基于动脉管壁的脂联素球状域保护机理

脂肪细胞分泌产物脂联素(adiponectin,APN)的发现是脂肪内分泌学研究领域的重大进展。它主要通过与相应受体结合,发挥相应的生物学效应,且其心血管保护作用目前已成为研究热点。动脉管壁上也存在其受体,在此基础上,将APN活性区域的脂联素球状域(globular domain of adiponectin,gAd)设计为新靶点,研究其对动脉管壁的保护作用及其相关机理,将为动脉粥样硬化疾病的防治提供新方案。     

Secondary structure prediction and folding of globular protein: refolding of ferredoxin

The physicochemical mechanism of protein folding has been elucidated by the island model, describing a growth type of folding. The folding pathway is closely related with nucleation on the polypeptide chain and thus the formation of small local structures or secondary structures at the earliest stage of folding is essential to all following steps. The island model is applicable to any protein, but a high precision of secondary structure prediction is indispensable to folding simulation. The secondary structures formed at the earliest stage of folding are supposed to be of standard form, but they are usually deformed during the folding process, especially at the last stage, although the degree of deformation is different for each protein. Ferredoxin is an example of a protein having this property. According to X-ray investigation (1FDX), ferredoxin is not supposed to have secondary structures. However, if we assumed that in ferredoxin all the residues are in a coil state, we could not attain the correct structure similar to the native one. Further, we found that some parts of the chain are not flexible, suggesting the presence of secondary structures, in agreement with the recent PDB data (1DUR). Assuming standard secondary structures (-helices and -strands) at the nonflexible parts at the early stage of folding, and deforming these at the final stage, a structure similar to the native one was obtained. Another peculiarity of ferredoxin is the absence of disulfide bonds, in spite of its having eight cysteines. The reason cysteines do not form disulfide bonds became clear by applying the lampshade criterion, but more importantly, the two groups of cysteines are ready to make iron complexes, respectively, at a rather later stage of folding. The reason for poor prediction accuracy of secondary structure with conventional methods is discussed.     

Characterization of the weak calcium binding of trimeric globular adiponectin

Adiponectin is secreted from adipose tissue and functions as a protein hormone in regulating glucose metabolism and fatty acid catabolism. Adiponectin plays an important role as a novel risk factor and potential diagnostic and prognostic biomarker in cancer. Crystal structures of globular adiponectin have been resolved with three calcium‐binding sites on the top of its central tunnel. However, the calcium‐binding property of adiponectin remains elusive. Mouse globular adiponectin was cloned into pET11a and expressed in Escherichia coli. The folding of adiponectin was indicated by the spread of resonances in HSQC spectrum. Luminescence resonance energy transfer was used to obtain the binding constant (K_d) of Tb³⁺ and the inhibitor constant (K_i) of Ca²⁺ for globular adiponectin. The obtained calcium‐binding affinity to adiponectin is relatively low (~2 mM), which indicates that the high concentration of adiponectin in circulating system may function as calcium storage bank and buffer the free calcium concentration. Copyright © 2012 John Wiley & Sons, Ltd.     

Accelerated simulation of unfolding and refolding of a large single chain globular protein

We have developed novel strategies for contracting simulation times in protein dynamics that enable us to study a complex protein with molecular weight in excess of 34 kDa. Starting from a crystal structure, we produce unfolded and then refolded states for the protein. We then compare these quantitatively using both established and new metrics for protein structure and quality checking. These include use of the programs Concoord and Darvols. Simulation of protein-folded structure well beyond the molten globule state and then recovery back to the folded state is itself new, and our results throw new light on the protein-folding process. We accomplish this using a novel cooling protocol developed for this work.     

Crystal structure of a single-chain trimer of human adiponectin globular domain

Adiponectin is increasingly recognized as a potential therapeutic agent for the treatment of diabetes and other metabolic diseases. It circulates in plasma as homotrimers and higher-order oliogomers of homotrimers. To facilitate the production of active recombinant adiponectin as a therapeutic tool, we designed a single-chain globular domain adiponectin (sc-gAd) in which three monomer sequences are linked together in tandem to form one contiguous polypeptide. Here, we present the crystal structure of human sc-gAd at 2.0 Å resolution. The structure reveals a similar trimeric topology to that of mouse gAd protein. Trimer formation is further rigidified by three calcium ions.     

Anti-inflammatory activity of a globular adiponectin function on RAW 264 cells stimulated by lipopolysaccharide from Aggregatibacter actinomycetemcomitans

Adiponectin is an adipokine with potent anti-inflammatory properties. We previously reported that a globular adiponectin (gAd) suppresses Aggregatibacter actinomycetemcomitans lipopolysaccharide-induced nuclear factor-κB activity, suggesting an anti-inflammatory effect of gAd. In this study, we investigated whether gAd is able to modulate the effect of A. actinomycetemcomitans lipopolysaccharide on cytokine induction in a murine macrophage cell line (RAW 264). The phosphorylation of p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, extracellular signal-regulated kinase, and IκB kinase α/β and the degradation of IκB, which were induced by A. actinomycetemcomitans lipopolysaccharide intoxication, were clearly reduced in gAd-pretreated RAW 264 cells compared with the untreated cells. Expression levels of tumor necrosis factor (TNF)-α and interleukin-10 (IL-10) mRNA were assessed by real-time PCR. Cell-free supernatants were collected after 12 h of stimulation and analyzed by enzyme-linked immunosorbent assay for TNF-α and IL-10. Pretreatment with gAd significantly inhibited the A. actinomycetemcomitans lipopolysaccharide-induced TNF-α mRNA expression and protein secretion. In contrast, pretreatment with gAd significantly enhanced the A. actinomycetemcomitans lipopolysaccharide-induced IL-10 mRNA expression and protein secretion. These data suggest a mechanism for the anti-inflammatory activity of gAd in local inflammatory lesions, such as periodontitis.     

Stimulated HSP90 binding to eNOS and activation of the PI3-Akt pathway contribute to globular adiponectin-induced NO production: vasorelaxation in response to globular adiponectin

The present study examined potential interactions between endothelial NO synthase (eNOS), heat shock protein (HSP)90, and Akt in vascular endothelial cells stimulated with globular adiponectin to produce nitric oxide (NO). Globular adiponectin-induced eNOS phosphorylation was accompanied by eNOS-HSP90-Akt complex formation, resulting in a dose-dependent increase in NO release. Globular adiponectin stimulated binding of HSP90 to eNOS, and inhibition of HSP90 significantly suppressed globular adiponectin-stimulated NO release. Globular adiponectin also caused Akt phosphorylation, and inhibition of PI3 kinase significantly suppressed globular adiponectin-stimulated NO release. This study also examined whether globular adiponectin really induces endothelial-dependent vasodilation using rings from rat thoracic aorta. It was observed that globular adiponectin caused dose-dependent vasorelaxation in the aorta. These results indicate that stimulated HSP90 binding to eNOS and activation of the PI3-Akt pathway contribute to globular adiponectin-induced eNOS phosphorylation and NO production, and to endothelium-dependent vasorelaxation.     

Protein refolding using chemical refolding additives

In laboratories and manufacturing settings, a rapid and inexpensive method for the preparation of a target protein is crucial for promoting resesrach in protein science and engineering. Inclusion-body-based protein production is a promising method because high yields are achieved in the upstream process, although the refolding of solubilized, unfolded proteins in downstream processes often leads to significantly lower yields. The most challenging problem is that the effective condition for refolding is protein dependent and is therefore difficult to select in a rational manner. Accordingly, considerable time and expense using trial-and-error approaches are often needed to increase the final protein yield. Furthermore, for certain target proteins, finding suitable conditions to achieve an adequate yield cannot be obtained by existing methods. Therefore, to convert such a troublesome refolding process into a routine one, a wide array of methods based on novel technologies and materials have been developed. These methods select refolding conditions where productive refolding dominates over unproductive aggregation in competitive refolding reactions. This review focuses on synthetic refolding additives and describes the concepts underlying the development of reported chemical additives or chemical-additive-b     

The mechanism of folding of globular proteins. Suitability of a penicillinase from Staphylococcus Aureus as a model for refolding studies.

1. A homogeneous preparation of penicillinase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) was isolated and purified from cultures of Staphylococcus aureus by a simple two-stage procedure. 2. The native protein contains 20-30% helix as determined by optical-rotatory-dispersion and circular-dichroism measurements. Some 54(+/-5)% of the 13 tyrosine residues are exposed to solvent molecules of diameter 0.44 and 0.94 nm. 3. Conditions that allow full recovery of enzymic activity and native conformation from the fully unfolded state in 4M-guanidinium chloride were defined. 4. Refolding of the protein was shown to be inhibited by intermolecular interaction, by small changes in ionization and by low concentrations (0.025 M) of phenol.     

Calcium-induced refolding of the calmodulin V136G mutant studied by NMR spectroscopy: evidence for interaction between the two globular domains

The Ca(2+) titration of the (15)N-labeled mutant V136G calmodulin has been monitored using (1)H-(15)N HSQC NMR spectra. Up to a [Ca(2+)]/[CaM] ratio of 2, the Ca(2+) ions bind predominantly to sites I and II on the N-domain in contrast with the behavior of the wild-type calmodulin where the C-terminal domain has the higher affinity for Ca(2+). Surprisingly, the Ca(2+)-binding affinity for the N-domain in the mutant calmodulin is greater than that for the N-domain in the wild-type protein. The mutated C-domain is observed as a mixture of unfolded, partially folded (site III occupied), and native-like folded (sites III and IV occupied) conformations, with relative populations dependent on the [Ca(2+)]/[CaM] ratio. The occupancy of site III independently of site IV in this mutant shows that the cooperativity of Ca(2+) binding in the C-domain is mediated by the integrity of the domain structure. Several NH signals from residues in the Ca(2+)-bound N-domain appear as two signals during the Ca(2+) titration indicating separate species in slow exchange, and it can be deduced that these result from the presence and absence of interdomain interactions in the mutant. It is proposed that an unfolded part of the mutated C-domain interacts with sites on the N-domain that normally bind to target proteins. This would also account for the increase in the Ca(2+) affinity for the N-domain in the mutant compared with the wild-type calmodulin. The results therefore show the wide-ranging effects of a point mutation in a single Ca(2+)-binding site, providing details of the involvement of individual residues in the calcium-induced folding reactions.     

Access to gram scale amounts of functional globular adiponectin from E. coli inclusion bodies by alkaline-shock solubilization

The adipose tissue derived protein adiponectin exerts anti-diabetic, anti-inflammatory and anti-atherosclerotic effects. Adiponectin serum concentrations are in the microgram per milliliter range in healthy humans and inversely correlate with obesity and metabolic disorders. Accordingly, raising circulating adiponectin levels by direct administration may be an intriguing strategy in the treatment of obesity-related metabolic disorders. However production of large amounts of recombinant adiponectin protein is a primary obstacle so far.Here, we report a novel method for large amount production of globular adiponectin from E. coli inclusion bodies utilizing an alkaline-shock solubilization method without chaotropic agents followed by precipitation of the readily renaturing protein. Precipitation of the mildly solubilized protein capitalizes on advantages of inclusion body formation. This approach of inclusion body protein recovery provides access to gram scale amounts of globular adiponectin with standard laboratory equipment avoiding vast dilution or dialysis steps to neutralize the pH and renature the protein, thus saving chemicals and time. The precipitated protein is readily renaturing in buffer, is of adequate purity without a chromatography step and shows biological activity in cultured MCF7 cells and significantly lowered blood glucose levels in mice with streptozotocin induced type 1 diabetes.     

Surfactant-induced refolding of lysozyme

The surfactant-lysozyme interaction was investigated by circular dichroism, fluorescence, UV, dynamic light scattering, surface tension, turbidity measurements and lysozyme activity assay. A new way of refolding of lysozyme was found. It was shown that the lysozyme unfolded by anionic surfactants could be renatured by adding cationic surfactants. That is, lysozyme formed precipitate with anionic surfactants, the precipitates could be dissolved by adding a cationic surfactant solution, and then the lysozyme was refolded to its native state spontaneously. Different couples of anionic surfactants and cationic surfactants including C10SO3/C10NE, C12SO3/C10NE, C10SO3/C12NE, C10SO3/C12NB, C10SO4/C10NE and C12SO4/C10NE (C(n)SO3, C(n)SO4, C(n)NE and C(n)NB represent sodium alkyl sulfonate/sulfate, alkyl triethyl/butyl ammonium bromide respectively) were investigated, all of them gave similar results. The results were explained in terms of the differences between the interaction of anionic-cationic surfactants and that of surfactant-lysozyme. It was thought that the formation of mixed micelles of anionic-cationic surfactants is a more favorable process than that of lysozyme-surfactant complexes, which induces the dissociation of lysozyme-surfactant complexes when cationic surfactants were added.     

Preparative protein refolding

The rapid provision of purified native protein underpins both structural biology and the development of new biopharmaceuticals. The dominance of Escherichia coli as a cellular biofactory depends on technology for solubilizing and refolding proteins that are expressed as insoluble inclusion bodies. Such technology must be scale invariant, easily automated, generic for a broad range of similar proteins and economical. Refolding methods relying on denaturant dilution and column-based approaches meet these criteria. Recent developments, particularly in column-based methods, promise to extend the range of proteins that can be refolded successfully. Developments in preparing denatured purified protein and in the analysis of protein refolding products promise to remove bottlenecks in the overall process. Combined, these developments promise to facilitate the rapid and automated determination of appropriate refolding conditions and to simplify scale-up.     

可溶性肿瘤坏死因子受体II-脂联素球部融合蛋白的真核表达及生物学活性检测

本研究设计和构建了一种人肿瘤坏死因子受体II胞外区与人脂联素球部的融合基因sTNFRII-gAD,且相应的融合蛋白在哺乳动物细胞BHK-21S的无血清培养体系中实现了表达,并对该融合蛋白进行了初步鉴定。首先,用RT-PCR方法从人的外周血淋巴细胞总RNA中扩增人肿瘤坏死因子II型受体胞外区基因片段,与脂联素球部基因片段融合,克隆至pAAV2neo表达载体中,构建成pAAV2neo-sTNFRII-gAD。随后,用pAAV2neo-sTNFRII-gAD转染BHK-21S细胞获得G418抗性细胞BHK-21S/pAAV2neo-sTNFRII-gAD;然后,将原来含有血清的培养液换成无血清的化学成分限定的培养液,细胞从贴壁培养方式转换成悬浮培养方式;最后,收集BHK-21S/pAAV2neo-sTNFRII-gAD无血清悬浮培养24h后的培养上清,进行sTNFRII-gAD融合蛋白的鉴定分析。酶切鉴定和测序结果显示,所构建的pAAV2neo-sTNFRII-gAD质粒结构正确,sTNFRII-gAD序列与预期一致;分别用抗人肿瘤坏死因子受体II和抗人脂联素球部的单克隆抗体检测pAAV2neo-sTNFRII-gAD瞬时转染的BHK-21S细胞,免疫荧光呈现阳性;免疫印迹分析在pAAV2neo-sTNFRII-gAD稳定转染的BHK-21S细胞上清中检测到sTNFRII-gAD融合蛋白的表达,并以单体、三聚体和三聚体以上的多聚体形式存在。活性测定结果表明,sTNFRII-gAD融合蛋白具有显著抑制TNFα杀伤L929细胞的活性。因此,本研究为下一步大量制备sTNFRII-gAD融合蛋白用于体内外功能研究提供了良好基础。     

Topology of globular proteins

This paper inquires whether it is reasonable to expect the native structure of proteins to be “knotted”. To this end, some topological properties of polypeptides containing disulfide bridges are discussed using notions from mathematical knot theory and graph theory. The probability of occurrence of knots in random cyclic polymers is calculated as a function of chain length by elementary Monte Carlo methods. The implications of this for protein renaturation and for determining the tertiary structure of proteins are discussed.     

Thermodynamics of globular proteins

The analysis of temperature-induced unfolding of proteins in aqueous solutions was performed. Based on the data of thermodynamic parameters of protein unfolding and using the method of semi-empirical calculations of hydration parameters at reference temperature 298 K, we obtained numerical values of enthalpy, free energy, and entropy which characterize the unfolding of proteins in the ‘gas phase’. It was shown that specific values of the energy of weak intramolecular bonds (?H^int), conformational free energy (?G^conf) and entropy (?S^conf) are the same for proteins with molecular weight 7–25 kDa. Using the energy value (?H^int) and the proposed approach for estimation of the conformational entropy of native protein (S^NC), numerical values of the absolute free energy (G^NC) were obtained.     

Copper refolding of prion protein

We have shown previously that normal mouse prion protein (MoPrP) binds copper ions during protein refolding and acquires antioxidant activity. In this report, we probe the structure of the copper refolded form of MoPrP to determine how copper binding alters the secondary and tertiary features of the protein. Circular dichroism showed that recombinant MoPrP prepared in the presence of copper (as Cu(++)) showed an increased signal in the 210-220 nm range of the spectrum. Changes in protein conformation were localised to the N-terminal region of MoPrP using a panel of antibodies to assess epitope accessibility. The copper refolded recombinant prion protein had reduced proteinase K (PK) sensitivity when compared to the non-copper liganded form. Reduced PK sensitivity was not due to aggregation however as high resolution electron microscopy showed a homogenous preparation with little aggregate when compared to the non-copper form. Finally, disruption of the single disulphide linkage in MoPrP significantly diminished the antioxidant activity of the copper refolded form suggesting that activity was not solely dependent on bound copper but also on a conformation enabled by the formation of the disulphide bond.     

Chaperone-mediated refolding of recombinant prochymosin

It has been verified that prochymosin is characterized by a two-stage refolding: dilution of unfolded protein into pH 11 buffer followed by neutralization at pH 8; the high-pH step is indispensable. Here we demonstrate that one-stage refolding around pH 8 can be achieved when GroE or 10-fold molar excess (rather than catalytic concentration) of protein disulfide isomerase (PDI) over prochymosin is present. The helping effect varies with the oxidation states of prochymosin. GroE and PDI increase the reactivation of the unfolded, partially reduced and the unfolded, oxidized prochymosin from 5% to 40% and from 50% to 100%, respectively. For the unfolded and fully reduced prochymosin, GroE does not have a positive effect, whereas PDI promotes renaturation from 2% to 28%. Based on our previous and present observations, we propose that at pH 8 there may be two kinds of incorrect interactions within and between prochymosin polypeptides leading to unproductive pathways: one prevents disulfide rearrangement, which can be avoided by high pH; the other interferes with acquisition of native conformation, which can be relieved by GroE and PDI.     

Autocrine and paracrine modulation of microRNA-155 expression by globular adiponectin in RAW 264.7 macrophages: Involvement of MAPK/NF-κB pathway

Adiponectin, a hormone produced from adipose tissue, regulates various biological responses, including inflammation and many metabolic processes. MicroRNAs control expression of diverse target genes and various physiological responses. Many of these responses are commonly regulated by adiponectin. However, effects of adiponectin on microRNAs regulation are largely unknown. Herein we demonstrated that globular adiponectin induces increase in miR-155 expression, which plays an important role in inflammatory response, in RAW 264.7 macrophages. We further showed that this effect was modulated by and MANK/NF-κB dependent mechanisms. These results suggest that miR-155 would be a novel promising target mediating adiponectin-induced various biological responses.     

Investigation of protein refolding: a special feature of native structure responsible for refolding ability

A possible origin of the refolding ability of globular proteins is discussed. It is shown that the structure of native proteins has a special feature, namely, that this is the only structure in which the short overlapping segments of the polypeptide chain are in one of the significantly stable conformations of the oligopeptides with the same amino acid sequences as segments themselves. It is shown that this special feature is responsible for the refolding ability of proteins. A simple formula is given for the estimation of the time, t, necessary for the spontaneous formation of a refolding nucleus by a certain segment and it is shown that the segment which has the smallest t value, will serve as a refolding nucleus. It is suggested that natural selection which ensures the maintenance of the native structure of globular proteins automatically results in the refolding ability of proteins regardless of the biological relevance of this ability.