Prediction of Protein Relative Enthalpic Stability from Molecular Dynamics Simulations of the Folded and Unfolded States

For proteins of known structure, the relative enthalpic stability with respect to wild-type, ΔΔH^U, can be estimated by direct computation of the folded and unfolded state energies. We propose a model by which the change in stability upon mutation can be predicted from all-atom molecular dynamics simulations for the folded state and a peptide-based model for the unfolded state. The unfolding enthalpies are expressed in terms of environmental and hydration-solvent reorganization contributions that readily allow a residue-specific analysis of ΔΔH^U. The method is applied to estimate the relative enthalpic stability of variants with buried charged groups in T4 lysozyme. The predicted relative stabilities are in good agreement with experimental data. Environmental factors are observed to contribute more than hydration to the overall ΔΔH^U. The residue-specific analysis finds that the effects of burying charge are both localized and long-range. The enthalpy for hydration-solvent reorganization varies considerably among different amino-acid types, but because the variant folded state structures are similar to those of the wild-type, the hydration-solvent reorganization contribution to ΔΔH^U is localized at the mutation site, in contrast to environmental contributions. Overall, mutation of apolar and polar amino acids to charged amino acids are destabilizing, but the reasons are complex and differ from site to site.     

Glutathione Ethylester, a Novel Protein Refolding Reagent, Enhances both the Efficiency of Refolding and Correct Disulfide Formation

Protein refolding constitutes a crucial process for recombinant proteins. We report here on the development of a multifunctional refolding additive, glutathione ethyl ester (GSHEE), prepared from a redox reagent glutathione and an amino acid ethyl ester, an aggregation suppressor. Compared to glutathione, GSHEE showed 3.2-fold higher efficiency for the refolding yield of hen egg lysozyme. More importantly, a low concentration of GSHEE is more effective for refolding than conventional additives, such as amino acid ethyl esters by two orders of magnitude. The high potency of GSHEE makes it a candidate for use as a refolding additive for use in conjunction with reduced and denatured proteins.     

Studies of Peroxidase Refolding in the Presence of Specific Antibodies

A panel of eight monoclonal antibodies raised against horseradish root peroxidase was assembled and characterized. Affinity constants were determined for all antibodies, and their specificity for various structural forms of the enzyme (native peroxidase, apoperoxidase, and denatured peroxidase) were assessed by competitive enzyme immunoassay. The effects of the antibodies on the process of refolding of peroxidase after its denaturing with 6.5 M guanidine chloride were studied spectrophotometrically, by the restoration of the enzymatic activity in the reaction of 2,2-azino-bis(3-ethylbenzthiazoline-6-sulfonate) oxidation. The yield of the active enzyme in the course of the refolding was increased by 1.5–1.7 times in the presence of antibody H1. Effects of the antibodies constituting the panel on the activity of native peroxidase and the stability of its dilute solutions were analyzed.     

Substrate-induced Unlocking of the Inner Gate Determines the Catalytic Efficiency of a Neurotransmitter:Sodium Symporter

Neurotransmitter:sodium symporters (NSSs) mediate reuptake of neurotransmitters from the synaptic cleft and are targets for several therapeutics and psychostimulants. The prokaryotic NSS homologue, LeuT, represents a principal structural model for Na⁺-coupled transport catalyzed by these proteins. Here, we used site-directed fluorescence quenching spectroscopy to identify in LeuT a substrate-induced conformational rearrangement at the inner gate conceivably leading to formation of a structural intermediate preceding transition to the inward-open conformation. The substrate-induced, Na⁺-dependent change required an intact primary substrate-binding site and involved increased water exposure of the cytoplasmic end of transmembrane segment 5. The findings were supported by simulations predicting disruption of an intracellular interaction network leading to a discrete rotation of transmembrane segment 5 and the adjacent intracellular loop 2. The magnitude of the spectroscopic response correlated inversely with the transport rate for different substrates, suggesting that stability of the intermediate represents an unrecognized rate-limiting barrier in the NSS transport mechanism.     

The Unfolded State of the Murine Prion Protein and Properties of Single-point Mutants Related to Human Prion Diseases

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

二硫键异构酶的纯化及其对重组蛋白体外折叠的影响

自牛肝中纯化了蛋白二硫键异构酶(PDI),并对重组蛋白的酶促折叠过程进行了探讨．结果表明。在等摩尔PDl的催化作用下,可使1mg／ml的IIJ-2的正确折叠率提高到58%以上,比活性由4×10⁶u／mg增加到8．2×10⁶u／mg,PDl还能部分纠正二硫键错配的IL-2异构体成为正确折叠的lL-2和防止IL-2通过cys的链问交联形成聚合体。GM-CSF在PDl催化下也有类似的结果。PDl作用的关键是它所催化的琉基一二硫键的交换反应。     

The Effect of Osmolytes on the Bioluminescent Reaction of Bacteria: Structural and Dynamic Properties

Biophysics - The effects of viscous media with glycerol and sucrose (10–40%) on the kinetics of the bacterial bioluminescent reaction have been investigated by stopped-flow technique....     

Recognition of the Different Structural Forms of the Capsid Protein Determines the Outcome following Infection with Porcine Circovirus Type 2

Porcine circovirus type 2 (PCV2) capsid protein (CP) is the only protein necessary for the formation of the virion capsid, and recombinant CP spontaneously forms virus-like particles (VLPs). Located within a single CP subunit is an immunodominant epitope consisting of residues 169 to 180 [CP(169–180)], which is exposed on the surface of the subunit, but, in the structural context of the VLP, the epitope is buried and inaccessible to antibody. High levels of anti-CP(169–180) activity are associated with porcine circovirus-associated disease (PCVAD). The purpose of this study was to investigate the role of the immune response to monomer CP in the development of PCVAD. The approach was to immunize pigs with CP monomer, followed by challenge with PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV). To maintain the CP immunogen as a stable monomer, CP(43–233) was fused to ubiquitin (Ub-CP). Size exclusion chromatography showed that Ub-CP was present as a single 33-kDa protein. Pigs immunized with Ub-CP developed a strong antibody response to PCV2, including antibodies against CP(169–180). However, only low levels of virus neutralizing activity were detected, and viremia levels were similar to those of nonimmunized pigs. As a positive control, immunization with baculovirus-expressed CP (Bac-CP) resulted in high levels of virus neutralizing activity, small amounts of anti-CP(169–180) activity, and the absence of viremia in pigs following virus challenge. The data support the role of CP(169–180) as an immunological decoy and illustrate the importance of the structural form of the CP immunogen in determining the outcome following infection.     

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

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

Precipitation of Protein in the Presence of Melanoidin

A melanoidin was prepared by the reaction of glycine-d-glucose system. The fraction of melanoidin between 10,000 and 50,000 in molecular weight was mainly used to clarify its interaction with protein. Remarkable precipitation was observed between the isoelectric points of melanoidin (pH 2.5) and ovalbumin (pH 4.6). An increase of ionic strength restricted those interaction. As the concentration of melanoidin rose against protein in the reaction system, the molar ratio (protein/melanoidin) in precipitate fell towards 1:1. When the melanoidin was in excess beyond a certain level, precipitation hardly happened. The composition in the reaction system to attain the maximum rate of precipitation was affected by molecular weight of melanoidin. Precipitation proceeded according to second order reaction in respect to both ovalbumin and melanoidin. Based on the analysis of Arrhenius plots, precipitation was found to be promoted by the two subsequent steps. A mechanism involved in this reaction was proposed.     

Role of Protein Stabilizers on the Conformation of the Unfolded State of Cytochrome c and Its Early Folding Kinetics: INVESTIGATION AT SINGLE MOLECULAR RESOLUTION*

An insight into the conformation and dynamics of unfolded and early intermediate states of a protein is essential to understand the mechanism of its aggregation and to design potent inhibitor molecules. Fluorescence correlation spectroscopy has been used to study the effects of several model protein stabilizers on the conformation of the unfolded state and early folding dynamics of tetramethyl rhodamine-labeled cytochrome c from Saccharomyces cerevisiae at single molecular resolution. Special attention has been given to arginine, which is a widely used stabilizer for improving refolding yield of different proteins. The value of the hydrodynamic radius (r_H) obtained by analyzing the intensity fluctuations of the diffusing molecules has been found to increase in a two-state manner as the protein is unfolded by urea. The results further show that the presence of arginine and other protein stabilizers favors a relatively structured conformation of the unfolded states (r_H of 29 Å) over an extended one (r_H of 40 Å), which forms in their absence. Also, the time constant of a kinetic component (τ_R) of about 30 μs has been observed by analyzing the correlation functions, which represents formation of a collapsed state. This time constant varies with urea concentration representing an inverted Chevron plot that shows a roll-over and behavior in the absence of arginine. To the best of our knowledge, this is one of the first applications of fluorescence correlation spectroscopy to study direct folding kinetics of a protein.     

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

All-trans retinoic acid (ATRA) induces cellular senescence via up-regulation of p16 and p21; however, the action mechanism of ATRA is unknown. Here, we show that ATRA induces promoter hypomethylation of p16 and p21 via down-regulation of DNA methyltransferases 1, 3a, and 3b to facilitate binding of Ets1/2 to the p16 promoter and p53 to the p21 promoter, resulting in up-regulation of their expression and subsequent induction of cellular senescence in HepG2 cells. These effects were mediated by retinoic acid receptor β₂ whose promoter was also hypomethylated in the presence of ATRA. Therefore, ATRA can be considered as an epi-drug in cancer therapy.     

未折叠蛋白反应在胰腺癌发生发展中的作用及意义

胰腺癌是一种致死率相当高的消化系统肿瘤，其起病隐蔽导致早期诊断困难。近期研究发现，内质网应激 (endoplasmic reticulum stress，ERS) 状态下的未折叠蛋白反应 (unfolded protein response，UPR) 通路的调节作用，对于胰腺癌发生发展至关重要。UPR通路伴侣蛋白 GRP78 抑制了胰腺导管腺癌 (pancreatic adenocarcinoma，PDAC)细胞的凋亡，并增强了其化学抗性和耐药性。而 UPR 途径及其调节因子对于血管内皮生长因子 (vascular endothelial growth factor，VEGF) 的调节作用，有助于胰腺癌抵抗缺血缺氧环境。尝试靶向 UPR 途径关键调节因子的药物来控制胰腺癌的研究，可以为胰腺癌的治疗开辟新的途径。本文通过对近年来 UPR 在胰腺癌发生发展中的作用及意义进行综述，希望为通过调控 UPR 通路作为针对治疗胰腺癌的关键过程的一种新型抗癌方法研究提供参考。     

未折叠蛋白反应在胰腺癌发生发展中的作用及意义

胰腺癌是一种致死率相当高的消化系统肿瘤,其起病隐蔽导致早期诊断困难。近期研究发现,内质网应激 (endoplasmic reticulum stress,ERS) 状态下的未折叠蛋白反应 (unfolded protein response,UPR) 通路的调节作用,对于胰腺癌发生发展至关重要。UPR通路伴侣蛋白 GRP78 抑制了胰腺导管腺癌 (pancreatic adenocarcinoma,PDAC)细胞的凋亡,并增强了其化学抗性和耐药性。而 UPR 途径及其调节因子对于血管内皮生长因子 (vascular endothelial growth factor,VEGF) 的调节作用,有助于胰腺癌抵抗缺血缺氧环境。尝试靶向 UPR 途径关键调节因子的药物来控制胰腺癌的研究,可以为胰腺癌的治疗开辟新的途径。本文通过对近年来 UPR 在胰腺癌发生发展中的作用及意义进行综述,希望为通过调控 UPR 通路作为针对治疗胰腺癌的关键过程的一种新型抗癌方法研究提供参考。     

Mitochondrial Sulfide Quinone Oxidoreductase Prevents Activation of the Unfolded Protein Response in Hydrogen Sulfide

Hydrogen sulfide (H₂S) is an endogenously produced gaseous molecule with important roles in cellular signaling. In mammals, exogenous H₂S improves survival of ischemia/reperfusion. We have previously shown that exposure to H₂S increases the lifespan and thermotolerance in Caenorhabditis elegans, and improves protein homeostasis in low oxygen. The mitochondrial SQRD-1 (sulfide quinone oxidoreductase) protein is a highly conserved enzyme involved in H₂S metabolism. SQRD-1 is generally considered important to detoxify H₂S. Here, we show that SQRD-1 is also required to maintain protein translation in H₂S. In sqrd-1 mutant animals, exposure to H₂S leads to phosphorylation of eIF2α and inhibition of protein synthesis. In contrast, global protein translation is not altered in wild-type animals exposed to lethally high H₂S or in hif-1(ia04) mutants that die when exposed to low H₂S. We demonstrate that both gcn-2 and pek-1 kinases are involved in the H₂S-induced phosphorylation of eIF2α. Both ER and mitochondrial stress responses are activated in sqrd-1 mutant animals exposed to H₂S, but not in wild-type animals. We speculate that SQRD-1 activity in H₂S may coordinate proteostasis responses in multiple cellular compartments.     

Homocysteine Supplementation Attenuates the Unfolded Protein Response in a Murine Nutritional Model of Steatohepatitis

Hyperhomocysteinemia has been correlated with hepatic steatosis and activation of the unfolded protein response (UPR), yet a causal relationship has not been established. Although methionine and choline are essential components of homocysteine metabolism, the role of homocysteine in the pathogenesis of a methionine- and choline-deficient (MCD) diet remains unknown. We explored the effects of homocysteine supplementation on hepatic steatosis and the UPR in mice fed a control or MCD diet. Mice fed the MCD diet developed severe hyperhomocysteinemia and activation of the hepatic UPR. Supplementing the MCD diet with homocysteine attenuated the MCD diet-induced hepatic UPR activation and other injurious effects of the MCD diet including hepatic cholesterol accumulation, weight loss, and plasma ALT elevation. Homocysteine supplementation replenished the MCD diet-induced depletion of hepatic S-adenosylmethionine (SAM). Depleting SAM in HepG2 cells using MAT1α siRNA or cycloleucine resulted in enhanced activation of the UPR upon exposure to thapsigargin. Mice fed a control diet supplemented with homocysteine had a 3-fold elevation in plasma homocysteine level by 2 weeks and 6-fold elevation by 6 weeks but demonstrated no other pathophysiologic change. In summary, we found that homocysteine attenuates MCD diet-induced hepatic UPR activation, likely via repletion of hepatic SAM. Furthermore, homocysteine supplementation alone does not cause hepatic steatosis or UPR activation despite inducing hyperhomocysteinemia. These studies indicate that although hyperhomocysteinemia is often associated with hepatic steatosis and UPR activation, these effects may be a secondary response rather than a direct effect of homocysteine.