Mechanism of pH‐sensitive polymer‐assisted protein refolding and its application in TGF‐β1 and KGF‐2

Refolding of proteins at high concentrations often results in non‐productive aggregation. This study, through a unique combination of spectroscopic and chromatographic analyzes, provides biomolecular evidence to demonstrate the ability of Eudragit S‐100, a pH‐responsive polymer, to enhance refolding of denatured‐reduced lysozyme at high concentrations. The addition of Eudragit in the refolding buffer significantly increases lysozyme refolding yield to 75%, when dilution refolding was conducted at 1 mg/mL lysozyme. This study shows evidence of an electrostatic interaction between oppositely charged lysozyme and the Eudragit polymer during refolding. This ionic complexing of Eudragit and lysozyme appears to shield exposed hydrophobic residues of the lysozyme refolding intermediates, thus minimizing hydrophobic‐driven aggregation of the molecules. Importantly, results from this study show that the Eudragit‐lysozyme bioconjugation does not compromise refolded protein structure, and that the polymer can be readily dissociated from the protein by ion exchange chromatography. The strategy was also applied to refolding of TGF‐β1 and KGF‐2. © 2009 American Institute of Chemical Engineers Biotechnol. Prog. 2009     

Integrin–TGF‐β crosstalk in fibrosis,cancer and wound healing

Accumulating evidence indicates that there is extensive crosstalk between integrins and TGF‐β signalling. TGF‐β affects integrin‐mediated cell adhesion and migration by regulating the expression of integrins, their ligands and integrin‐associated proteins. Conversely, several integrins directly control TGF‐β activation. In addition, a number of integrins can interfere with both Smad‐dependent and Smad‐independent TGF‐β signalling in different ways, including the regulation of the expression of TGF‐β signalling pathway components, the physical association of integrins with TGF‐β receptors and the modulation of downstream effectors. Reciprocal TGF‐β–integrin signalling is implicated in normal physiology, as well as in a variety of pathological processes including systemic sclerosis, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease and cancer; thus, integrins could provide attractive therapeutic targets to interfere with TGF‐β signalling in these processes.     

GC Separation of Enantiomers of Alkyl Esters of 2‐Bromo Substituted Carboxylic Acids Enantiomers on 6‐TBDMS‐2,3‐di‐alkyl‐ β‐ and γ‐Cyclodextrin Stationary Phases

The gas chromatographic separation of enantiomers of 2‐Br carboxylic acid derivatives was studied on four different 6‐TBDMS‐2,3‐di‐O‐alkyl‐ β‐ and ‐γ‐CD stationary phases. The differences in thermodynamic data {ΔH and –ΔS} for the 15 structurally related racemates were evaluated. The influence of structure differences in the alkyl substituents covalently attached to the stereogenic carbon atom, as well as in the ester group of the homologous analytes, and the selectivity of modified β‐ and γ‐ cyclodextrin derivatives was studied in detail. The cyclodextrin cavity size, as well as elongation of alkyl substituents in positions 2 and 3 of 6‐TBDMS‐β‐CD, also affected their selectivity. The quality of enantiomeric separations is influenced mainly by alkyl chains of the ester group of the molecule and this appears to be independent of the CD stationary phase used. In some cases the separations occur as the result of external adsorption rather than inclusion complexations with the chiral selector. It was found that the temperature dependencies of the selectivity factor were nonlinear. Chirality 26:279–285, 2014. © 2014 Wiley Periodicals, Inc.     

A novel approach to predicting protein structural classes in a (20–1)-D amino acid composition space

The development of prediction methods based on statistical theory generally consists of two parts: one is focused on the exploration of new algorithms, and the other on the improvement of a training database. The current study is devoted to improving the prediction of protein structural classes from both of the two aspects. To explore a new algorithm, a method has been developed that makes allowance for taking into account the coupling effect among different amino acid components of a protein by a covariance matrix. To improve the training database, the selection of proteins is carried out so that they have (1) as many non-homologous structures as possible, and (2) a good quality of structure. Thus, 129 representative proteins are selected. They are classified into 30 α, 30 β, 30 α + β, 30 α/β, and 9 ζ (irregular) proteins according to a new criterion that better reflects the feature of the structural classes concerned. The average accuracy of prediction by the current method for the 4 × 30 regular proteins is 99.2%, and that for 64 independent testing proteins not included in the training database is 95.3%. To further validate its efficiency, a jackknife analysis has been performed for the current method as well as the previous ones, and the results are also much in favor of the current method. To complete the mathematical basis, a theorem is presented and proved in Appendix A that is instructive for understanding the novel method at a deeper level. © 1995 Wiley-Liss, Inc.     

Association of bovine β‐casein protein variant I with milk production and milk protein composition

The aim of this study was to detect new polymorphisms in the bovine β‐casein (β‐CN) gene and to evaluate association of (new) β‐CN protein variants with milk production traits and milk protein composition. Screening of the β‐CN gene in genomic DNA from 72 Holstein Friesian (HF) bulls resulted in detection of 19 polymorphisms and revealed the presence of β‐CN protein variant I in the Dutch HF population. Studies of association of β‐CN protein variants with milk composition usually do not discriminate protein variant I from variant A2. Association of β‐CN protein variants with milk composition was studied in 1857 first‐lactation HF cows and showed that associations of protein variants A2 and I were quite different for several traits. β‐CN protein variant I was significantly associated with protein percentage and protein yield, and with α_s1‐casein (α_s1‐CN), α_s2‐casein (α_s2‐CN), κ‐casein (κ‐CN), α‐lactalbumin (α‐LA), β‐lactoglobulin (β‐LG), casein index and casein yield. Inferring β‐κ‐CN haplotypes showed that β‐CN protein variant I occurred only with κ‐CN variant B. Consequently, associations of β‐κ‐CN haplotype IB with protein percentage, κ‐CN, α‐LA, β‐LG and casein index are likely resulting from associations of κ‐CN protein variant B, while associations of β‐κ‐CN haplotype IB with α_s1‐CN and α_s2‐CN seem to be resulting from associations of β‐CN variant I.     

Posttranslational modification impact on the mechanism by which amyloid‐β induces synaptic dysfunction

Oligomeric amyloid‐β (Aβ) 1‐42 disrupts synaptic function at an early stage of Alzheimer's disease (AD). Multiple posttranslational modifications of Aβ have been identified, among which N‐terminally truncated forms are the most abundant. It is not clear, however, whether modified species can induce synaptic dysfunction on their own and how altered biochemical properties can contribute to the synaptotoxic mechanisms. Here, we show that a prominent isoform, pyroglutamated Aβ3(pE)‐42, induces synaptic dysfunction to a similar extent like Aβ1‐42 but by clearly different mechanisms. In contrast to Aβ1‐42, Aβ3(pE)‐42 does not directly associate with synaptic membranes or the prion protein but is instead taken up by astrocytes and potently induces glial release of the proinflammatory cytokine TNFα. Moreover, Aβ3(pE)‐42‐induced synaptic dysfunction is not related to NMDAR signalling and Aβ3(pE)‐42‐induced impairment of synaptic plasticity cannot be rescued by D1‐agonists. Collectively, the data point to a scenario where neuroinflammatory processes together with direct synaptotoxic effects are caused by posttranslational modification of soluble oligomeric Aβ and contribute synergistically to the onset of synaptic dysfunction in AD.     

The N‐terminal tripeptide of insulin‐like growth factor‐I protects against β‐amyloid‐induced somatostatin depletion by calcium and glycogen synthase kinase 3β modulation

The protective effects of insulin‐like growth factor I on the somatostatin (SRIF) system in the temporal cortex after β‐amyloid (Aβ) injury may be mediated through its N‐terminal tripeptide glycine‐proline‐glutamate (GPE). GPE is cleaved to cyclo[Pro‐Gly] (cPG), a metabolite suggested to mediate in neuroprotective actions. We evaluated the effects of GPE and cPG in the temporal cortex of Aβ25–35‐treated rats on SRIF and SRIF receptor protein and mRNA levels, adenylyl cyclase activity, cell death, Aβ25–35 accumulation, cytosolic calcium levels ([Ca²⁺]_c) and the intracellular signaling mechanisms involved. GPE and cPG did not change Aβ25–35 levels, but GPE partially restored SRIF and SRIF receptor 2 protein content and mRNA levels and protected against cell death after Aβ25–35 insult, which was coincident with Akt activation and glycogen synthase kinase 3β inhibition. In addition, GPE displaced glutamate from NMDA receptors and blocked the glutamate induced rise in cytosolic calcium in isolated rat neurons and moderately increased Ca²⁺ influx per se. Our findings suggest that GPE, but not its metabolite, mimics insulin‐like growth factor I effects on the SRIF system through a mechanism independent of Aβ clearance that involves modulation of calcium and glycogen synthase kinase 3β signaling.     

Regulation of interleukin‐1β by interferon‐γ is species specific,limited by suppressor of cytokine signalling 1 and influences interleukin‐17 production

Reports describing the effect of interferon‐γ (IFNγ) on interleukin‐1β (IL‐1β) production are conflicting. We resolve this controversy by showing that IFNγ potentiates IL‐1β release from human cells, but transiently inhibits the production of IL‐1β from mouse cells. Release from this inhibition is dependent on suppressor of cytokine signalling 1. IL‐1β and Th17 cells are pathogenic in mouse models for autoimmune disease, which use Mycobacterium tuberculosis (MTB), in which IFNγ and IFNβ are anti‐inflammatory. We observed that these cytokines suppress IL‐1β production in response to MTB, resulting in a reduced number of IL‐17‐producing cells. In human cells, IFNγ increased IL‐1β production, and this might explain why IFNγ is detrimental for multiple sclerosis. In mice, IFNγ decreased IL‐1β and subsequently IL‐17, indicating that the adaptive immune response can provide a systemic, but transient, signal to limit inflammation.     

Oligoethylene glycols prevent thermal aggregation of α‐chymotrypsin in a temperature‐dependent manner: Implications for design guidelines

Protein aggregation is problematic in various fields, where aggregation can frequently occur during routine experiments. This study showed that tetraethylene glycol (TEG) and tetraethylene glycol dimethyl ether (TEGDE) act as aggregation suppressors that have different unique properties from typical additives to prevent protein aggregation, such as arginine (Arg) and NaCl. Thermal aggregation of α‐chymotrypsin was well suppressed with the addition of both TEG and TEGDE. Interestingly, the suppressive effects of Arg and NaCl on thermal aggregation were almost unchanged when temperature was shifted from 65°C to 85°C, whereas both TEG and TEGDE significantly decreased the aggregation rate with increasing temperature. Note that the effects of TEG and TEGDE were higher than Arg above 75°C. This temperature‐dependent behavior of TEG and TEGDE provides a novel design guideline to develop aggregation suppressors for use at high temperature, i.e., the importance of the ethylene oxide group. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1325–1330, 2013     

Kinetics of the competitive reactions of isomerization and peptide bond cleavage at l‐α‐ and d‐β‐aspartyl residues in an αA‐crystallin fragment

d ‐β‐aspartyl (Asp) residue has been found in a living body such as aged lens crystallin, although l ‐α‐amino acids are constituents in natural proteins. Isomerization from l ‐α‐ to d ‐β‐Asp probably modulates structures to affect biochemical reactions. At Asp residue, isomerization and peptide bond cleavage compete with each other. To gain insight into how fast each reaction proceeds, the analysis requires the consideration of both pathways simultaneously and independently. No information has been provided, however, about these competitive processes because each reaction has been studied separately. The contribution of Asp isomers to the respective pathways has still been veiled. In this work, the two competitive reactions, isomerization and spontaneous peptide bond cleavage at Asp residue, were simultaneously observed and compared in an αA‐crystallin fragment, S⁵¹LFRTVLD⁵⁸SG⁶⁰ containing l ‐α‐ and d ‐β‐Asp58 isomers. The kinetics showed that the formation of l ‐ and d ‐succinimide (Suc) intermediate, as a first step of isomerization, was comparable at l ‐α‐ and d ‐β‐Asp. Although l ‐Suc was converted to l ‐β‐Asp, d ‐Suc was liable to return to the original d ‐β‐Asp, the reverse reaction marked enough to consider d ‐β‐Asp as apparently stable. d ‐β‐Asp was also resistant to the peptide bond cleavage. Such apparent less reactivity is probably the reason for gradual and abnormal accumulation of d ‐β‐Asp in a living body under physiological conditions. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Epitope structure and binding affinity of single chain llama anti‐β‐amyloid antibodies revealed by proteolytic excision affinity‐mass spectrometry

ß‐Amyloid (Aß) immunotherapy has become a promising strategy for reducing the level of Aß in brain. New immunological approaches have been recently proposed for rapid, early diagnosis, and molecular treatment of neurodegenerative diseases related to Alzheimer's Disease (AD). The combination of proteolytic epitope excision and extraction and mass spectrometry using digestion with various proteases has been shown to be an efficient tool for the identification and molecular characterization of antigenic determinants. Here, we report the identification of the Aβ epitope recognized by the variable domain of single chain llama anti‐Aβ‐antibodies, termed Aβ‐nanobodies, that have been discovered in the blood of camelids and found to be promising candidates for immunotherapy of AD. The epitope recognized by two Aβ‐specific nanobodies was identified by proteolytic epitope extraction‐ and excision‐mass spectrometry using a series of proteases (trypsin, chymotrypsin, GluC‐protease, and LysC‐protease). Matrix‐assisted laser desorption ionization – mass spectrometric analysis of the affinity – elution fraction provided the epitope, Aβ(17–28), in the mid‐ to carboxy‐terminal domain of Aβ, which has been shown to exert an Aß‐fibril inhibiting effect. Affinity studies of the synthetic epitope confirmed that the Aβ(17–28) peptide is the minimal fragment that binds to the nanobodies. The interactions between the nanobodies and full length Aβ(1–40) or Aβ‐peptides containing or lacking the epitope sequence were further characterized by enzyme linked immunosorbent assay and bioaffinity analysis. Determinations of binding affinities between the Aβ‐nanobodies and Aβ(1–40) and the Aβ(17–28) epitope provided K_D values of approximately 150 and 700 nmol, respectively. Thus, the knowledge of the epitope may be highly useful for future studies of Aβ‐aggregation (oligomerization and fibril formation) and for designing new aggregation inhibitors. Copyright © 2012 John Wiley & Sons, Ltd.     

17α-Ethyl-5β-estrane-3α,17β-diol, a biological marker for the abuse of norethandrolone and ethylestrenol in slaughter cattle

The metabolism of the illegal growth promoter ethylestrenol (EES) was evaluated in bovine liver cells and subcellular fractions of bovine liver preparations. Incubations with bovine microsomal preparations revealed that EES is extensively biotransformed into norethandrolone (NE), another illegal growth promoter. Furthermore, incubations of monolayer cultures of hepatocytes with NE indicated that NE itself is rapidly reduced to 17α-ethyl-5β-estrane-3α,17β-diol (EED). In vivo tests confirmed that, after administration of either EES or NE, EED is excreted as a major metabolite. Therefore, it was concluded that, both in urine and faeces samples, EED can be used as a biological marker for the illegal use of EES and/or NE. Moreover, by monitoring EED in urine or faeces samples, the detection period after NE administration is significantly prolonged. These findings were further confirmed by three cases of norethandrolone abuse in a routine screening program for forbidden growth promoters.     

SHC–α5β1 Integrin Interactions Regulate Breast Cancer Cell Adhesion and Motility

The oncogenic SHC proteins are signaling substrates for most receptor and cytoplasmic tyrosine kinases (TKs) and have been implicated in cellular growth, transformation, and differentiation. In tumor cells overexpressing TKs, the levels of tyrosine phosphorylated SHC are chronically elevated. The significance of amplified SHC signaling in breast tumorigenesis and metastasis remains unknown. Here we demonstrate that seven- to ninefold overexpression of SHC significantly altered interactions of cells with fibronectin (FN). Specifically, in human breast cancer cells overexpressing SHC (MCF-7/SHC) the association of SHC with α5β1 integrin (FN receptor) was increased, spreading on FN was accelerated, and basal growth on FN was reduced. These effects coincided with an early decline of adhesion-dependent MAP kinase activity. Basal motility of MCF-7/SHC cells on FN was inhibited relative to that in several cell lines with normal SHC levels. However, when EGF or IGF-I was used as the chemoattractant, the locomotion of MCF-7/SHC cells was greatly (approx fivefold) stimulated, while it was only minimally altered in the control cells. These data suggest that SHC is a mediator of the dynamic regulation of cell adhesion and motility on FN in breast cancer cells.     

Nanotubular structures developed from whey‐based α‐lactalbumin fractions for food applications

Whey proteins have high nutritional value providing use in dietary purposes and improvement of technological properties in processed foods. Functionality of the whey‐based α‐lactalbumin (α‐La) may be increased when assembled in the form of nanotubes, promising novel potential applications subject to investigation. The purpose of this study was to extract highly pure α‐La from whey protein isolate (WPI) and whey powder (WP) and to construct protein nanotubes from them for industrial applications. For protein fractionation, WPI was directly fed to chromatography, however, WP was first subjected to membrane filtration and the retentate fraction, whey protein concentrate (WPC), was obtained and then used for chromatographic separation. α‐La and, additionally β‐Lg, were purified at the same batches with the purities in the range of 95%–99%. After enzymatic hydrolysis, WPI‐based α‐La produced chain‐like and long nanotubules with ～20 nm width while WPC‐based α‐La produced thinner, miscellaneous, and fibril‐like nanostructures by self‐assembly. Raman and FT‐IR spectroscopies revealed that α‐La fractions, obtained from both sources and the nanostructures, developed using both fractions have some structural differences due to conformation of secondary structure elements. Nanotube formation induced gelation and nanotubular gel network entrapped a colorant uniformly with a transparent appearance. Dairy‐based α‐La protein nanotubules could be served as alternative gelling agents and the carriers of natural colorants in various food processes. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1301–1310, 2014