a response matrix, the means of linear algebra fail if the matrix is ill conditioned. In such cases, unfolding could be accomplished by means of a mathematical method based on a priori knowledge of the photon spectrum to be expected. The method which belongs to the class of regularization techniques was tested on in-situ gamma-ray spectra of 16 N recorded in a nuclear power plant near the primary circuit, using an 1.5 x 1.5 in. NaI(Tl) scintillation detector. For one regularized unfolding the results were presented in the form of an energy and a dose-rate spectrum. (author)
Molecular dynamics of surfactant protein C
DEFF Research Database (Denmark)
Ramírez, Eunice; Santana, Alberto; Cruz, Anthony
2006-01-01
Surfactant protein C (SP-C) is a membrane-associated protein essential for normal respiration. It has been found that the alpha-helix form of SP-C can undergo, under certain conditions, a transformation from an alpha-helix to a beta-strand conformation that closely resembles amyloid fibrils, which...... are possible contributors to the pathogenesis of pulmonary alveolar proteinosis. Molecular dynamics simulations using the NAMD2 package were performed for systems containing from one to seven SP-C molecules to study their behavior in water. The results of our simulations show that unfolding of the protein...
Dynamic prestress in a globular protein.
Directory of Open Access Journals (Sweden)
Scott A Edwards
Full Text Available A protein at equilibrium is commonly thought of as a fully relaxed structure, with the intra-molecular interactions showing fluctuations around their energy minimum. In contrast, here we find direct evidence for a protein as a molecular tensegrity structure, comprising a balance of tensed and compressed interactions, a concept that has been put forward for macroscopic structures. We quantified the distribution of inter-residue prestress in ubiquitin and immunoglobulin from all-atom molecular dynamics simulations. The network of highly fluctuating yet significant inter-residue forces in proteins is a consequence of the intrinsic frustration of a protein when sampling its rugged energy landscape. In beta sheets, this balance of forces is found to compress the intra-strand hydrogen bonds. We estimate that the observed magnitude of this pre-compression is enough to induce significant changes in the hydrogen bond lifetimes; thus, prestress, which can be as high as a few 100 pN, can be considered a key factor in determining the unfolding kinetics and pathway of proteins under force. Strong pre-tension in certain salt bridges on the other hand is connected to the thermodynamic stability of ubiquitin. Effective force profiles between some side-chains reveal the signature of multiple, distinct conformational states, and such static disorder could be one factor explaining the growing body of experiments revealing non-exponential unfolding kinetics of proteins. The design of prestress distributions in engineering proteins promises to be a new tool for tailoring the mechanical properties of made-to-order nanomaterials.
International Nuclear Information System (INIS)
Sugibayashi, Keiichi; Abe, Yoshiteru; Suga, Yutaka
1996-01-01
To improve the quantification of the left ventricular surface area (LVSA) by unfolded map method, we evaluated the cut-off value for determining the left ventricular border. The LVSA measured by unfolded map was compared with those measured using myocardial phantom and autopsy findings. The relative error (RE) was calculated as difference between LVSA in phantom and area of unfolded map. In phantom study, the cut-off value was calculated as 73.3±0.5% when the RE was zero. In autopsy study, the cut-off value was 74.0±7.2%. The area of unfolded map had good correlation with LVSA at autopsy when the cut-off value was 74% (r=0.83, p<0.003). The diameter of left ventricle at autopsy was compared with that of beating heart obtained by two-dimensional echocardiography, because the area of unfolded map was greater than LVSA at autopsy. The ratio of LVSA at autopsy to beating heart was calculated as 1.37. The suitable cut-off value was evaluated as 55.6% when the unfolded map area obtained by autopsy was increased 1.37 magnifications. There was a good correlation between LVSA of unfolded map (cut-off=56%) and the LVSA at autopsy (r=0.90, p<0.001). These results suggest that the cut-off value for determining the left ventricular border in vivo is 56%. (author)
Zhang, Yi; Berghaus, Melanie; Klein, Sean; Jenkins, Kelly; Zhang, Siwen; McCallum, Scott A; Morgan, Joel E; Winter, Roland; Barrick, Doug; Royer, Catherine A
2018-04-27
Many repeat proteins contain capping motifs, which serve to shield the hydrophobic core from solvent and maintain structural integrity. While the role of capping motifs in enhancing the stability and structural integrity of repeat proteins is well documented, their contribution to folding cooperativity is not. Here we examined the role of capping motifs in defining the folding cooperativity of the leucine-rich repeat protein, pp32, by monitoring the pressure- and urea-induced unfolding of an N-terminal capping motif (N-cap) deletion mutant, pp32-∆N-cap, and a C-terminal capping motif destabilization mutant pp32-Y131F/D146L, using residue-specific NMR and small-angle X-ray scattering. Destabilization of the C-terminal capping motif resulted in higher cooperativity for the unfolding transition compared to wild-type pp32, as these mutations render the stability of the C-terminus similar to that of the rest of the protein. In contrast, deletion of the N-cap led to strong deviation from two-state unfolding. In both urea- and pressure-induced unfolding, residues in repeats 1-3 of pp32-ΔN-cap lost their native structure first, while the C-terminal half was more stable. The residue-specific free energy changes in all regions of pp32-ΔN-cap were larger in urea compared to high pressure, indicating a less cooperative destabilization by pressure. Moreover, in contrast to complete structural disruption of pp32-ΔN-cap at high urea concentration, its pressure unfolded state remained compact. The contrasting effects of the capping motifs on folding cooperativity arise from the differential local stabilities of pp32, whereas the contrasting effects of pressure and urea on the pp32-ΔN-cap variant arise from their distinct mechanisms of action. Copyright © 2018 Elsevier Ltd. All rights reserved.
Directional Unfolded Source Term (DUST) for Compton Cameras.
Energy Technology Data Exchange (ETDEWEB)
Mitchell, Dean J.; Mitchell, Dean J.; Horne, Steven M.; O' Brien, Sean; Thoreson, Gregory G
2018-03-01
A Directional Unfolded Source Term (DUST) algorithm was developed to enable improved spectral analysis capabilities using data collected by Compton cameras. Achieving this objective required modification of the detector response function in the Gamma Detector Response and Analysis Software (GADRAS). Experimental data that were collected in support of this work include measurements of calibration sources at a range of separation distances and cylindrical depleted uranium castings.
Geometrically engineering the standard model: Locally unfolding three families out of E8
International Nuclear Information System (INIS)
Bourjaily, Jacob L.
2007-01-01
This paper extends and builds upon the results of [J. L. Bourjaily, arXiv:0704.0444.], in which we described how to use the tools of geometrical engineering to deform geometrically engineered grand unified models into ones with lower symmetry. This top-down unfolding has the advantage that the relative positions of singularities giving rise to the many 'low-energy' matter fields are related by only a few parameters which deform the geometry of the unified model. And because the relative positions of singularities are necessary to compute the superpotential, for example, this is a framework in which the arbitrariness of geometrically engineered models can be greatly reduced. In [J. L. Bourjaily, arXiv:0704.0444.], this picture was made concrete for the case of deforming the representations of an SU 5 model into their standard model content. In this paper we continue that discussion to show how a geometrically engineered 16 of SO 10 can be unfolded into the standard model, and how the three families of the standard model uniquely emerge from the unfolding of a single, isolated E 8 singularity
Protein oxidation and peroxidation
DEFF Research Database (Denmark)
Davies, Michael Jonathan
2016-01-01
Proteins are major targets for radicals and two-electron oxidants in biological systems due to their abundance and high rate constants for reaction. With highly reactive radicals damage occurs at multiple side-chain and backbone sites. Less reactive species show greater selectivity with regard...... to the residues targeted and their spatial location. Modification can result in increased side-chain hydrophilicity, side-chain and backbone fragmentation, aggregation via covalent cross-linking or hydrophobic interactions, protein unfolding and altered conformation, altered interactions with biological partners...... and modified turnover. In the presence of O2, high yields of peroxyl radicals and peroxides (protein peroxidation) are formed; the latter account for up to 70% of the initial oxidant flux. Protein peroxides can oxidize both proteins and other targets. One-electron reduction results in additional radicals...
Protein folding and translocation : single-molecule investigations
Leeuwen, Rudolphus Gerardus Henricus van
2006-01-01
This thesis describes experiments, in which we used an optical-tweezers setup to study a number of biological systems. We studied the interaction between the E. coli molecular chaperone SecB and a protein that was being unfolded and refolded using our optical tweezers setup. Our measurements clearly
AMPylation matches BiP activity to client protein load in the endoplasmic reticulum.
Preissler, Steffen; Rato, Cláudia; Chen, Ruming; Antrobus, Robin; Ding, Shujing; Fearnley, Ian M; Ron, David
2015-12-17
The endoplasmic reticulum (ER)-localized Hsp70 chaperone BiP affects protein folding homeostasis and the response to ER stress. Reversible inactivating covalent modification of BiP is believed to contribute to the balance between chaperones and unfolded ER proteins, but the nature of this modification has so far been hinted at indirectly. We report that deletion of FICD, a gene encoding an ER-localized AMPylating enzyme, abolished detectable modification of endogenous BiP enhancing ER buffering of unfolded protein stress in mammalian cells, whilst deregulated FICD activity had the opposite effect. In vitro, FICD AMPylated BiP to completion on a single residue, Thr(518). AMPylation increased, in a strictly FICD-dependent manner, as the flux of proteins entering the ER was attenuated in vivo. In vitro, Thr(518) AMPylation enhanced peptide dissociation from BiP 6-fold and abolished stimulation of ATP hydrolysis by J-domain cofactor. These findings expose the molecular basis for covalent inactivation of BiP.
Energy Technology Data Exchange (ETDEWEB)
Schollmeier, M.; Geissel, M.; Sefkow, A. B. [Sandia National Laboratories, Albuquerque, New Mexico 87185 (United States); Flippo, K. A. [Los Alamos National Laboratory, Los Alamos, New Mexico 87545 (United States)
2014-04-15
An improved method to unfold the space-resolved proton energy distribution function of laser-accelerated proton beams using a layered, radiochromic film (RCF) detector stack has been developed. The method takes into account the reduced RCF response near the Bragg peak due to a high linear energy transfer (LET). This LET dependence of the active RCF layer has been measured, and published data have been re-interpreted to find a nonlinear saturation scaling of the RCF response with stopping power. Accounting for the LET effect increased the integrated particle yield by 25% after data unfolding. An iterative, analytical, space-resolved deconvolution of the RCF response functions from the measured dose was developed that does not rely on fitting. After the particle number unfold, three-dimensional interpolation is performed to determine the spatial proton beam distribution for proton energies in-between the RCF data points. Here, image morphing has been implemented as a novel interpolation method that takes into account the energy-dependent, changing beam topology.
The cardiokine story unfolds: ischemic stress-induced protein secretion in the heart.
Doroudgar, Shirin; Glembotski, Christopher C
2011-04-01
Intercellular communication depends on many factors, including proteins released via the classical or non-classical secretory pathways, many of which must be properly folded to be functional. Owing to their adverse effects on the secretion machinery, stresses such as ischemia can impair the folding of secreted proteins. Paradoxically, cells rely on secreted proteins to mount a response designed to resist stress-induced damage. This review examines this paradox using proteins secreted from the heart, cardiokines, as examples, and focuses on how the ischemic heart maintains or even increases the release of select cardiokines that regulate important cellular processes in the heart, including excitation-contraction coupling, hypertrophic growth, myocardial remodeling and stem cell function, in ways that moderate ischemic damage and enhance cardiac repair. Copyright © 2010 Elsevier Ltd. All rights reserved.
Directory of Open Access Journals (Sweden)
Qiangqiang Wang
2016-01-01
Full Text Available Since excessive reactive oxygen species (ROS is known to be associated with aging and age-related diseases, strategies modulating ROS level and antioxidant defense systems may contribute to the delay of senescence. Here we show that the protein hydrolyzate from Angelica sinensis was capable of increasing oxidative survival of the model animal Caenorhabditis elegans intoxicated by paraquat. The hydrolyzate was then fractionated by ultrafiltration, and the antioxidant fraction (<3 kDa was purified by gel filtration to obtain the antioxidant A. sinensis peptides (AsiPeps, which were mostly composed of peptides with <20 amino acid residues. Further studies demonstrate that AsiPeps were able to reduce the endogenous ROS level, increase the activities of the antioxidant enzymes superoxide dismutase and catalase, and decrease the content of the lipid peroxidation product malondialdehyde in nematodes treated with paraquat or undergoing senescence. AsiPeps were also shown to reduce age pigments accumulation and extend lifespan but did not affect the food-intake behavior of the nematodes. Taken together, our results demonstrate that A. sinensis peptides (AsiPeps are able to delay aging process in C. elegans through antioxidant activities independent of dietary restriction.
Acosta-Sampson, Ligia; Döring, Kristina; Lin, Yuping; Yu, Vivian Y; Bukau, Bernd; Kramer, Günter; Cate, Jamie H D
2017-12-01
Targeting of most integral membrane proteins to the endoplasmic reticulum is controlled by the signal recognition particle, which recognizes a hydrophobic signal sequence near the protein N terminus. Proper folding of these proteins is monitored by the unfolded protein response and involves protein degradation pathways to ensure quality control. Here, we identify a new pathway for quality control of major facilitator superfamily transporters that occurs before the first transmembrane helix, the signal sequence recognized by the signal recognition particle, is made by the ribosome. Increased rates of translation elongation of the N-terminal sequence of these integral membrane proteins can divert the nascent protein chains to the ribosome-associated complex and stress-seventy subfamily B chaperones. We also show that quality control of integral membrane proteins by ribosome-associated complex-stress-seventy subfamily B couples translation rate to the unfolded protein response, which has implications for understanding mechanisms underlying human disease and protein production in biotechnology. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Nonintegrability of the unfolding of the fold-Hopf bifurcation
Yagasaki, Kazuyuki
2018-02-01
We consider the unfolding of the codimension-two fold-Hopf bifurcation and prove its meromorphic nonintegrability in the meaning of Bogoyavlenskij for almost all parameter values. Our proof is based on a generalized version of the Morales-Ramis-Simó theory for non-Hamiltonian systems and related variational equations up to second order are used.
Descriptive and Computer Aided Drawing Perspective on an Unfolded Polyhedral Projection Surface
Dzwierzynska, Jolanta
2017-10-01
The aim of the herby study is to develop a method of direct and practical mapping of perspective on an unfolded prism polyhedral projection surface. The considered perspective representation is a rectilinear central projection onto a surface composed of several flat elements. In the paper two descriptive methods of drawing perspective are presented: direct and indirect. The graphical mapping of the effects of the representation is realized directly on the unfolded flat projection surface. That is due to the projective and graphical connection between points displayed on the polyhedral background and their counterparts received on the unfolded flat surface. For a significant improvement of the construction of line, analytical algorithms are formulated. They draw a perspective image of a segment of line passing through two different points determined by their coordinates in a spatial coordinate system of axis x, y, z. Compared to other perspective construction methods that use information about points, for computer vision and the computer aided design, our algorithms utilize data about lines, which are applied very often in architectural forms. Possibility of drawing lines in the considered perspective enables drawing an edge perspective image of an architectural object. The application of the changeable base elements of perspective as a horizon height and a station point location enable drawing perspective image from different viewing positions. The analytical algorithms for drawing perspective images are formulated in Mathcad software, however, they can be implemented in the majority of computer graphical packages, which can make drawing perspective more efficient and easier. The representation presented in the paper and the way of its direct mapping on the flat unfolded projection surface can find application in presentation of architectural space in advertisement and art.
Unfolding and smoothing applied to the quality enhancement of neutron tomographic images
International Nuclear Information System (INIS)
Almeida, Gevaldo L. de; Silvani, Maria I.; Lopes, Ricardo T.
2008-01-01
Resolution and contrast are the major parameters defining the quality of a computer-aided tomographic image. These parameters depend upon several features of the image acquisition system, such as detector resolution, geometrical arrangement of the source-object-detector, beam divergence, source strength, detector efficiency and counting time. Roughly, the detector finite resolution is the main source of systematic errors affecting the separation power of the image acquisition system, while the electronic noise and statistical fluctuation are responsible for the data dispersion, which spoils the contrast. An algorithm has been developed in this work aiming at the improvement of the image quality through the minimization of both types of errors. The systematic ones are reduced by a mathematical unfolding of the position spectra - used as projections to reconstruct the 2D-images - using the Line Spread Function - LSF of the neutron tomographic system. The principle behind this technique is that every single channel contains information about all channels of the spectrum, but it is concealed due to the automatic integration carried out by the detector. Therefore, knowing the shape of this curve, it is possible to retrieve the original spectra. These spectra are unfortunately corrupted by the unavoidable statistical fluctuation, and by oscillations arising from the unfolding process, which strongly affects the quality of the final unfolded image. In order to reduce this impact, the spectra have been filtered by a Fourier transform technique or smoothed with a least square fitting procedure. The algorithm has been applied to spectra of some test-bodies generated by an earlier developed tomographic simulator, which reproduces the spectra furnished by a thermal neutron tomographic system employing a position sensitive detector. The obtained results have shown that the unfolded spectra produce final images capable to resolve features otherwise not achievable with the
Colin, Didier J; Hain, Karolina O; Allan, Lindsey A; Clarke, Paul R
2015-03-01
Anti-cancer drugs that disrupt mitosis inhibit cell proliferation and induce apoptosis, although the mechanisms of these responses are poorly understood. Here, we characterize a mitotic stress response that determines cell fate in response to microtubule poisons. We show that mitotic arrest induced by these drugs produces a temporally controlled DNA damage response (DDR) characterized by the caspase-dependent formation of γH2AX foci in non-apoptotic cells. Following exit from a delayed mitosis, this initial response results in activation of DDR protein kinases, phosphorylation of the tumour suppressor p53 and a delay in subsequent cell cycle progression. We show that this response is controlled by Mcl-1, a regulator of caspase activation that becomes degraded during mitotic arrest. Chemical inhibition of Mcl-1 and the related proteins Bcl-2 and Bcl-xL by a BH3 mimetic enhances the mitotic DDR, promotes p53 activation and inhibits subsequent cell cycle progression. We also show that inhibitors of DDR protein kinases as well as BH3 mimetics promote apoptosis synergistically with taxol (paclitaxel) in a variety of cancer cell lines. Our work demonstrates the role of mitotic DNA damage responses in determining cell fate in response to microtubule poisons and BH3 mimetics, providing a rationale for anti-cancer combination chemotherapies.
Energy landscape, structure and rate effects on strength properties of alpha-helical proteins
International Nuclear Information System (INIS)
Bertaud, Jeremie; Hester, Joshua; Jimenez, Daniel D; Buehler, Markus J
2010-01-01
The strength of protein domains is crucial to identify the mechanical role of protein domains in biological processes such as mechanotransduction, tissue mechanics and tissue remodeling. Whereas the concept of strength has been widely investigated for engineered materials, the strength of fundamental protein material building blocks and how it depends on structural parameters such as the chemical bonding, the protein filament length and the timescale of observation or deformation velocity remains poorly understood. Here we report a systematic analysis of the influence of key parameters that define the energy landscape of the strength properties of alpha-helical protein domains, including energy barriers, unfolding and refolding distances, the locations of folded and unfolded states, as well as variations of the length and pulling velocity of alpha-helical protein filaments. The analysis is facilitated by the development of a double-well mesoscale potential formulation, utilized here to carry out a systematic numerical analysis of the behavior of alpha-helices. We compare the results against widely used protein strength models based on the Bell model, one of the simplest models used to characterize the strength of protein filaments. We find that, whereas Bell-type models are a reasonable approximation to describe the rupture of alpha-helical protein domains for a certain range of pulling speeds and values of energy barriers, the model ceases to hold for very large energy barriers and for very small pulling speeds, in agreement with earlier findings. We conclude with an application of our mesoscale model to investigate the effect of the length of alpha-helices on their mechanical strength. We find a weakening effect as the length of alpha-helical proteins increases, followed by an asymptotic regime in which the strength remains constant. We compare strand lengths found in biological proteins with the scaling law of strength versus alpha-helix filament length. The
Liyanage, Rohana; Devarapalli, Nagarjuna; Pyland, Derek B; Puckett, Latisha M; Phan, N H; Starch, Joel A; Okimoto, Mark R; Gidden, Jennifer; Stites, Wesley E; Lay, Jackson O
2012-12-15
Protein equilibrium snapshot by hydrogen/deuterium exchange electrospray ionization mass spectrometry (PEPS-HDX-ESI-MS or PEPS) is a method recently introduced for estimating protein folding energies and rates. Herein we describe the basis for this method using both theory and new experiments. Benchmark experiments were conducted using ubiquitin because of the availability of reference data for folding and unfolding rates from NMR studies. A second set of experiments was also conducted to illustrate the surprising resilience of the PEPS to changes in HDX time, using staphylococcal nuclease and time frames ranging from a few seconds to several minutes. Theory suggests that PEPS experiments should be conducted at relatively high denaturant concentrations, where the protein folding/unfolding rates are slow with respect to HDX and the life times of both the closed and open states are long enough to be sampled experimentally. Upon deliberate denaturation, changes in folding/unfolding are correlated with associated changes in the ESI-MS signal upon fast HDX. When experiments are done quickly, typically within a few seconds, ESI-MS signals, corresponding to the equilibrium population of the native (closed) and denatured (open) states can both be detected. The interior of folded proteins remains largely un-exchanged. Amongst MS methods, the simultaneous detection of both states in the spectrum is unique to PEPS and provides a "snapshot" of these populations. The associated ion intensities are used to estimate the protein folding equilibrium constant (or the free energy change, ΔG). Linear extrapolation method (LEM) plots of derived ΔG values for each denaturant concentration can then be used to calculate ΔG in the absence of denaturant, ΔG(H(2)O). In accordance with the requirement for detection of signals for both the folded and unfolded states, this theoretical framework predicts that PEPS experiments work best at the middle of the denaturation curve where natured
An Auto sequence Code to Integrate a Neutron Unfolding Code with thePC-MCA Accuspec
International Nuclear Information System (INIS)
Darsono
2000-01-01
In a neutron spectrometry using proton recoil method, the neutronunfolding code is needed to unfold the measured proton spectrum to become theneutron spectrum. The process of the unfolding neutron in the existingneutron spectrometry which was successfully installed last year was doneseparately. This manuscript reports that the auto sequence code to integratethe neutron unfolding code UNFSPEC.EXE with the software facility of thePC-MCA Accuspec has been made and run successfully so that the new neutronspectrometry become compact. The auto sequence code was written based on therules in application program facility of PC-MCA Accuspec and then it wascompiled using AC-EXE. Result of the test of the auto sequence code showedthat for binning width 20, 30, and 40 giving a little different spectrumshape. The binning width around 30 gives a better spectrum in mean of givingsmall error compared to the others. (author)
Unfolding measurement of the atmospheric muon neutrino spectrum using IceCube
Energy Technology Data Exchange (ETDEWEB)
Boerner, Mathis; Ruhe, Tim; Meier, Maximilian; Schlunder, Philipp; Menne, Thorben; Fuchs, Tomasz [Dept. of Physics, Technical University of Dortmund, 44227 Dortmund (Germany); Collaboration: IceCube-Collaboration
2016-07-01
IceCube is a cubic kilometer neutrino observatory located at the geographic South Pole. With its huge volume, the detector is well suited for measurements of the atmospheric muon neutrino energy spectrum. Over the last years, several unfolding analyses for single years were able to provide model independent measurements for the northern hemisphere in an energy region between 200 GeV and 3.2 PeV. In this talk, the extension of the analyses to four additional years of data is presented. With this significant enlargement of the data basis, it is possible to reanalyze the full northern hemisphere with smaller statistical errors. Moreover, the spectrum can be unfolded in several small zenith bands. Measurements of the energy spectrum for different zenith regions provide further information on the composition and the shape of the flux.
Situated peer coaching and unfolding cases in the fundamentals skills laboratory.
Himes, Deborah O; Ravert, Patricia K
2012-09-03
Using unfolding case studies and situated peer coaching for the Fundamentals Skills Laboratory provides students with individualized feedback and creates a realistic clinical learning experience. A quasi-experimental design with pre- and post-intervention data was used to evaluate changes in student ratings of the course. An instrument was used to examine students' self-ratings and student comments about each lab. We found that students' ratings of the lab remained high with the new method and self-evaluations of their performance were higher as the semester progressed. Students appreciated the personalized feedback associated with peer coaching and demonstrated strong motivation and self-regulation in learning. By participating in unfolding case studies with situated peer coaching, students focus on safety issues, practice collaborative communication, and critical thinking in addition to performing psychomotor skills.
Folding and unfolding of large-size shell construction for application in Earth orbit
Kondyurin, Alexey; Pestrenina, Irena; Pestrenin, Valery; Rusakov, Sergey
2016-07-01
A future exploration of space requires a technology of large module for biological, technological, logistic and other applications in Earth orbits [1-3]. This report describes the possibility of using large-sized shell structures deployable in space. Structure is delivered to the orbit in the spaceship container. The shell is folded for the transportation. The shell material is either rigid plastic or multilayer prepreg comprising rigid reinforcements (such as reinforcing fibers). The unfolding process (bringing a construction to the unfolded state by loading the internal pressure) needs be considered at the presence of both stretching and bending deformations. An analysis of the deployment conditions (the minimum internal pressure bringing a construction from the folded state to the unfolded state) of large laminated CFRP shell structures is formulated in this report. Solution of this mechanics of deformable solids (MDS) problem of the shell structure is based on the following assumptions: the shell is made of components whose median surface has a reamer; in the separate structural element relaxed state (not stressed and not deformed) its median surface coincides with its reamer (this assumption allows choose the relaxed state of the structure correctly); structural elements are joined (sewn together) by a seam that does not resist rotation around the tangent to the seam line. The ways of large shell structures folding, whose median surface has a reamer, are suggested. Unfolding of cylindrical, conical (full and truncated cones), and large-size composite shells (cylinder-cones, cones-cones) is considered. These results show that the unfolding pressure of such large-size structures (0.01-0.2 atm.) is comparable to the deploying pressure of pneumatic parts (0.001-0.1 atm.) [3]. It would be possible to extend this approach to investigate the unfolding process of large-sized shells with ruled median surface or for non-developable surfaces. This research was
Some properties of circular proteins
International Nuclear Information System (INIS)
Prosselkov, P.; John, P.; Dixon, N.E.; Liepinsh, E.; Williams, N.K.; University of Sydney, NSW; Matthews, J.M.; Otting, G.; Karolinska Institutet, Stockholm,
2002-01-01
Full text: Protein backbone cyclization can be achieved by use of a circularly-permuted split mini-intein. We have used the small N-terminal domain of the E coli DnaB helicase (DnaB-N, residues 24-136) as a model protein for cyclization because its structure has been determined both by NMR spectroscopy and X-ray crystallography, and its ends are close together. Joining of the ends of DnaB-N' via a 9-amino acid linker occurs efficiently in vivo, and the circular (cz-) protein is stabilized in comparison to the linear (Hn-) protein against thermal denaturation (ΔΔG ∼2 kcal/mol). DnaB-N exists as a dimer in the crystalline state and in solution at high concentrations. To produce linear and cyclized versions that could not dimerize, Phe102 (at the dimer interface) was changed to Glu. NMR spectra showed that the F102E mutants remained monomeric at high concentrations but otherwise had essentially the same structures as the wild-type domains. Individual rate constants for proton exchange at the amide groups in lin- and cz-DnaB-N were determined at 10 C. Although they varied as expected depending on exposure to solvent, the ratios of rates between corresponding amides in the two proteins were constant. In the same buffer, lin- and cz-DnaB-N both unfolded reversibly, with transition temperatures of 37.9 and 48.5 deg C, respectively. Correlation of the (constant) ratio of amide exchange rates with measured thermodynamic parameters suggests that amide exchange in DnaB-N occurs predominantly in a globally unfolded state. Similar studies with other proteins are underway
Bennion, Brian J; Daggett, Valerie
2004-04-27
Proteins are very sensitive to their solvent environments. Urea is a common chemical denaturant of proteins, yet some animals contain high concentrations of urea. These animals have evolved an interesting mechanism to counteract the effects of urea by using trimethylamine N-oxide (TMAO). The molecular basis for the ability of TMAO to act as a chemical chaperone remains unknown. Here, we describe molecular dynamics simulations of a small globular protein, chymotrypsin inhibitor 2, in 8 M urea and 4 M TMAO/8 M urea solutions, in addition to other control simulations, to investigate this effect at the atomic level. In 8 M urea, the protein unfolds, and urea acts in both a direct and indirect manner to achieve this effect. In contrast, introduction of 4 M TMAO counteracts the effect of urea and the protein remains well structured. TMAO makes few direct interactions with the protein. Instead, it prevents unfolding of the protein by structuring the solvent. In particular, TMAO orders the solvent and discourages it from competing with intraprotein H bonds and breaking up the hydrophobic core of the protein.
Theoretical aspects of pressure and solute denaturation of proteins: A Kirkwood-buff-theory approach
Ben-Naim, Arieh
2012-12-01
A new approach to the problem of pressure-denaturation (PD) and solute-denaturation (SD) of proteins is presented. The problem is formulated in terms of Le Chatelier principle, and a solution is sought in terms of the Kirkwood-Buff theory of solutions. It is found that both problems have one factor in common; the excluded volumes of the folded and the unfolded forms with respect to the solvent molecules. It is shown that solvent-induced effects operating on hydrophilic groups along the protein are probably the main reason for PD. On the other hand, the SD depends on the preferential solvation of the folded and the unfolded forms with respect to solvent and co-solvent molecules.
The impact of urea-induced unfolding on the redox process of immobilised cytochrome c.
Monari, Stefano; Millo, Diego; Ranieri, Antonio; Di Rocco, Giulia; van der Zwan, Gert; Gooijer, Cees; Peressini, Silvia; Tavagnacco, Claudio; Hildebrandt, Peter; Borsari, Marco
2010-11-01
We have studied the effect of urea-induced unfolding on the electron transfer process of yeast iso-1-cytochrome c and its mutant K72AK73AK79A adsorbed on electrodes coated by mixed 11-mercapto-1-undecanoic acid/11-mercapto-1-undecanol self-assembled monolayers. Electrochemical measurements, complemented by surface enhanced resonance Raman studies, indicate two distinct states of the adsorbed proteins that mainly differ with respect to the ligation pattern of the haem. The native state, in which the haem is axially coordinated by Met80 and His18, displays a reduction potential that slightly shifts to negative values with increasing urea concentration. At urea concentrations higher than 6 M, a second state prevails in which the Met80 ligand is replaced by an additional histidine residue. This structural change in the haem pocket is associated with an approximately 0.4 V shift of the reduction potential to negative values. These two states were found for both the wild-type protein and the mutant in which lysine residues 72, 73 and 79 had been substituted by alanines. The analysis of the reduction potentials, the reaction enthalpies and entropies as well as the rate constants indicates that these three lysine residues have an important effect on stabilising the protein structure in the adsorbed state and facilitating the electron transfer dynamics.
Using fluorescence correlation spectroscopy to study conformational changes in denatured proteins.
Sherman, Eilon; Itkin, Anna; Kuttner, Yosef Yehuda; Rhoades, Elizabeth; Amir, Dan; Haas, Elisha; Haran, Gilad
2008-06-01
Fluorescence correlation spectroscopy (FCS) is a sensitive analytical tool that allows dynamics and hydrodynamics of biomolecules to be studied under a broad range of experimental conditions. One application of FCS of current interest is the determination of the size of protein molecules in the various states they sample along their folding reaction coordinate, which can be accessed through the measurement of diffusion coefficients. It has been pointed out that the analysis of FCS curves is prone to artifacts that may lead to erroneous size determination. To set the stage for FCS studies of unfolded proteins, we first show that the diffusion coefficients of small molecules as well as proteins can be determined accurately even in the presence of high concentrations of co-solutes that change the solution refractive index significantly. Indeed, it is found that the Stokes-Einstein relation between the measured diffusion coefficient and solution viscosity holds even in highly concentrated glycerol or guanidinium hydrochloride (GuHCl) solutions. These measurements form the basis for an investigation of the structure of the denatured state of two proteins, the small protein L and the larger, three-domain protein adenylate kinase (AK). FCS is found useful for probing expansion in the denatured state beyond the unfolding transition. It is shown that the denatured state of protein L expands as the denaturant concentration increases, in a process akin to the transition from a globule to a coil in polymers. This process continues at least up to 5 M GuHCl. On the other hand, the denatured state of AK does not seem to expand much beyond 2 M GuHCl, a result that is in qualitative accord with single-molecule fluorescence histograms. Because both the unfolding transition and the coil-globule transition of AK occur at a much lower denaturant concentration than those of protein L, a possible correlation between the two phenomena is suggested.
Membrane proteins bind lipids selectively to modulate their structure and function.
Laganowsky, Arthur; Reading, Eamonn; Allison, Timothy M; Ulmschneider, Martin B; Degiacomi, Matteo T; Baldwin, Andrew J; Robinson, Carol V
2014-06-05
Previous studies have established that the folding, structure and function of membrane proteins are influenced by their lipid environments and that lipids can bind to specific sites, for example, in potassium channels. Fundamental questions remain however regarding the extent of membrane protein selectivity towards lipids. Here we report a mass spectrometry approach designed to determine the selectivity of lipid binding to membrane protein complexes. We investigate the mechanosensitive channel of large conductance (MscL) from Mycobacterium tuberculosis and aquaporin Z (AqpZ) and the ammonia channel (AmtB) from Escherichia coli, using ion mobility mass spectrometry (IM-MS), which reports gas-phase collision cross-sections. We demonstrate that folded conformations of membrane protein complexes can exist in the gas phase. By resolving lipid-bound states, we then rank bound lipids on the basis of their ability to resist gas phase unfolding and thereby stabilize membrane protein structure. Lipids bind non-selectively and with high avidity to MscL, all imparting comparable stability; however, the highest-ranking lipid is phosphatidylinositol phosphate, in line with its proposed functional role in mechanosensation. AqpZ is also stabilized by many lipids, with cardiolipin imparting the most significant resistance to unfolding. Subsequently, through functional assays we show that cardiolipin modulates AqpZ function. Similar experiments identify AmtB as being highly selective for phosphatidylglycerol, prompting us to obtain an X-ray structure in this lipid membrane-like environment. The 2.3 Å resolution structure, when compared with others obtained without lipid bound, reveals distinct conformational changes that re-position AmtB residues to interact with the lipid bilayer. Our results demonstrate that resistance to unfolding correlates with specific lipid-binding events, enabling a distinction to be made between lipids that merely bind from those that modulate membrane
Directory of Open Access Journals (Sweden)
D. L. Fehl
2010-12-01
Full Text Available An algorithm for spectral reconstructions (unfolds and spectrally integrated flux estimates from data obtained by a five-channel, filtered x-ray-detector array (XRD is described in detail and characterized. This diagnostic is a broad-channel spectrometer, used primarily to measure time-dependent soft x-ray flux emitted by z-pinch plasmas at the Z pulsed-power accelerator (Sandia National Laboratories, Albuquerque, New Mexico, USA, and serves as both a plasma probe and a gauge of accelerator performance. The unfold method, suitable for online analysis, arises naturally from general assumptions about the x-ray source and spectral properties of the channel responses; a priori constraints control the ill-posed nature of the inversion. The unfolded spectrum is not assumed to be Planckian. This study is divided into two consecutive papers. This paper considers three major issues: (a Formulation of the unfold method.—The mathematical background, assumptions, and procedures leading to the algorithm are described: the spectral reconstruction S_{unfold}(E,t—five histogram x-ray bins j over the x-ray interval, 137≤E≤2300 eV at each time step t—depends on the shape and overlap of the calibrated channel responses and on the maximum electrical power delivered to the plasma. The x-ray flux F_{unfold} is estimated as ∫S_{unfold}(E,tdE. (b Validation with simulations.—Tests of the unfold algorithm with known static and time-varying spectra are described. These spectra included—but were not limited to—Planckian spectra S_{bb}(E,T (25≤T≤250 eV, from which noise-free channel data were simulated and unfolded. For Planckian simulations with 125≤T≤250 eV and typical responses, the binwise unfold values S_{j} and the corresponding binwise averages ⟨S_{bb}⟩_{j} agreed to ∼20%, except where S_{bb}≪max{S_{bb}}. Occasionally, unfold values S_{j}≲0 (artifacts were encountered. The algorithm recovered ≳90% of the x
Makabe, Koki; Nakamura, Takashi; Dhar, Debanjan; Ikura, Teikichi; Koide, Shohei; Kuwajima, Kunihiro
2018-04-27
Although many naturally occurring proteins consist of multiple domains, most studies on protein folding to date deal with single-domain proteins or isolated domains of multi-domain proteins. Studies of multi-domain protein folding are required for further advancing our understanding of protein folding mechanisms. Borrelia outer surface protein A (OspA) is a β-rich two-domain protein, in which two globular domains are connected by a rigid and stable single-layer β-sheet. Thus, OspA is particularly suited as a model system for studying the interplays of domains in protein folding. Here, we studied the equilibria and kinetics of the urea-induced folding-unfolding reactions of OspA probed with tryptophan fluorescence and ultraviolet circular dichroism. Global analysis of the experimental data revealed compelling lines of evidence for accumulation of an on-pathway intermediate during kinetic refolding and for the identity between the kinetic intermediate and a previously described equilibrium unfolding intermediate. The results suggest that the intermediate has the fully native structure in the N-terminal domain and the single layer β-sheet, with the C-terminal domain still unfolded. The observation of the productive on-pathway folding intermediate clearly indicates substantial interactions between the two domains mediated by the single-layer β-sheet. We propose that a rigid and stable intervening region between two domains creates an overlap between two folding units and can energetically couple their folding reactions. Copyright © 2018. Published by Elsevier Ltd.
Spectrum unfolding by the least-squares methods
International Nuclear Information System (INIS)
Perey, F.G.
1977-01-01
The method of least squares is briefly reviewed, and the conditions under which it may be used are stated. From this analysis, a least-squares approach to the solution of the dosimetry neutron spectrum unfolding problem is introduced. The mathematical solution to this least-squares problem is derived from the general solution. The existence of this solution is analyzed in some detail. A chi 2 -test is derived for the consistency of the input data which does not require the solution to be obtained first. The fact that the problem is technically nonlinear, but should be treated in general as a linear one, is argued. Therefore, the solution should not be obtained by iteration. Two interpretations are made for the solution of the code STAY'SL, which solves this least-squares problem. The relationship of the solution to this least-squares problem to those obtained currently by other methods of solving the dosimetry neutron spectrum unfolding problem is extensively discussed. It is shown that the least-squares method does not require more input information than would be needed by current methods in order to estimate the uncertainties in their solutions. From this discussion it is concluded that the proposed least-squares method does provide the best complete solution, with uncertainties, to the problem as it is understood now. Finally, some implications of this method are mentioned regarding future work required in order to exploit its potential fully
The unfolding effects of transfer functions and processing of the pulse height distributions
Directory of Open Access Journals (Sweden)
Avdić Senada
2010-01-01
Full Text Available This paper deals with the improvements of the linear artificial neural network unfolding approach aimed at accurately determining the incident neutron spectrum. The effects of the transfer functions and pre-processing of the simulated pulse height distributions from liquid scintillation detectors on the artificial neural networks performance have been studied. A better energy resolution and higher reliability of the linear artificial neural network technique have been achieved after implementation of the results of this study. The optimized structure of the network was used to unfold both monoenergetic and continuous neutron energy spectra, such as the spectra of 252Cf and 241Am-Be sources, traditionally used in the nuclear safeguards experiments. We have demonstrated that the artificial neural network energy resolution of 0.1 MeV is comparable with the one obtained by the reference maximum likelihood expectation-maximization method which was implemented by using the one step late algorithm. Although the maximum likelihood algorithm provides the unfolded results of higher accuracy, especially for continuous neutron sources, the artificial neural network approach with the improved performances is more suitable for fast and robust determination of the neutron spectra with sufficient accuracy.
Guanidinium chloride induction of partial unfolding in amide proton exchange in RNase A.
Mayo, S L; Baldwin, R L
1993-11-05
Amide (NH) proton exchange rates were measured in 0.0 to 0.7 M guanidinium chloride (GdmCl) for 23 slowly exchanging peptide NH protons of ribonuclease A (RNase A) at pH* 5.5 (uncorrected pH measured in D2O), 34 degrees C. The purpose was to find out whether GdmCl induces exchange through binding to exchange intermediates that are partly or wholly unfolded. It was predicted that, when the logarithm of the exchange rate is plotted as a function of the molarity of GdmCl, the slope should be a measure of the amount of buried surface area exposed to GdmCl in the exchange intermediate. The results indicate that these concentrations of GdmCl do induce exchange by means of a partial unfolding mechanism for all 23 protons; this implies that exchange reactions can be used to study the unfolding and stability of local regions. Of the 23 protons, nine also show a second mechanism of exchange at lower concentrations of GdmCl, a mechanism that is nearly independent of GdmCl concentration and is termed "limited structural fluctuation."
Energy Technology Data Exchange (ETDEWEB)
Hosseini, Seyed Abolfazl, E-mail: sahosseini@sharif.edu [Department of Energy Engineering, Sharif University of Technology, Tehran 8639-11365 (Iran, Islamic Republic of); Afrakoti, Iman Esmaili Paeen [Faculty of Engineering & Technology, University of Mazandaran, Pasdaran Street, P.O. Box: 416, Babolsar 47415 (Iran, Islamic Republic of)
2017-04-11
Accurate unfolding of the energy spectrum of a neutron source gives important information about unknown neutron sources. The obtained information is useful in many areas like nuclear safeguards, nuclear nonproliferation, and homeland security. In the present study, the energy spectrum of a poly-energetic fast neutron source is reconstructed using the developed computational codes based on the Group Method of Data Handling (GMDH) and Decision Tree (DT) algorithms. The neutron pulse height distribution (neutron response function) in the considered NE-213 liquid organic scintillator has been simulated using the developed MCNPX-ESUT computational code (MCNPX-Energy engineering of Sharif University of Technology). The developed computational codes based on the GMDH and DT algorithms use some data for training, testing and validation steps. In order to prepare the required data, 4000 randomly generated energy spectra distributed over 52 bins are used. The randomly generated energy spectra and the simulated neutron pulse height distributions by MCNPX-ESUT for each energy spectrum are used as the output and input data. Since there is no need to solve the inverse problem with an ill-conditioned response matrix, the unfolded energy spectrum has the highest accuracy. The {sup 241}Am-{sup 9}Be and {sup 252}Cf neutron sources are used in the validation step of the calculation. The unfolded energy spectra for the used fast neutron sources have an excellent agreement with the reference ones. Also, the accuracy of the unfolded energy spectra obtained using the GMDH is slightly better than those obtained from the DT. The results obtained in the present study have good accuracy in comparison with the previously published paper based on the logsig and tansig transfer functions. - Highlights: • The neutron pulse height distribution was simulated using MCNPX-ESUT. • The energy spectrum of the neutron source was unfolded using GMDH. • The energy spectrum of the neutron source was
Küberl, Andreas; Polen, Tino; Bott, Michael
2016-04-26
The balance of sufficient iron supply and avoidance of iron toxicity by iron homeostasis is a prerequisite for cellular metabolism and growth. Here we provide evidence that, in Actinobacteria, pupylation plays a crucial role in this process. Pupylation is a posttranslational modification in which the prokaryotic ubiquitin-like protein Pup is covalently attached to a lysine residue in target proteins, thus resembling ubiquitination in eukaryotes. Pupylated proteins are recognized and unfolded by a dedicated AAA+ ATPase (Mycobacterium proteasomal AAA+ ATPase; ATPase forming ring-shaped complexes). In Mycobacteria, degradation of pupylated proteins by the proteasome serves as a protection mechanism against several stress conditions. Other bacterial genera capable of pupylation such as Corynebacterium lack a proteasome, and the fate of pupylated proteins is unknown. We discovered that Corynebacterium glutamicum mutants lacking components of the pupylation machinery show a strong growth defect under iron limitation, which was caused by the absence of pupylation and unfolding of the iron storage protein ferritin. Genetic and biochemical data support a model in which the pupylation machinery is responsible for iron release from ferritin independent of degradation.
Redox Thermodynamics of Cytochromes c Subjected to Urea Induced Unfolding
Monari, S.; Ranieri, A.; Di Rocco, G.; van der Zwan, G.; Peressini, S.; Tavagnacco, C.; Millo, D.; Borsari, M.
2009-01-01
The thermodynamics of the electron transfer (ET) process for beef heart and yeast cytochromes c and the Lys72Ala/Lys73Ala/Lys79Ala mutant of the latter species subjected to progressive urea-induced unfolding was determined electrochemically. The results indicate the presence of at least three
CalFitter: a web server for analysis of protein thermal denaturation data.
Mazurenko, Stanislav; Stourac, Jan; Kunka, Antonin; Nedeljkovic, Sava; Bednar, David; Prokop, Zbynek; Damborsky, Jiri
2018-05-14
Despite significant advances in the understanding of protein structure-function relationships, revealing protein folding pathways still poses a challenge due to a limited number of relevant experimental tools. Widely-used experimental techniques, such as calorimetry or spectroscopy, critically depend on a proper data analysis. Currently, there are only separate data analysis tools available for each type of experiment with a limited model selection. To address this problem, we have developed the CalFitter web server to be a unified platform for comprehensive data fitting and analysis of protein thermal denaturation data. The server allows simultaneous global data fitting using any combination of input data types and offers 12 protein unfolding pathway models for selection, including irreversible transitions often missing from other tools. The data fitting produces optimal parameter values, their confidence intervals, and statistical information to define unfolding pathways. The server provides an interactive and easy-to-use interface that allows users to directly analyse input datasets and simulate modelled output based on the model parameters. CalFitter web server is available free at https://loschmidt.chemi.muni.cz/calfitter/.
A linear iterative unfolding method
International Nuclear Information System (INIS)
László, András
2012-01-01
A frequently faced task in experimental physics is to measure the probability distribution of some quantity. Often this quantity to be measured is smeared by a non-ideal detector response or by some physical process. The procedure of removing this smearing effect from the measured distribution is called unfolding, and is a delicate problem in signal processing, due to the well-known numerical ill behavior of this task. Various methods were invented which, given some assumptions on the initial probability distribution, try to regularize the unfolding problem. Most of these methods definitely introduce bias into the estimate of the initial probability distribution. We propose a linear iterative method (motivated by the Neumann series / Landweber iteration known in functional analysis), which has the advantage that no assumptions on the initial probability distribution is needed, and the only regularization parameter is the stopping order of the iteration, which can be used to choose the best compromise between the introduced bias and the propagated statistical and systematic errors. The method is consistent: 'binwise' convergence to the initial probability distribution is proved in absence of measurement errors under a quite general condition on the response function. This condition holds for practical applications such as convolutions, calorimeter response functions, momentum reconstruction response functions based on tracking in magnetic field etc. In presence of measurement errors, explicit formulae for the propagation of the three important error terms is provided: bias error (distance from the unknown to-be-reconstructed initial distribution at a finite iteration order), statistical error, and systematic error. A trade-off between these three error terms can be used to define an optimal iteration stopping criterion, and the errors can be estimated there. We provide a numerical C library for the implementation of the method, which incorporates automatic
Energy Technology Data Exchange (ETDEWEB)
Jeon, Jong Ho, E-mail: jhjeon07@ibs.re.kr; Nakajima, Kazuhisa, E-mail: naka115@dia-net.ne.jp; Pathak, Vishwa Bandhu; Cho, Myung Hoon; Yoo, Byung Ju; Shin, Kang Woo [Center for Relativistic Laser Science, Institute for Basic Science (IBS), Gwangju 500-712 (Korea, Republic of); Kim, Hyung Taek; Sung, Jae Hee; Lee, Seung Ku; Choi, Il Woo [Center for Relativistic Laser Science, Institute for Basic Science (IBS), Gwangju 500-712 (Korea, Republic of); Advanced Photonics Research Institute, GIST, Gwangju 500-712 (Korea, Republic of); Rhee, Yong Joo [Nuclear Data Center, Korea Atomic Energy Research Institute, Daejeon 305-353 (Korea, Republic of); Shin, Jung Hun; Jo, Sung Ha [Advanced Photonics Research Institute, GIST, Gwangju 500-712 (Korea, Republic of); Hojbota, Calin; Cho, Byeoung Ick; Nam, Chang Hee [Center for Relativistic Laser Science, Institute for Basic Science (IBS), Gwangju 500-712 (Korea, Republic of); Department of Physics and Photon Science, GIST, Gwangju 500-712 (Korea, Republic of)
2015-12-15
We present a high-flux, broadband gamma-ray spectrometry capable of characterizing the betatron radiation spectrum over the photon energy range from 10 keV to 20 MeV with respect to the peak photon energy, spectral bandwidth, and unique discrimination from background radiations, using a differential filtering spectrometer and the unfolding procedure based on the Monte Carlo code GEANT4. These properties are experimentally verified by measuring betatron radiation from a cm-scale laser wakefield accelerator (LWFA) driven by a 1-PW laser, using a differential filtering spectrometer consisting of a 15-filter and image plate stack. The gamma-ray spectra were derived by unfolding the photostimulated luminescence (PSL) values recorded on the image plates, using the spectrometer response matrix modeled with the Monte Carlo code GEANT4. The accuracy of unfolded betatron radiation spectra was assessed by unfolding the test PSL data simulated with GEANT4, showing an ambiguity of less than 20% and clear discrimination from the background radiation with less than 10%. The spectral analysis of betatron radiation from laser wakefield-accelerated electron beams with energies up to 3 GeV revealed radiation spectra characterized by synchrotron radiation with the critical photon energy up to 7 MeV. The gamma-ray spectrometer and unfolding method presented here facilitate an in-depth understanding of betatron radiation from LWFA process and a novel radiation source of high-quality photon beams in the MeV regime.
NEWSPEC: A computer code to unfold neutron spectra from Bonner sphere data
International Nuclear Information System (INIS)
Lemley, E.C.; West, L.
1996-01-01
A new computer code, NEWSPEC, is in development at the University of Arkansas. The NEWSPEC code allows a user to unfold, fold, rebin, display, and manipulate neutron spectra as applied to Bonner sphere measurements. The SPUNIT unfolding algorithm, a new rebinning algorithm, and the graphical capabilities of Microsoft (MS) Windows and MS Excel are utilized to perform these operations. The computer platform for NEWSPEC is a personal computer (PC) running MS Windows 3.x or Win95, while the code is written in MS Visual Basic (VB) and MS VB for Applications (VBA) under Excel. One of the most useful attributes of the NEWSPEC software is the link to Excel allowing additional manipulation of program output or creation of program input
DEFF Research Database (Denmark)
Teilum, Kaare; Poulsen, F. M.; Akke, M.
2006-01-01
those from stopped-flow kinetics and define an "inverted chevron" plot. The combination of NMR relaxation and stopped-flow kinetic measurements allowed determination of k f and k u in the range from 0.48 M GuHCl to 1.28 M GuHCl. Individually, the stopped-flow and NMR data fit two-state models...... for folding. However, although the values of k f determined by the two methods agree, the values of k u do not. As a result, a combined analysis of all data does not comply with a two-state model but indicates that an unfolding intermediate exists on the native side of the dominant energy barrier...
Protein folding and wring resonances
DEFF Research Database (Denmark)
Bohr, Jakob; Bohr, Henrik; Brunak, Søren
1997-01-01
The polypeptide chain of a protein is shown to obey topological contraints which enable long range excitations in the form of wring modes of the protein backbone. Wring modes of proteins of specific lengths can therefore resonate with molecular modes present in the cell. It is suggested that prot......The polypeptide chain of a protein is shown to obey topological contraints which enable long range excitations in the form of wring modes of the protein backbone. Wring modes of proteins of specific lengths can therefore resonate with molecular modes present in the cell. It is suggested...... that protein folding takes place when the amplitude of a wring excitation becomes so large that it is energetically favorable to bend the protein backbone. The condition under which such structural transformations can occur is found, and it is shown that both cold and hot denaturation (the unfolding...
Two for the Price of One: A Neuroprotective Chaperone Kit within NAD Synthase Protein NMNAT2.
Directory of Open Access Journals (Sweden)
Angela Lavado-Roldán
2016-07-01
Full Text Available One of the most fascinating properties of the brain is the ability to function smoothly across decades of a lifespan. Neurons are nondividing mature cells specialized in fast electrical and chemical communication at synapses. Often, neurons and synapses operate at high levels of activity through sophisticated arborizations of long axons and dendrites that nevertheless stay healthy throughout years. On the other hand, aging and activity-dependent stress strike onto the protein machineries turning proteins unfolded and prone to form pathological aggregates associated with neurodegeneration. How do neurons protect from those insults and remain healthy for their whole life? Ali and colleagues now present a molecular mechanism by which the enzyme nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2 acts not only as a NAD synthase involved in axonal maintenance but as a molecular chaperone helping neurons to overcome protein unfolding and protein aggregation.
Neff, H.; Laborde, H. M.; Lima, A. M. N.
2016-11-01
An oscillatory molecular adsorption pattern of the protein neutravidin from aqueous solution onto gold, in presence of a pre-deposited self assembled mono-molecular biotin film, is reported. Real time surface Plasmon resonance sensing was utilized for evaluation of the adsorption kinetics. Two different fractions were identified: in the initial phase, protein molecules attach irreversibly onto the Biotin ligands beneath towards the jamming limit, forming a neutravidin-biotin fraction. Afterwards, the growth rate exhibits distinct, albeit damped adsorption-desorption oscillations over an extended time span, assigned to a quasi reversibly bound fraction. These findings agree with, and firstly confirm a previously published model, proposing macro-molecular adsorption with time delay. The non-linear dynamic model is applicable to and also resembles non-damped oscillatory binding features of the hetero-catalytic oxidation of carbon monoxide molecules on platinum in the gas phase. An associated surface residence time can be linked to the dynamics and time scale required for self-organization.
Knoch, Fabian; Schäfer, Ken; Diezemann, Gregor; Speck, Thomas
2018-01-01
We present a dynamic coarse-graining technique that allows one to simulate the mechanical unfolding of biomolecules or molecular complexes on experimentally relevant time scales. It is based on Markov state models (MSMs), which we construct from molecular dynamics simulations using the pulling coordinate as an order parameter. We obtain a sequence of MSMs as a function of the discretized pulling coordinate, and the pulling process is modeled by switching among the MSMs according to the protocol applied to unfold the complex. This way we cover seven orders of magnitude in pulling speed. In the region of rapid pulling, we additionally perform steered molecular dynamics simulations and find excellent agreement between the results of the fully atomistic and the dynamically coarse-grained simulations. Our technique allows the determination of the rates of mechanical unfolding in a dynamical range from approximately 10-8/ns to 1/ns thus reaching experimentally accessible time regimes without abandoning atomistic resolution.
Effects of caffeine on cleavage delay of sea urchin eggs induced by ethidium bromide or puromycin
International Nuclear Information System (INIS)
Kimura, Hiroshi
1975-01-01
The mitotic delay was induced when sea urchin eggs were treated with either ethidium bromide or puromycin, as observed with eggs fertilized with x- or UV-irradiated sperm. Treatment of these eggs with caffeine during the period of early prophase block resulted in the reduction of the mitotic delay. Protein synthesis of these eggs was not affected by x-irradiation but inhibited by ethidium bromide or puromycin. Caffeine was almost ineffective in changing the protein synthesis of eggs inseminated with x-irradiated sperm or treated with ethidium bromide. These facts mean that additive synthesis of protein is not required for the reduction by caffeine of the mitotic delay. Some role of protein synthesis in the reduction by caffeine of the cleavage delay is not excluded for puromycin treated eggs, since caffeine counteracted the inhibitory effect of puromycin on protein synthesis. (author)
A neutron spectrum unfolding code based on iterative procedures
International Nuclear Information System (INIS)
Ortiz R, J. M.; Vega C, H. R.
2012-10-01
In this work, the version 3.0 of the neutron spectrum unfolding code called Neutron Spectrometry and Dosimetry from Universidad Autonoma de Zacatecas (NSDUAZ), is presented. This code was designed in a graphical interface under the LabVIEW programming environment and it is based on the iterative SPUNIT iterative algorithm, using as entrance data, only the rate counts obtained with 7 Bonner spheres based on a 6 Lil(Eu) neutron detector. The main features of the code are: it is intuitive and friendly to the user; it has a programming routine which automatically selects the initial guess spectrum by using a set of neutron spectra compiled by the International Atomic Energy Agency. Besides the neutron spectrum, this code calculates the total flux, the mean energy, H(10), h(10), 15 dosimetric quantities for radiation protection porpoises and 7 survey meter responses, in four energy grids, based on the International Atomic Energy Agency compilation. This code generates a full report in html format with all relevant information. In this work, the neutron spectrum of a 241 AmBe neutron source on air, located at 150 cm from detector, is unfolded. (Author)
Antibody-Unfolding and Metastable-State Binding in Force Spectroscopy and Recognition Imaging
Kaur, Parminder; Qiang-Fu; Fuhrmann, Alexander; Ros, Robert; Kutner, Linda Obenauer; Schneeweis, Lumelle A.; Navoa, Ryman; Steger, Kirby; Xie, Lei; Yonan, Christopher; Abraham, Ralph; Grace, Michael J.; Lindsay, Stuart
2011-01-01
Force spectroscopy and recognition imaging are important techniques for characterizing and mapping molecular interactions. In both cases, an antibody is pulled away from its target in times that are much less than the normal residence time of the antibody on its target. The distribution of pulling lengths in force spectroscopy shows the development of additional peaks at high loading rates, indicating that part of the antibody frequently unfolds. This propensity to unfold is reversible, indicating that exposure to high loading rates induces a structural transition to a metastable state. Weakened interactions of the antibody in this metastable state could account for reduced specificity in recognition imaging where the loading rates are always high. The much weaker interaction between the partially unfolded antibody and target, while still specific (as shown by control experiments), results in unbinding on millisecond timescales, giving rise to rapid switching noise in the recognition images. At the lower loading rates used in force spectroscopy, we still find discrepancies between the binding kinetics determined by force spectroscopy and those determined by surface plasmon resonance—possibly a consequence of the short tethers used in recognition imaging. Recognition imaging is nonetheless a powerful tool for interpreting complex atomic force microscopy images, so long as specificity is calibrated in situ, and not inferred from equilibrium binding kinetics. PMID:21190677
BiP clustering facilitates protein folding in the endoplasmic reticulum.
Directory of Open Access Journals (Sweden)
Marc Griesemer
2014-07-01
Full Text Available The chaperone BiP participates in several regulatory processes within the endoplasmic reticulum (ER: translocation, protein folding, and ER-associated degradation. To facilitate protein folding, a cooperative mechanism known as entropic pulling has been proposed to demonstrate the molecular-level understanding of how multiple BiP molecules bind to nascent and unfolded proteins. Recently, experimental evidence revealed the spatial heterogeneity of BiP within the nuclear and peripheral ER of S. cerevisiae (commonly referred to as 'clusters'. Here, we developed a model to evaluate the potential advantages of accounting for multiple BiP molecules binding to peptides, while proposing that BiP's spatial heterogeneity may enhance protein folding and maturation. Scenarios were simulated to gauge the effectiveness of binding multiple chaperone molecules to peptides. Using two metrics: folding efficiency and chaperone cost, we determined that the single binding site model achieves a higher efficiency than models characterized by multiple binding sites, in the absence of cooperativity. Due to entropic pulling, however, multiple chaperones perform in concert to facilitate the resolubilization and ultimate yield of folded proteins. As a result of cooperativity, multiple binding site models used fewer BiP molecules and maintained a higher folding efficiency than the single binding site model. These insilico investigations reveal that clusters of BiP molecules bound to unfolded proteins may enhance folding efficiency through cooperative action via entropic pulling.
Directory of Open Access Journals (Sweden)
Risako Kato
2018-03-01
Full Text Available General anesthetics decrease the frequency and density of spike firing. This effect makes it difficult to detect spike regularity. To overcome this problem, we developed a method utilizing the unfolding transformation which analyzes the energy level statistics in the random matrix theory. We regarded the energy axis as time axis of neuron spike and analyzed the time series of cortical neural firing in vivo. Unfolding transformation detected regularities of neural firing while changes in firing densities were associated with pentobarbital. We found that unfolding transformation enables us to compare firing regularity between awake and anesthetic conditions on a universal scale. Keywords: Unfolding transformation, Spike-timing, Regularity
Directory of Open Access Journals (Sweden)
Amit Kumar Singh Gautam
Full Text Available Eukaryotic 26S proteasomes are structurally organized to recognize, unfold and degrade globular proteins. However, all existing model substrates of the 26S proteasome in addition to ubiquitin or adaptor proteins require unstructured regions in the form of fusion tags for efficient degradation. We report for the first time that purified 26S proteasome can directly recognize and degrade apomyoglobin, a globular protein, in the absence of ubiquitin, extrinsic degradation tags or adaptor proteins. Despite a high affinity interaction, absence of a ligand and presence of only helices/loops that follow the degradation signal, apomyoglobin is degraded slowly by the proteasome. A short floppy F-helix exposed upon ligand removal and in conformational equilibrium with a disordered structure is mandatory for recognition and initiation of degradation. Holomyoglobin, in which the helix is buried, is neither recognized nor degraded. Exposure of the floppy F-helix seems to sensitize the proteasome and primes the substrate for degradation. Using peptide panning and competition experiments we speculate that initial encounters through the floppy helix and additional strong interactions with N-terminal helices anchors apomyoglobin to the proteasome. Stabilizing helical structure in the floppy F-helix slows down degradation. Destabilization of adjacent helices accelerates degradation. Unfolding seems to follow the mechanism of helix unraveling rather than global unfolding. Our findings while confirming the requirement for unstructured regions in degradation offers the following new insights: a origin and identification of an intrinsic degradation signal in the substrate, b identification of sequences in the native substrate that are likely to be responsible for direct interactions with the proteasome, and c identification of critical rate limiting steps like exposure of the intrinsic degron and destabilization of an unfolding intermediate that are presumably
Inclusive neutral current ep cross sections with HERA II and two-dimensional unfolding
International Nuclear Information System (INIS)
Fischer, David-Johannes
2011-06-01
In this thesis, the inclusive neutral current ep → eX cross section at small e - scattering angles has been measured using the electromagnetic SpaCal calorimeter in the backward region of the H1 detector. This calorimeter constructed of lead and scintillating fiber was designed to measure the scattered electron with high resolution in both energy and polar angle. The analysis comprises the kinematic range of 0.06 e 2 e 2 2 for the squared momentum exchange. The data sample consists of positron proton collisions of the years 2006 and 2007, adding up to an integrated luminosity of ∝141 pb -1 . Due to the high luminosity of the HERA II run phase the accuracy is no longer limited by the data statistics but rather by the detector resolution and systematics. The migration becomes increasingly influential; an effect which leads to distortions of the measured distribution as well as to statistical correlations between adjacent data points. At this stage, the correction of detector effects as well as the precise determination of statistical correlations become important features of a rigorous error treatment. In this analysis two-dimensional unfolding has been applied. This is a novel approach to H1 inclusive cross section measurements, which are usually based on a bin-by-bin efficiency correction (bin-by-bin method). With unfolding, the detector effect to the measurements is modelled by a linear transformation (''response matrix'') which is used to correct any distortion of the data. The inclusion of off-diagonal elements results in a coherent assessment of the statistical uncertainties and correlations. The model dependence can be optimally evaluated. In this context, the bin-by-bin method can be viewed as an approximation based on a diagonal response matrix. In a scenario of limited detector resolution, the unfolded data distributions will typically exhibit strong fluctuations and correlations between the data points. This issue can be addressed by smoothing
Inclusive neutral current ep cross sections with HERA II and two-dimensional unfolding
Energy Technology Data Exchange (ETDEWEB)
Fischer, David-Johannes
2011-06-15
In this thesis, the inclusive neutral current ep {yields} eX cross section at small e{sup -} scattering angles has been measured using the electromagnetic SpaCal calorimeter in the backward region of the H1 detector. This calorimeter constructed of lead and scintillating fiber was designed to measure the scattered electron with high resolution in both energy and polar angle. The analysis comprises the kinematic range of 0.06 < y{sub e} < 0.6 for the inelasticity and 14 GeV{sup 2} < Q{sub e}{sup 2} < 110 GeV{sup 2} for the squared momentum exchange. The data sample consists of positron proton collisions of the years 2006 and 2007, adding up to an integrated luminosity of {proportional_to}141 pb{sup -1}. Due to the high luminosity of the HERA II run phase the accuracy is no longer limited by the data statistics but rather by the detector resolution and systematics. The migration becomes increasingly influential; an effect which leads to distortions of the measured distribution as well as to statistical correlations between adjacent data points. At this stage, the correction of detector effects as well as the precise determination of statistical correlations become important features of a rigorous error treatment. In this analysis two-dimensional unfolding has been applied. This is a novel approach to H1 inclusive cross section measurements, which are usually based on a bin-by-bin efficiency correction (bin-by-bin method). With unfolding, the detector effect to the measurements is modelled by a linear transformation (''response matrix'') which is used to correct any distortion of the data. The inclusion of off-diagonal elements results in a coherent assessment of the statistical uncertainties and correlations. The model dependence can be optimally evaluated. In this context, the bin-by-bin method can be viewed as an approximation based on a diagonal response matrix. In a scenario of limited detector resolution, the unfolded data distributions will
Photo-reduction on the rupture of disulfide bonds and the related protein assembling
Wang, Wei
It has been found that many proteins can self-assemble into nanoscale assemblies when they unfold or partially unfold under harsh conditions, such as low pH, high temperature, or the presence of denaturants, and so on. These nanoscale assemblies can have some applications such as the drug-delivery systems (DDSs). Here we report a study that a very physical way, the UV illumination, can be used to facilitate the formation of protein fibrils and nanoparticles under native conditions by breaking disulfide bonds in some disulfide-containing proteins. By controlling the intensity of UV light and the illumination time, we realized the preparation of self-assembly nanoparticles which encapsulate the anticancer drug doxorubicin (DOX) and can be used as the DDS for inhibiting the growth of tumor. The formation of fibrillary assemblies was also observed. The rupture of disulfide bonds through photo-reduction process due to the effect of tryptophan and tyrosine was studied, and the physical mechanism of the assembling of the related disulfide-containing proteins was also discussed. We thank the financial support from NSF of China and the 973 project.
Hilario, Eric C; Stern, Alan; Wang, Charlie H; Vargas, Yenny W; Morgan, Charles J; Swartz, Trevor E; Patapoff, Thomas W
2017-01-01
Concentration determination is an important method of protein characterization required in the development of protein therapeutics. There are many known methods for determining the concentration of a protein solution, but the easiest to implement in a manufacturing setting is absorption spectroscopy in the ultraviolet region. For typical proteins composed of the standard amino acids, absorption at wavelengths near 280 nm is due to the three amino acid chromophores tryptophan, tyrosine, and phenylalanine in addition to a contribution from disulfide bonds. According to the Beer-Lambert law, absorbance is proportional to concentration and path length, with the proportionality constant being the extinction coefficient. Typically the extinction coefficient of proteins is experimentally determined by measuring a solution absorbance then experimentally determining the concentration, a measurement with some inherent variability depending on the method used. In this study, extinction coefficients were calculated based on the measured absorbance of model compounds of the four amino acid chromophores. These calculated values for an unfolded protein were then compared with an experimental concentration determination based on enzymatic digestion of proteins. The experimentally determined extinction coefficient for the native proteins was consistently found to be 1.05 times the calculated value for the unfolded proteins for a wide range of proteins with good accuracy and precision under well-controlled experimental conditions. The value of 1.05 times the calculated value was termed the predicted extinction coefficient. Statistical analysis shows that the differences between predicted and experimentally determined coefficients are scattered randomly, indicating no systematic bias between the values among the proteins measured. The predicted extinction coefficient was found to be accurate and not subject to the inherent variability of experimental methods. We propose the use of a
Stretching of macromolecules and proteins
International Nuclear Information System (INIS)
Strick, T R; Dessinges, M-N; Charvin, G; Dekker, N H; Allemand, J-F; Bensimon, D; Croquette, V
2003-01-01
In this paper we review the biophysics revealed by stretching single biopolymers. During the last decade various techniques have emerged allowing micromanipulation of single molecules and simultaneous measurements of their elasticity. Using such techniques, it has been possible to investigate some of the interactions playing a role in biology. We shall first review the simplest case of a non-interacting polymer and then present the structural transitions in DNA, RNA and proteins that have been studied by single-molecule techniques. We shall explain how these techniques permit a new approach to the protein folding/unfolding transition
DEFF Research Database (Denmark)
Neess, Ditte; Marcher, Ann-Britt; Bloksgaard, Maria
The acyl-CoA binding protein/diazepam binding inhibitor (ACBP/DBI) is an evolutionary conserved intracellular protein that binds C14-C22 acyl-CoA esters with very high affinity. ACBP is thought to act as an acyl-CoA transporter, and in vitro analyses have indicated that ACBP can transport acyl......-CoA esters between different enzymatic systems. However, little is known about the in vivo function in mammalian cells. We have generated mice with targeted disruption of ACBP (ACBP-/-). These mice are viable and fertile and develop normally. However, around weaning the ACBP-/- mice show decreased growth......) family, around the weaning period. As a result, the hepatic de novo cholesterogenesis is significantly decreased at weaning. The delayed induction of SREBP target genes around weaning is caused by a compromised processing and decreased expression of SREBP precursors leading to reduced binding of SREBP...
Directory of Open Access Journals (Sweden)
Bani Kumar Pathak
Full Text Available BACKGROUND: Molecular chaperones that support de novo folding of proteins under non stress condition are classified as chaperone 'foldases' that are distinct from chaperone' holdases' that provide high affinity binding platform for unfolded proteins and prevent their aggregation specifically under stress conditions. Ribosome, the cellular protein synthesis machine can act as a foldase chaperone that can bind unfolded proteins and release them in folding competent state. The peptidyl transferase center (PTC located in the domain V of the 23S rRNA of Escherichia coli ribosome (bDV RNA is the chaperoning center of the ribosome. It has been proposed that via specific interactions between the RNA and refolding proteins, the chaperone provides information for the correct folding of unfolded polypeptide chains. RESULTS: We demonstrate using Escherichia coli ribosome and variants of its domain V RNA that the ribosome can bind to partially folded intermediates of bovine carbonic anhydrase II (BCAII and lysozyme and suppress aggregation during their refolding. Using mutants of domain V RNA we demonstrate that the time for which the chaperone retains the bound protein is an important factor in determining its ability to suppress aggregation and/or support reactivation of protein. CONCLUSION: The ribosome can behave like a 'holdase' chaperone and has the ability to bind and hold back partially folded intermediate states of proteins from participating in the aggregation process. Since the ribosome is an essential organelle that is present in large numbers in all living cells, this ability of the ribosome provides an energetically inexpensive way to suppress cellular aggregation. Further, this ability of the ribosome might also be crucial in the context that the ribosome is one of the first chaperones to be encountered by a large nascent polypeptide chains that have a tendency to form partially folded intermediates immediately following their synthesis.
Structure of haze forming proteins in white wines: Vitis vinifera thaumatin-like proteins.
Marangon, Matteo; Van Sluyter, Steven C; Waters, Elizabeth J; Menz, Robert I
2014-01-01
Grape thaumatin-like proteins (TLPs) play roles in plant-pathogen interactions and can cause protein haze in white wine unless removed prior to bottling. Different isoforms of TLPs have different hazing potential and aggregation behavior. Here we present the elucidation of the molecular structures of three grape TLPs that display different hazing potential. The three TLPs have very similar structures despite belonging to two different classes (F2/4JRU is a thaumatin-like protein while I/4L5H and H2/4MBT are VVTL1), and having different unfolding temperatures (56 vs. 62°C), with protein F2/4JRU being heat unstable and forming haze, while I/4L5H does not. These differences in properties are attributable to the conformation of a single loop and the amino acid composition of its flanking regions.
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
Nonresonant Double Hopf Bifurcation in Toxic Phytoplankton-Zooplankton Model with Delay
Yuan, Rui; Jiang, Weihua; Wang, Yong
This paper investigates a toxic phytoplankton-zooplankton model with Michaelis-Menten type phytoplankton harvesting. The model has rich dynamical behaviors. It undergoes transcritical, saddle-node, fold, Hopf, fold-Hopf and double Hopf bifurcation, when the parameters change and go through some of the critical values, the dynamical properties of the system will change also, such as the stability, equilibrium points and the periodic orbit. We first study the stability of the equilibria, and analyze the critical conditions for the above bifurcations at each equilibrium. In addition, the stability and direction of local Hopf bifurcations, and the completion bifurcation set by calculating the universal unfoldings near the double Hopf bifurcation point are given by the normal form theory and center manifold theorem. We obtained that the stable coexistent equilibrium point and stable periodic orbit alternate regularly when the digestion time delay is within some finite value. That is, we derived the pattern for the occurrence, and disappearance of a stable periodic orbit. Furthermore, we calculated the approximation expression of the critical bifurcation curve using the digestion time delay and the harvesting rate as parameters, and determined a large range in terms of the harvesting rate for the phytoplankton and zooplankton to coexist in a long term.
Simulation study on unfolding methods for diagnostic X-rays and mixed gamma rays
International Nuclear Information System (INIS)
Hashimoto, Makoto; Ohtaka, Masahiko; Ara, Kuniaki; Kanno, Ikuo; Imamura, Ryo; Mikami, Kenta; Nomiya, Seiichiro; Onabe, Hideaki
2009-01-01
A photon detector operating in current mode that can sense X-ray energy distribution has been reported. This detector consists of a row of several segment detectors. The energy distribution is derived using an unfolding technique. In this paper, comparisons of the unfolding techniques among error reduction, spectrum surveillance, and neural network methods are discussed through simulation studies on the detection of diagnostic X-rays and gamma rays emitted by a mixture of 137 Cs and 60 Co. For diagnostic X-ray measurement, the spectrum surveillance and neural network methods appeared promising, while the error reduction method yielded poor results. However, in the case of measuring mixtures of gamma rays, the error reduction method was both sufficient and effective. (author)
Thermodynamic properties of an extremely rapid protein folding reaction.
Schindler, T; Schmid, F X
1996-12-24
The cold-shock protein CspB from Bacillus subtilis is a very small beta-barrel protein, which folds with a time constant of 1 ms (at 25 degrees C) in a U reversible N two-state reaction. To elucidate the energetics of this extremely fast reaction we investigated the folding kinetics of CspB as a function of both temperature and denaturant concentration between 2 and 45 degrees C and between 1 and 8 M urea. Under all these conditions unfolding and refolding were reversible monoexponential reactions. By using transition state theory, data from 327 kinetic curves were jointly analyzed to determine the thermodynamic activation parameters delta H H2O++, delta S H2O++, delta G H2O++, and delta C p H2O++ for unfolding and refolding and their dependences on the urea concentration. 90% of the total change in heat capacity and 96% of the change in the m value (m = d delta G/d[urea]) occur between the unfolded state and the activated state. This suggests that for CspB the activated state of folding is unusually well structured and almost equivalent to the native protein in its interactions with the solvent. As a consequence of this native-like activated state a strong temperature-dependent enthalpy/entropy compensation is observed for the refolding kinetics, and the barrier to refolding shifts from being largely enthalpic at low temperature to largely entropic at high temperature. This shift originates not from the changes in the folding protein chains itself, but from the changes in the protein-solvent interactions. We speculate that the absence of intermediates and the native-like activated state in the folding of CspB are correlated with the small size and the structural type of this protein. The stabilization of a small beta-sheet as in CspB requires extensive non-local interactions, and therefore incomplete sheets are unstable. As a consequence, the critical activated state is reached only very late in folding. The instability of partially folded structure is a means to
Evaluation of spectral unfolding techniques for neutron spectroscopy
International Nuclear Information System (INIS)
Sunden, Erik Andersson; Conroy, S.; Ericsson, G.; Johnson, M. Gatu; Giacomelli, L.; Hellesen, C.; Hjalmarsson, A.; Ronchi, E.; Sjoestrand, H.; Weiszflog, M.; Kaellne, J.; Gorini, G.; Tardocchi, M.
2008-01-01
The precision of the JET installations of MAXED, GRAVEL and the L-curve version of MAXED has been evaluated by using synthetic neutron spectra. We have determined the number of counts needed for the detector systems NE213 and MPR to get an error below 10% of the MAXED unfolded neutron spectra is determined to be ∼10 6 and ∼10 4 , respectively. For GRAVEL the same number is ∼10 7 and ∼3·10 4 for NE213 and MPR, respectively
Lack of Dependence of the Sizes of the Mesoscopic Protein Clusters on Electrostatics.
Vorontsova, Maria A; Chan, Ho Yin; Lubchenko, Vassiliy; Vekilov, Peter G
2015-11-03
Protein-rich clusters of steady submicron size and narrow size distribution exist in protein solutions in apparent violation of the classical laws of phase equilibrium. Even though they contain a minor fraction of the total protein, evidence suggests that they may serve as essential precursors for the nucleation of ordered solids such as crystals, sickle-cell hemoglobin polymers, and amyloid fibrils. The cluster formation mechanism remains elusive. We use the highly basic protein lysozyme at nearly neutral and lower pH as a model and explore the response of the cluster population to the electrostatic forces, which govern numerous biophysical phenomena, including crystallization and fibrillization. We tune the strength of intermolecular electrostatic forces by varying the solution ionic strength I and pH and find that despite the weaker repulsion at higher I and pH, the cluster size remains constant. Cluster responses to the presence of urea and ethanol demonstrate that cluster formation is controlled by hydrophobic interactions between the peptide backbones, exposed to the solvent after partial protein unfolding that may lead to transient protein oligomers. These findings reveal that the mechanism of the mesoscopic clusters is fundamentally different from those underlying the two main classes of ordered protein solid phases, crystals and amyloid fibrils, and partial unfolding of the protein chain may play a significant role. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.
Olou, Appolinaire A; Sarkar, Aniruddha; Bele, Aditya; Gurumurthy, C B; Mir, Riyaz A; Ammons, Shalis A; Mirza, Sameer; Saleem, Irfana; Urano, Fumihiko; Band, Hamid; Band, Vimla
2017-09-15
Mammalian Ecdysoneless (ECD) is a highly conserved ortholog of the Drosophila Ecd gene product whose mutations impair the synthesis of Ecdysone and produce cell-autonomous survival defects, but the mechanisms by which ECD functions are largely unknown. Here we present evidence that ECD regulates the endoplasmic reticulum (ER) stress response. ER stress induction led to a reduced ECD protein level, but this effect was not seen in PKR-like ER kinase knockout (PERK-KO) or phosphodeficient eukaryotic translation initiation factor 2α (eIF2α) mouse embryonic fibroblasts (MEFs); moreover, ECD mRNA levels were increased, suggesting impaired ECD translation as the mechanism for reduced protein levels. ECD colocalizes and coimmunoprecipitates with PERK and GRP78. ECD depletion increased the levels of both phospho-PERK (p-PERK) and p-eIF2α, and these effects were enhanced upon ER stress induction. Reciprocally, overexpression of ECD led to marked decreases in p-PERK, p-eIF2α, and ATF4 levels but robust increases in GRP78 protein levels. However, GRP78 mRNA levels were unchanged, suggesting a posttranscriptional event. Knockdown of GRP78 reversed the attenuating effect of ECD overexpression on PERK signaling. Significantly, overexpression of ECD provided a survival advantage to cells upon ER stress induction. Taken together, our data demonstrate that ECD promotes survival upon ER stress by increasing GRP78 protein levels to enhance the adaptive folding protein in the ER to attenuate PERK signaling. Copyright © 2017 Olou et al.
Directory of Open Access Journals (Sweden)
Rui Li
2016-03-01
Full Text Available For reducing protein aggregation in foam fractionation, the role of pH-induced structural change in the interface-induced protein aggregation was analyzed using bovine serum albumin (BSA as a model protein. The results show that the decrease in pH from 7.0 to 3.0 gradually unfolded the BSA structure to increase the molecular size and the relative content of β-sheet and thus reduced the stability of BSA in the aqueous solution. At the isoelectric point (pH 4.7, BSA suffered the lowest level in protein aggregation induced by the gas–liquid interface. In the pH range from 7.0 to 4.7, most BSA aggregates were formed in the defoaming process while in the pH range from 4.7 to 3.0, the BSA aggregates were formed at the gas–liquid interface due to the unfolded BSA structure and they further aggregated to form insoluble ones in the desorption process.
Large-scale biophysical evaluation of protein PEGylation effects
DEFF Research Database (Denmark)
Vernet, Erik; Popa, Gina; Pozdnyakova, Irina
2016-01-01
PEGylation is the most widely used method to chemically modify protein biopharmaceuticals, but surprisingly limited public data is available on the biophysical effects of protein PEGylation. Here we report the first large-scale study, with site-specific mono-PEGylation of 15 different proteins...... of PEGylation on the thermal stability of a protein based on data generated by circular dichroism (CD), differential scanning calorimetry (DSC), or differential scanning fluorimetry (DSF). In addition, DSF was validated as a fast and inexpensive screening method for thermal unfolding studies of PEGylated...... proteins. Multivariate data analysis revealed clear trends in biophysical properties upon PEGylation for a subset of proteins, although no universal trends were found. Taken together, these findings are important in the consideration of biophysical methods and evaluation of second...
High-resolution nuclear magnetic resonance studies of proteins.
Jonas, Jiri
2002-03-25
The combination of advanced high-resolution nuclear magnetic resonance (NMR) techniques with high-pressure capability represents a powerful experimental tool in studies of protein folding. This review is organized as follows: after a general introduction of high-pressure, high-resolution NMR spectroscopy of proteins, the experimental part deals with instrumentation. The main section of the review is devoted to NMR studies of reversible pressure unfolding of proteins with special emphasis on pressure-assisted cold denaturation and the detection of folding intermediates. Recent studies investigating local perturbations in proteins and the experiments following the effects of point mutations on pressure stability of proteins are also discussed. Ribonuclease A, lysozyme, ubiquitin, apomyoglobin, alpha-lactalbumin and troponin C were the model proteins investigated.
Structural and kinetic mapping of side-chain exposure onto the protein energy landscape.
Bernstein, Rachel; Schmidt, Kierstin L; Harbury, Pehr B; Marqusee, Susan
2011-06-28
Identification and characterization of structural fluctuations that occur under native conditions is crucial for understanding protein folding and function, but such fluctuations are often rare and transient, making them difficult to study. Native-state hydrogen exchange (NSHX) has been a powerful tool for identifying such rarely populated conformations, but it generally reveals no information about the placement of these species along the folding reaction coordinate or the barriers separating them from the folded state and provides little insight into side-chain packing. To complement such studies, we have performed native-state alkyl-proton exchange, a method analogous to NSHX that monitors cysteine modification rather than backbone amide exchange, to examine the folding landscape of Escherichia coli ribonuclease H, a protein well characterized by hydrogen exchange. We have chosen experimental conditions such that the rate-limiting barrier acts as a kinetic partition: residues that become exposed only upon crossing the unfolding barrier are modified in the EX1 regime (alkylation rates report on the rate of unfolding), while those exposed on the native side of the barrier are modified predominantly in the EX2 regime (alkylation rates report on equilibrium populations). This kinetic partitioning allows for identification and placement of partially unfolded forms along the reaction coordinate. Using this approach we detect previously unidentified, rarely populated conformations residing on the native side of the barrier and identify side chains that are modified only upon crossing the unfolding barrier. Thus, in a single experiment under native conditions, both sides of the rate-limiting barrier are investigated.
Xiao, Naidong; Chen, Yinguang; Chen, Aihui; Feng, Leiyu
2014-01-01
Enhanced bio-hydrogen production from protein wastewater by altering protein structure and amino acids acidification type via pH control was investigated. The hydrogen production reached 205.2 mL/g-protein when protein wastewater was pretreated at pH 12 and then fermented at pH 10. The mechanism studies showed that pH 12 pretreatment significantly enhanced protein bio-hydrolysis during the subsequent fermentation stage as it caused the unfolding of protein, damaged the protein hydrogen bonding networks, and destroyed the disulfide bridges, which increased the susceptibility of protein to protease. Moreover, pH 10 fermentation produced more acetic but less propionic acid during the anaerobic fermentation of amino acids, which was consistent with the theory of fermentation type affecting hydrogen production. Further analyses of the critical enzymes, genes, and microorganisms indicated that the activity and abundance of hydrogen producing bacteria in the pH 10 fermentation reactor were greater than those in the control. PMID:24495932
Xiao, Naidong; Chen, Yinguang; Chen, Aihui; Feng, Leiyu
2014-02-05
Enhanced bio-hydrogen production from protein wastewater by altering protein structure and amino acids acidification type via pH control was investigated. The hydrogen production reached 205.2 mL/g-protein when protein wastewater was pretreated at pH 12 and then fermented at pH 10. The mechanism studies showed that pH 12 pretreatment significantly enhanced protein bio-hydrolysis during the subsequent fermentation stage as it caused the unfolding of protein, damaged the protein hydrogen bonding networks, and destroyed the disulfide bridges, which increased the susceptibility of protein to protease. Moreover, pH 10 fermentation produced more acetic but less propionic acid during the anaerobic fermentation of amino acids, which was consistent with the theory of fermentation type affecting hydrogen production. Further analyses of the critical enzymes, genes, and microorganisms indicated that the activity and abundance of hydrogen producing bacteria in the pH 10 fermentation reactor were greater than those in the control.
How Chain Length and Charge Affect Surfactant Denaturation of Acyl Coenzyme A Binding Protein (ACBP)
DEFF Research Database (Denmark)
Andersen, Kell Kleiner; Otzen, Daniel
2009-01-01
maltoside (DDM). The aim has been to determine how surfactant chain length and micellar charge affect the denaturation mechanism. ACBP denatures in two steps irrespective of surfactant chain length, but with increasing chain length, the potency of the denaturant rises more rapidly than the critical micelle......Using intrinsic tryptophan fluorescence, equilibria and kinetics of unfolding of acyl coenzyme A binding protein (ACBP) have been investigated in sodium alkyl sulfate surfactants of different chain length (8-16 carbon atoms) and with different proportions of the nonionic surfactant dodecyl...... constants increases linearly with denaturant concentration below the cmc but declines at higher concentrations. Both shortening chain length and decreasing micellar charge reduce the overall kinetics of unfolding and makes the dependence of unfolding rate constants on surfactant concentration more complex...
International Nuclear Information System (INIS)
Banerjee, Tuhina; Kishore, Nand
2008-01-01
Calorimetry has been employed to investigate the quantitative energetic aspects and mechanism underlying protein-tetraethylammonium bromide (TEAB) interactions. Differential scanning calorimetry and UV-Visible spectroscopy have been used to study the thermal unfolding of three proteins of different structure and function (bovine serum albumin, α-lactalbumin, and bovine pancreatic ribonuclease A). The mode of interaction has been studied by using isothermal titration calorimetry, which demonstrates the absence of appreciable specific binding of TEAB to the protein. This suggests the involvement of solvent mediated effects and, possibly weak non-specific binding. The thermal unfolding transitions were found to be calorimetrically reversible for α-lactalbumin and bovine pancreatic ribonuclease A and partially reversible in the case of bovine serum albumin. The results indicate protein destabilization promoted by the TEAB interaction. The preferential interaction parameters of TEAB with α-lactalbumin and ribonuclease A confirm that an increased interaction of the hydrophobic groups of the TEAB with that of the protein upon denaturation is responsible for the reduced thermal stability of the protein. The decrease in the thermal stability of proteins in the presence of TEAB is well supported by a red shift in the intrinsic fluorescence of these proteins leading to conformational change thereby shifting the native ↔ denatured equilibrium towards right. The forces responsible for the thermal denaturation of the proteins of different structure and function in the presence of TEAB are discussed
Network Unfolding Map by Vertex-Edge Dynamics Modeling.
Verri, Filipe Alves Neto; Urio, Paulo Roberto; Zhao, Liang
2018-02-01
The emergence of collective dynamics in neural networks is a mechanism of the animal and human brain for information processing. In this paper, we develop a computational technique using distributed processing elements in a complex network, which are called particles, to solve semisupervised learning problems. Three actions govern the particles' dynamics: generation, walking, and absorption. Labeled vertices generate new particles that compete against rival particles for edge domination. Active particles randomly walk in the network until they are absorbed by either a rival vertex or an edge currently dominated by rival particles. The result from the model evolution consists of sets of edges arranged by the label dominance. Each set tends to form a connected subnetwork to represent a data class. Although the intrinsic dynamics of the model is a stochastic one, we prove that there exists a deterministic version with largely reduced computational complexity; specifically, with linear growth. Furthermore, the edge domination process corresponds to an unfolding map in such way that edges "stretch" and "shrink" according to the vertex-edge dynamics. Consequently, the unfolding effect summarizes the relevant relationships between vertices and the uncovered data classes. The proposed model captures important details of connectivity patterns over the vertex-edge dynamics evolution, in contrast to the previous approaches, which focused on only vertex or only edge dynamics. Computer simulations reveal that the new model can identify nonlinear features in both real and artificial data, including boundaries between distinct classes and overlapping structures of data.
Anumalla, Bramhini; Prabhu, N Prakash
2018-01-25
When organisms are subjected to stress conditions, one of their adaptive responses is accumulation of small organic molecules called osmolytes. These osmolytes affect the structure and stability of the biological macromolecules including proteins. The present study examines the effect of a negatively charged amino acid osmolyte, glutamate (Glu), on two model proteins, ribonuclease A (RNase A) and α-lactalbumin (α-LA), which have positive and negative surface charges at pH 7, respectively. These proteins follow two-state unfolding transitions during both heat and chemical induced denaturation processes. The addition of Glu stabilizes the proteins against temperature and induces an early equilibrium intermediate during unfolding. The stability is found to be enthalpy-driven, and the free energy of stabilization is more for α-LA compared to RNase A. The decrease in the partial molar volume and compressibility of both of the proteins in the presence of Glu suggests that the proteins attain a more compact state through surface hydration which could provide a more stable conformation. This is also supported by molecule dynamic simulation studies which demonstrate that the water density around the proteins is increased upon the addition of Glu. Further, the intermediates could be completely destabilized by lower concentrations (∼0.5 M) of guanidinium chloride and salt. However, urea subverts the Glu-induced intermediate formed by α-LA, whereas it only slightly destabilizes in the case of RNase A which has a positive surface charge and could possess charge-charge interactions with Glu. This suggests that, apart from hydration, columbic interactions might also contribute to the stability of the intermediate. Gdm-induced denaturation of RNase A and α-LA in the absence and the presence of Glu at different temperatures was carried out. These results also show the Glu-induced stabilization of both of the proteins; however, all of the unfolding transitions followed two
Li, Jiahuang; Chen, Yuan; Yang, Jie; Hua, Zichun
2015-05-01
The Schistosoma juponicum 26 kDa glutathione S-transferase (sj26GST) consists of the N-terminal domain (N-domain), containing three alpha-helices (named H1-H3) and four anti-parallel beta-strands (S1-S4), and the C-terminal domain (C-domain), comprising five alpha-helices (named H4-H8). In present work, molecular dynamics simulations and fluorescence spectroscopic were used to gain insights into the unfolding process of sj26GST. The molecular dynamics simulations on sj26GST subunit both in water and in 8 M urea were carried out at 300 K, 400 K and 500 K, respectively. Spectroscopic measurements were employed to monitor structural changes. Molecular dynamics simulations of sj26GST subunit induced by urea and temperature showed that the initial unfolding step of sj26GST both in water and urea occurred on N-domain, involving the disruption of helices H2, H3 and strands S3 and S4, whereas H6 was the last region exposed to solution and was the last helix to unfold. Moreover, simulations analyses combining with fluorescence and circular dichroism spectra indicated that N-domain could not fold independent, suggesting that correct folding of N-domain depended on its interactions with C-domain. We further proposed that the folding of GSTs could begin with the hydrophobic collapse of C-domain whose H4, H5, H6 and H7 could move close to each other and form a hydrophobic core, especially H6 wrapped in the hydrophobic center and beginning spontaneous formation of the helix. S3, S4, H3, and H2 could form in the wake of the interaction between C-domain and N-domain. The paper can offer insights into the molecular mechanism of GSTs unfolding. © 2014 Wiley Periodicals, Inc.
Chatty Mitochondria: Keeping Balance in Cellular Protein Homeostasis.
Topf, Ulrike; Wrobel, Lidia; Chacinska, Agnieszka
2016-08-01
Mitochondria are multifunctional cellular organelles that host many biochemical pathways including oxidative phosphorylation (OXPHOS). Defective mitochondria pose a threat to cellular homeostasis and compensatory responses exist to curtail the source of stress and/or its consequences. The mitochondrial proteome comprises proteins encoded by the nuclear and mitochondrial genomes. Disturbances in protein homeostasis may originate from mistargeting of nuclear encoded mitochondrial proteins. Defective protein import and accumulation of mistargeted proteins leads to stress that triggers translation alterations and proteasomal activation. These cytosolic pathways are complementary to the mitochondrial unfolded protein response (UPRmt) that aims to increase the capacity of protein quality control mechanisms inside mitochondria. They constitute putative targets for interventions aimed at increasing the fitness, stress resistance, and longevity of cells and organisms. Copyright © 2016 Elsevier Ltd. All rights reserved.
Energy Technology Data Exchange (ETDEWEB)
Benavente C, J. A.; Lacerda, M. A. S.; Guimaraes, A. M.; Da Silva, T. A. [Universidade Federal de Minas Gerais, Departamento de Engenharia Nuclear, Programa de Pos-graduacao em Ciencias e Tecnicas Nucleares, Pte. Antonio Carlos 6627, Belo Horizonte 31270-901, Minas Gerais (Brazil); Vega C, H. R. [Universidad Autonoma de Zacatecas, Unidad Academica de Estudios Nucleares, Cipres No. 10, Fracc. La Penuela, 98060 Zacatecas, Zac. (Mexico)
2015-10-15
In a cyclotron facility is strongly advised the use of spectrometry techniques to support workplace neutron dosimetry. Bonner sphere spectrometer (Bss) is the most used for radiation protection applications. Bss data must be unfolded to determine the spectral particle fluence. Some computer codes have been utilized for this purpose. These codes allow unfolding the spectrum from the Bss count rates through different algorithms. Some iterative routines need an initial guess spectrum to start the unfolding. The adequate choice of this initial spectrum is a critical part of the process and can affect the final solution. In this work, we evaluate the influence of the initial guess spectrum in the unfolding of Bss data obtained in four points inside the bunker of a PET cyclotron. The measurements were done utilizing a modified Bss system with thermoluminescent detectors (TLDs). Codes BUNKIUT and NSDUAZ were utilized to unfold the Bss data. For the NSDUAZ the starting spectrum is automatically obtained from a library initial guess spectra. For the BUNKIUT code were utilized two different initial guess spectra: (a) a Maxwellian spectrum with temperature of 1.4 MeV and shape factor of 0.1, created with the MAXIET algorithm and; (b) the spectra obtained through simulation with the MCNPX code version 2.7. Spectra obtained with both unfold codes and with the different initial guess spectra presented epithermal and thermal neutrons due to room-return effects. However, the contribution of the fast neutron to the total fluence were quite different for the different cases studied. These differences highlight the importance of an appropriate choice of an initial guess spectra for the quality of the results. (Author)
International Nuclear Information System (INIS)
Benavente C, J. A.; Lacerda, M. A. S.; Guimaraes, A. M.; Da Silva, T. A.; Vega C, H. R.
2015-10-01
In a cyclotron facility is strongly advised the use of spectrometry techniques to support workplace neutron dosimetry. Bonner sphere spectrometer (Bss) is the most used for radiation protection applications. Bss data must be unfolded to determine the spectral particle fluence. Some computer codes have been utilized for this purpose. These codes allow unfolding the spectrum from the Bss count rates through different algorithms. Some iterative routines need an initial guess spectrum to start the unfolding. The adequate choice of this initial spectrum is a critical part of the process and can affect the final solution. In this work, we evaluate the influence of the initial guess spectrum in the unfolding of Bss data obtained in four points inside the bunker of a PET cyclotron. The measurements were done utilizing a modified Bss system with thermoluminescent detectors (TLDs). Codes BUNKIUT and NSDUAZ were utilized to unfold the Bss data. For the NSDUAZ the starting spectrum is automatically obtained from a library initial guess spectra. For the BUNKIUT code were utilized two different initial guess spectra: (a) a Maxwellian spectrum with temperature of 1.4 MeV and shape factor of 0.1, created with the MAXIET algorithm and; (b) the spectra obtained through simulation with the MCNPX code version 2.7. Spectra obtained with both unfold codes and with the different initial guess spectra presented epithermal and thermal neutrons due to room-return effects. However, the contribution of the fast neutron to the total fluence were quite different for the different cases studied. These differences highlight the importance of an appropriate choice of an initial guess spectra for the quality of the results. (Author)
Small-angle X-ray scattering studies of thermally-induced globular protein gels
International Nuclear Information System (INIS)
Clark, A.H.; Tuffnell, C.D.
1980-01-01
Small-angle X-ray scattering has been applied to gels formed by heating globular proteins, in aqueous solution, above their unfolding temperatures. A number of BSA gels, previously characterised by electron microscopy, have been studied, and by setting up theoretical models for the scattering process, the X-ray data have been shown to be consistent with the microscope conclusions regarding network structure. It is concluded that the networks form by a linearly-directed aggregation of unfolded, disc-like, protein molecules, three-dimensional geometry being achieved by occasional branching, and/or cross-linking. Long-range inhomogeneities in network structure, easily observed by electron microscopy, and correlated with variations in pH or ionic strength, have an effect on X-ray scattering, and hence the X-ray method is sensitive not only to different network strand thicknesses, but to different degrees of uniformity as well. (author)
Structure of haze forming proteins in white wines: Vitis vinifera thaumatin-like proteins.
Directory of Open Access Journals (Sweden)
Matteo Marangon
Full Text Available Grape thaumatin-like proteins (TLPs play roles in plant-pathogen interactions and can cause protein haze in white wine unless removed prior to bottling. Different isoforms of TLPs have different hazing potential and aggregation behavior. Here we present the elucidation of the molecular structures of three grape TLPs that display different hazing potential. The three TLPs have very similar structures despite belonging to two different classes (F2/4JRU is a thaumatin-like protein while I/4L5H and H2/4MBT are VVTL1, and having different unfolding temperatures (56 vs. 62°C, with protein F2/4JRU being heat unstable and forming haze, while I/4L5H does not. These differences in properties are attributable to the conformation of a single loop and the amino acid composition of its flanking regions.