A SELDI‐TOF MS procedure for the detection,quantitation, and preliminary characterization of low‐molecular‐weight recombinant proteins expressed in transgenic plants

We describe a SELDI‐TOF MS procedure for the rapid detection and quantitation of low‐molecular‐weight recombinant proteins expressed in plants. Transgenic lines of potato (Solanum tuberosum L.) expressing the clinically useful protein bovine aprotinin or the cysteine protease inhibitor corn cystatin II were generated by Agrobacterium tumefaciens‐mediated transformation, and then used as test material for the analyses. Real‐time RT‐PCR amplifications and detection of the recombinant proteins by immunoblotting were first conducted for transformed potato lines accumulating the proteins in different cell compartments. Both proteins were found at varying levels in leaves, depending on their final cellular destination and transgene expression rate. These conclusions drawn from standard immunodetection assays were easily confirmed by SELDI‐TOF MS comparative profiling, after immobilizing the leaf proteins of control and transformed lines on protein biochips for weak cationic exchange. This procedure, carried out in less than 2 h, allows for the rapid comparison of recombinant protein levels in transgenic plant lines. The molecular weight of immobilized proteins can also be determined directly from the MS spectra, thus providing a simple way to assess the structural integrity and homogeneity of recombinant proteins in planta, and to identify the most suitable cellular compartments for their heterologous production.     

The role of the Val57 amino‐acid residue in the hinge loop of the human cystatin C. Conformational studies of the beta2‐L1‐beta3 segments of wild‐type human cystatin C and its mutants

Human cystatin C (HCC) is one of the amyloidogenic proteins to be shown to oligomerize via a three‐dimensional domain swapping mechanism. This process precedes the formation of a stable dimer and proceeds particularly easily in the case of the L68Q mutant. According to the proposed mechanism, dimerization of the HCC precedes conformational changes within the β2 and β3 strands. In this article, we present conformational studies, using circular dichroism and MD methods, of the β2‐L1‐β3 (His43‐Thr72) fragment of the HCC involved in HCC dimer formation. We also carried out studies of the β2‐L1‐β3 peptide, in which the Val57 residue was replaced by residues promoting β‐turn structure formation (Asp, Asn, or Pro). The present study established that point mutation could modify the structure of the L1 loop in the β‐hairpin peptide. Our results showed that the L1 loop in the peptide excised from human cystatin C is broader than that in cystatin C. In the HCC protein, broadening of the L1 loop together with the unfavorable L68Q mutation in the hydrophobic pocket could be a force sufficient to cause the partial unfolding and then the opening of HCC or its L68Q mutant structure for further dimerization. We presume further that the Asp57 and Asn57 mutations in the L1 loop of HCC could stabilize the closed form of HCC, whereas the Pro57 mutation could lead to the opening of the HCC structure and then to dimer/oligomer formation. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 373–383, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

过表达PDI对毕赤酵母分泌淀粉样蛋白的影响——以胱抑素为例

[目的] 本研究旨在结合酵母菌蛋白质二硫键异构酶（protein disulfide isomerase,PDI）与其底物蛋白鸡胱抑素C （chicken cystatin C,cC）在酵母中的共表达,理解PDI影响外源蛋白合成与表达的调控规律。运用转录组深度测序技术（RNA-Seq）筛选差异基因,调取并鉴定影响cC表达的关键基因,为解析外源蛋白高效表达机制,改造工程菌株提供理论支撑。[方法] 以巴斯德毕赤酵母GS115、GS115-cC为出发菌株,采用电转的方法将携带PDI编码基因的载体pPIC3.5K转入到GS115/GS115-cC菌株,使其在菌株中过表达,研究过表达PDI对cC表达的影响。采用RNA-Seq深度测序方法,研究重组毕赤酵母基因表达差异情况。并结合KEGG注释结果对数据进行分析,挑选差异显著表达基因进行验证,初步明确其在蛋白表达调控方面的功能。[结果] 本研究通过构建过表达PDI重组毕赤酵母菌株,使得外源蛋白cC的表达量显著增加。利用RNA-seq技术分析过表达PDI菌株与正常菌株的差异,最终筛选了373个差异表达基因,其中有122个差异基因注释到KEGG生物通路,包括12个基因注释到蛋白质转运和分解代谢途径,21个基因注释到蛋白质折叠分选和降解途径,以及24个基因参与蛋白质的翻译途径等。[结论] 在毕赤酵母中过表达PDI能显著增加外源蛋白cC的表达量。通过对过表达与正常表达PDI的毕赤酵母基因的表达谱分析,初步确定了其中一些转录情况变化显著的基因,明确了它们参与的细胞途径和信号通路,为改造具有高效率表达淀粉样蛋白的酵母菌株奠定基础。     

Steered molecular dynamics simulations on the binding of the appendant structure and helix-β2 in domain-swapped human cystatin C dimer

We have performed steered molecular dynamics (SMD) simulations to investigate the dissociation process between the appendant structure (AS) and helix-β2 in human cystatin C dimer. Energy change during SMD showed that electrostatic interactions, including hydrogen bonds and salt bridges, were the dominant interactions to stabilize the two parts of the dimer. Furthermore, our data indicated that residues, Asn35, Asp40, Ser44, Lys75, and Arg93 play significant roles in the formation of these electrostatic interactions. Docking studies suggested that the interactions between AS and β2-helix were formed following domain swapping and were responsible for stabilizing the structure of the domain-swapped dimer.     

Cathepsin L Plays an Important Role in the Lysosomal Degradation of L-Lactate Dehydrogenase

A cystatin α-sensitive cysteine proteinase that plays an important role in the lysosomal inactivation and degradation of L-lactate dehydrogenase (LDH) was purified by column chromatography from an ammonium sulfate precipitate of lysosome extract prepared from rat livers. It was eluted with marked delay from cathepsins B and H in a Sephacryl S-200 column by its specific interaction with the gel, and then effectively separated from cathepsins B and H and other proteins. It was eluted with 0.5 M NaCl after washing with 0.2 M NaCl in a CM-Sephadex column, indicating that it showed the same elution behavior as cathepsin L from the CM-Sephadex column. It had activity to hydrolyze z-Phe-Arg-NH-Mec, a synthetic substrate for cysteine proteinases, including cathepsins B and L. The N-terminal sequences of the final preparation of LDH-inactivating enzyme were identical with those of rat cathepsin L. Inactivation and degradation of LDH by the final preparation were observed and effectively inhibited by a low level of cystatin α as well as a general cysteine proteinase inhibitor, leupeptin or (L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine (3-methylbutyl)amide (E-64-c). From these results, it is concluded that cathepsin L plays a critical role in the lysosomal degradation of native LDH.     

抗胱抑素C鸡卵黄IgY抗体的制备及鉴定

目的:制备高效价、高特异性的抗人胱抑素 C 鸡卵黄 IgY 抗体,并对其基本特性进行分析和鉴定.方法:以人胱抑素 C 为抗免疫产蛋的罗曼鸡,采用水稀释-盐析法提取及纯化 IgY 抗体,采用蛋白质定量、SDS-PAGE、West?ern 印迹和 ELISA 法对 IgY 抗体进行分析和鉴定.结果:免疫后14 d 即可从鸡冠血中检测出抗胱抑素 C 的特异性抗体,抗体效价在28 d 达最高峰(1∶32000),并可维持2个月以上;收集高效价时的免疫鸡蛋,制备鸡卵黄抗体 IgY;还性 SDS-PAGE 显示抗体 IgY 为相对分子质量分别为65×103和21×103的2条带,抗体纯度可达92%,得率为每个鸡蛋36.5 mg,抗体检出敏感度为15.63 ng/mL;Western 印迹证明该抗体具有高度特异性.结论:制备了抗胱抑素 C 的高效价、高特异性 IgY 抗体.     

Tumor necrosis factor alpha promotes the expression of immunosuppressive proteins and enhances the cell growth in a human bone marrow-derived stem cell culture

Mesenchymal stem cells (MSCs) are widely used in experimental treatments for various conditions that involve normal tissue regeneration via inflammatory repair. It is known that MSCs can secrete multiple soluble factors and suppress inflammation. Even though the effect of MSCs on inflammation has been extensively studied, the effect of inflammation on MSCs is poorly understood. One of the major cytokines released at the site of inflammation is tumor necrosis factor alpha (TNF-α) which is known to induce MSC invasion and proliferation. Therefore, we wanted to test the effects of TNF-α exposure on MSCs derived from human bone marrow. We found, as expected, that cell proliferation was significantly enhanced during TNF-α exposure. However, according to the cell surface marker analysis, the intensity of several antigens in the minimum criteria panel for MSCs proposed by International Society of Cellular Therapy (ISCT) was decreased dramatically, and in certain cases, the criteria for MSCs were not fulfilled. In addition, TNF-α exposure resulted in a significant but transient increase in human leukocyte antigen and CD54 expression. Additional proteomic analysis by two-dimensional difference gel electrophoresis and mass spectrometry revealed three proteins whose expression levels decreased and 8 proteins whose expression levels increased significantly during TNF-α exposure. The majority of these proteins could be linked to immunosuppressive and signalling pathways. These results strongly support reactive and immunosuppressive activation of MSCs during TNF-α exposure, which might influence MSC differentiation stage and capacity.     

Probing the structural interactions between methotrexate and dexamethasone with muscle cystatin: a biophysical study

Abstract

Drug protein interactions have gained considerable attention over the past many years. In the current communication the association of muscle cystatin (MC) with anti-rheumatic drugs methotrexate and dexamethasone was studied by thiol proteinase inhibitor assay, ultra violet (UV) absorption, fluorescence spectroscopy, and fluorescence transform infra-red spectroscopy (FTIR). A static pattern of quenching was noticed between muscle cystatin and methotrexate (MTX). Binding constant (K_a) of methotrexate to muscle cystatin was found to be 1?×?10^?7 M^?1 and the stoichiometry of binding was calculated to be one. Fluorescence measurement of the emission quenching reveals that the quenching process of cystatin by dexamethasone (DXN) was also static. The stoichiometry of binding and binding constant was also obtained. Additional evidence regarding MTX–MC and DXN–MC was obtained from UV spectroscopy and FTIR spectroscopic results. Such spectroscopic studies would help in modelling new candidate drugs for rheumatoid arthritis based on their cystatin binding profile.

Communicated by Ramaswamy H. Sarma     

Mechanism of Unfolding of Goat Lung Cystatin During Urea and Guanidine Hydrochloride Induced Denaturation

Cystatins essentially regulate lysosomal cysteine protease besides affecting several physiological processes. In the present study, denaturation of a high molecular weight cystatin (Mr 66.4 kDa) purified from goat lung (GLC-I) has been studied by monitoring its inhibitory activity, intrinsic fluorescence, circular dichroism (CD), and binding of ANS. It was found that increasing concentration of GdnHCl significantly enhances the inactivation and unfolding of the purified inhibitor (GLC-I) with complete loss of inhibitory activity at 4 M GdnHCl. Denaturation of GLC-I in the presence of GdnHCl is accompanied by red shift (15 nm) of the emission maximum as shown by intrinsic fluorescence. The inhibitory activity of GLC-I was increased by 1.5 fold at 2 M urea; however, it decreased with further increased of the urea concentration. Intrinsic fluorescence studies of GLC-I in the presence of 0–3 M urea shows blue shift of 5 nm, suggesting stabilization of the inhibitor followed by 5 nm red shift at higher concentration. ANS binding studies in the presence of urea indicate significant changes in the tertiary structure of the inhibitor. Thus, our result shows denaturation profile of GLC-I following simple two state transitions in the presence of GdnHCl while it proceeds through an intermediate state in the presence of urea.