Proteomic analysis of the 14-3-3 family in Arabidopsis

In this study, various proteomics-based methods were utilized to examine the 14-3-3 protein family in Arabidopsis thaliana. A protein extract was prepared from an Arabidopsis hypocotyl suspension culture and analyzed by two-dimensional gel electrophoresis and immunoblotting with a 14-3-3 monoclonal antibody that recognizes multiple Arabidopsis isoforms. Protein spots that cross-reacted with the monoclonal antibody as well as the surrounding spots were analyzed by high performance liquid chromatography in conjunction with electrospray-tandem mass spectrometry. Nine separate spots contained 14-3-3s and each spot contained multiple 14-3-3 isoforms. Every isoform observed was verified by the identification of at least one isoform-specific peptide. Further analysis by mass spectrometry revealed that the isoforms Chi, Upsilon, Omega, Phi, and Lambda were acetylated on their N termini and no non-acetylated N termini were recovered. These data, together with the distribution of isoforms and the confirmation that 14-3-3s are not complexed during urea denaturing isoelectric focusing, supports the conclusion that Arabidopsis 14-3-3s are acetylated in vivo and are significantly affected by other post-translational modifications.     

Two‐compartment processing as a tool to boost recombinant protein production

Pichia pastoris is used extensively as a production platform for many recombinant proteins. The dissolved oxygen (DO) is one of the most important factors influencing protein production. The influence of the DO on productivity has not been studied independent from the feed rate. In this work, various DO levels were investigated independent from the feed rate. The model system was recombinant P. pastoris under the control of methanol‐induced alcohol oxidase promoter, which expressed HRP as the target protein. No significant effect was observed in terms of titer and specific productivity, which is a confirmation of the fact that the DO in a one‐compartment system cannot boost productivity for the model system under study. Hence, a two‐compartment system (a single reactor coupled with a plug flow reactor) was designed and implemented in order to apply oxygen‐related stress in the plug flow reactor and allow the cells to be recovered in the main reactor. Doing so, more than two‐fold increase in the titer and productivity and three‐fold increase in protein‐specific activity were achieved. Hence, partial application of oxygen‐related stress in the two‐compartment system was proposed as a process technology to enhance protein production.     

Investigating the in vivo activity of the DeaD protein using protein-protein interactions and the translational activity of structured chloramphenicol acetyltransferase mRNAs

Here, we report the use of an in vivo protein-protein interaction detection approach together with focused follow-up experiments to study the function of the DeaD protein in Escherichia coli. In this method, functions are assigned to proteins based on the interactions they make with others in the living cell. The assigned functions are further confirmed using follow-up experiments. The DeaD protein has been characterized in vitro as a putative prokaryotic factor required for the formation of translation initiation complexes on structured mRNAs. Although the RNA helicase activity of DeaD has been demonstrated in vitro, its in vivo activity remains controversial. Here, using a method called sequential peptide affinity (SPA) tagging, we show that DeaD interacts with certain ribosomal proteins as well as a series of other nucleic acid binding proteins. Focused follow-up experiments provide evidence for the mRNA helicase activity of the DeaD protein complex during translation initiation. DeaD overexpression compensates for the reduction of the translation activity caused by a structure placed at the initiation region of a chloramphenicol acetyltransferase gene (cat) used as a reporter. Deletion of the deaD gene, encoding DeaD, abolishes the translation activity of the mRNA with an inhibitory structure at its initiation region. Increasing the growth temperature disrupts RNA secondary structures and bypasses the DeaD requirement. These observations suggest that DeaD is involved in destabilizing mRNA structures during translation initiation. This study also provides further confirmation that large-scale protein-protein interaction data can be suitable to study protein functions in E. coli.     

A bioanalytical method for the proteome wide display and analysis of protein complexes from whole plant cell lysates

While protein interaction studies and protein network modeling come to the forefront, the isolation and identification of protein complexes in a cellular context remains a major challenge for plant science. To this end, a nondenaturing extraction procedure was optimized for plant whole cell matrices and the combined use of gel filtration and BN‐PAGE for the separation of protein complexes was studied. Hyphenation to denaturing electrophoresis and mass spectrometric analysis allows for the simultaneous identification of multiple (previously unidentified) protein interactions in single samples. The reliability and efficacy of the technique was confirmed (i) by the identification of well‐studied plant protein complexes, (ii) by the presence of nonplant interologs for several of the novel complexes (iii) by presenting physical evidence of previously hypothetical plant protein interactions and (iv) by the confirmation of found interactions using co‐IP. Furthermore practical issues concerning the use of this 2‐D BN/SDS‐PAGE display method for the analysis of protein–protein interactions are discussed.     

Prevalence of human T‐lymphotropic virus type 1 carriers among pregnant women in Hokkaido,Japan

As there is a risk of MTCT of HTLV‐1, the HSGP HTLV‐1 MTCT was organized in 2011. To determine how many pregnant women are infected with HTLV‐1 in Hokkaido, which is the northernmost and the second largest island in Japan with a population of 5 467 000 and 39 392 newborns in 2011, the HSGP HTLV‐1 MTCT asked all facilities that may care for pregnant women in Hokkaido in July 2013 to provide information on the number of pregnant women who underwent screening for anti‐HTLV‐1 antibody using particle agglutination or chemiluminescent enzyme immunoassay, and the numbers of those with positive, equivocal, and negative test results in the screening and confirmation tests using western blotting or PCR methods in 2012, respectively. A total of 111 facilities participated in this study and provided information on 33 617 pregnant women who underwent screening in 2012, corresponding to approximately 85% of all pregnant women who gave birth in Hokkaido in 2012. Of 81 candidates for a confirmation test because of positive (n = 77) or equivocal (n = 4) results on screening, 63 (78%) underwent the confirmation test and, finally, 34 (0.1%) and 33 563 (99.8%) women were judged to be HTLV‐1 carriers and non‐carriers, respectively. It was concluded that the prevalence rate of HTLV‐1 carriers was low, one per 1000 pregnant women in Hokkaido. Approximately 40 infants are born yearly to mothers infected with HTLV‐1 in Hokkaido.     

Identification of novel biomarker candidates for immunohistochemical diagnosis to distinguish low‐grade chondrosarcoma from enchondroma

Chondrosarcoma is the third most common primary bone cancer, requiring surgical resection. However, differentiation of low‐grade chondrosarcoma (grade 1) from enchondroma that is benign and only requires regular follow‐up is one of the most frequent diagnostic dilemmas facing orthopedic oncologists in clinical management. Although multiple techniques are applied to make the distinction, immunohistochemistry is an important ancillary technique, especially when a histopathological stain of specimen must be obtained in order to guarantee an accurate confirmation. Currently, no adequate immunohistochemical diagnostic protein biomarkers are available to distinguish low‐grade chondrosarcoma from enchondroma. To discover novel protein biomarker candidates, an LC‐MS/MS approach was applied to directly compare formalin‐fixed, paraffin‐embedded low‐grade chondrosarcoma with enchondroma tissue samples. The proteomics analysis revealed 17 protein biomarker candidates. A principle was developed to prioritize the candidates using category and ranking. An algorithm, prioritization index of biomarker candidates for immunohistochemistry on tissue specimens, was developed to rank the candidates inside each category. Using the proteomics data and bioinformatics results, the prioritization index of biomarker candidates for immunohistochemistry on tissue revealed periostin as a top candidate. Immunohistochemical staining of periostin in 23 low‐grade chondrosarcoma and 31 enchondroma tissue specimens disclosed 87% specificity and 70% sensitivity.     

Properties of integral membrane protein structures: derivation of an implicit membrane potential

Distributions of each amino acid in the trans-membrane domain were calculated as a function of the membrane normal using all currently available alpha-helical membrane protein structures with resolutions better than 4 A. The results were compared with previous sequence- and structure-based analyses. Calculation of the average hydrophobicity along the membrane normal demonstrated that the protein surface in the membrane domain is in fact much more hydrophobic than the protein core. While hydrophobic residues dominate the membrane domain, the interfacial regions of membrane proteins were found to be abundant in the small residues glycine, alanine, and serine, consistent with previous studies on membrane protein packing. Charged residues displayed nonsymmetric distributions with a preference for the intracellular interface. This effect was more prominent for Arg and Lys resulting in a direct confirmation of the positive inside rule. Potentials of mean force along the membrane normal were derived for each amino acid by fitting Gaussian functions to the residue distributions. The individual potentials agree well with experimental and theoretical considerations. The resulting implicit membrane potential was tested on various membrane proteins as well as single trans-membrane alpha-helices. All membrane proteins were found to be at an energy minimum when correctly inserted into the membrane. For alpha-helices both interfacial (i.e. surface bound) and inserted configurations were found to correspond to energy minima. The results demonstrate that the use of trans-membrane amino acid distributions to derive an implicit membrane representation yields meaningful residue potentials.     

Immunogenicity of plant‐produced porcine circovirus‐like particles in mice

Porcine circovirus type 2 (PCV‐2) is the main causative agent associated with a group of diseases collectively known as porcine circovirus‐associated disease (PCAD). There is a significant economic strain on the global swine industry due to PCAD and the production of commercial PCV‐2 vaccines is expensive. Plant expression systems are increasingly regarded as a viable technology to produce recombinant proteins for use as pharmaceutical agents and vaccines. However, successful production and purification of PCV‐2 capsid protein (CP) from plants is an essential first step towards the goal of a plant‐produced PCV‐2 vaccine candidate. In this study, the PCV‐2 CP was transiently expressed in Nicotiana benthamiana plants via agroinfiltration and PCV‐2 CP was successfully purified using sucrose gradient ultracentrifugation. The CP self‐assembled into virus‐like particles (VLPs) resembling native virions and up to 6.5 mg of VLPs could be purified from 1 kg of leaf wet weight. Mice immunized with the plant‐produced PCV‐2 VLPs elicited specific antibody responses to PCV‐2 CP. This is the first report describing the expression of PCV‐2 CP in plants, the confirmation of its assembly into VLPs and the demonstration of their use to elicit a strong immune response in a mammalian model.     

酵母双杂交筛选锥虫早老素蛋白相互作用蛋白

目的筛选寻找锥虫早老素蛋白相互作用蛋白,以了解锥虫早老素蛋白功能。方法体外表达锥虫早老素蛋白片段,装入pGBKT7诱饵质粒,与随机肽库系统共转化酵母,筛选阳性克隆并测序,通过与基因库锥虫功能序列比较,推导可能的相互作用蛋白。结果获得108个阳性克隆,对其中50个进行了序列测定和比较,获得最有可能的4个候选基因,分别为：丝/苏氨酸性磷酸酶2b催化亚基A2;钙激活蛋白,含锚蛋白重复序列蛋白以及一个具有与APP跨膜区结合位点特征序列的功能未知蛋白。结论成功利用随机肽库酵母双杂交系统筛选锥虫早老素蛋白相互作用基因,其相互作用仍有待进一步确认。     

蛋白质结构研究现状与展望

本文综述了近年来蛋白质结构的确定、预测、比较、分类和功能五个问题的研究概况,并分析了解决每个问题的方法,最后指出蛋白质结构研究的发展前景。     

Identification and characterization of two chitin-binding proteins from the peritrophic membrane of the silkworm, Bombyx mori L

The peritrophic membrane (PM) is a semi‐permeable lining of the insect midgut, broadly analogous to the mucous lining of vertebrate gut. The PM proteins are important achievements for the function of the PM. In this study, two chitin‐binding proteins (BmPM‐P43 and BmPM‐P41) from the PM of the silkworm, Bombyx mori, were identified and cloned. These proteins showed the molecular mass of 43 and 41 kDa, respectively. The deduced amino acid sequences codes for a protein of 381 amino acid residues and 364 amino acid residues, containing 12 and 14 cysteine residues followed by similar domain, both of them have 5 cysteine residues in similar position in the C‐terminal. The confirmation of these proteins was performed by western blot analysis of recombinant BmPM‐P43 and BmPM‐P41. The chitin‐binding activity analysis showed that the BmPM‐P43 and BmPM‐P41 could bind to chitin strongly. It is concluded that BmPM‐P43 and BmPM‐P41 contains a polysaccharide deacetylase domain instead of peritrophin domain, indicated that these two proteins may belong to a new chitin‐binding protein family. © 2010 Wiley Periodicals, Inc.     

Analyzing self‐controlled case series data when case confirmation rates are estimated from an internal validation sample

Vaccine safety studies are often electronic health record (EHR)‐based observational studies. These studies often face significant methodological challenges, including confounding and misclassification of adverse event. Vaccine safety researchers use self‐controlled case series (SCCS) study design to handle confounding effect and employ medical chart review to ascertain cases that are identified using EHR data. However, for common adverse events, limited resources often make it impossible to adjudicate all adverse events observed in electronic data. In this paper, we considered four approaches for analyzing SCCS data with confirmation rates estimated from an internal validation sample: (1) observed cases, (2) confirmed cases only, (3) known confirmation rate, and (4) multiple imputation (MI). We conducted a simulation study to evaluate these four approaches using type I error rates, percent bias, and empirical power. Our simulation results suggest that when misclassification of adverse events is present, approaches such as observed cases, confirmed case only, and known confirmation rate may inflate the type I error, yield biased point estimates, and affect statistical power. The multiple imputation approach considers the uncertainty of estimated confirmation rates from an internal validation sample, yields a proper type I error rate, largely unbiased point estimate, proper variance estimate, and statistical power.     

Sirtuin inhibitor Ex‐527 causes neural tube defects,ventral edema formations,and gastrointestinal malformations in Xenopus laevis embryos

Chemical reagent Ex‐527 is widely used as a major inhibitor of Sirtuin enzymes, which are a family of highly conserved protein deacetylases and have been linked with caloric restriction and aging by modulating energy metabolism, genomic stability, and stress resistance. However, the extent to which Ex‐527 controls early developmental events of vertebrate embryos remains to be understood. Here, we report an examination of Ex‐527 effects during Xenopus early development, followed by a confirmation of expressions of xSirt1 and xSirt2 in embryonic stages and enhancement of acetylation by Ex‐527. First, we found that reductions in size of neural plate at neurula stages were induced by Ex‐527 treatment. Second, tadpoles with short body length and large edematous swellings in the ventral side were frequently observed. Moreover, Ex‐527‐treated embryos showed severe gastrointestinal malformations in late tadpole stages. Taken together with these results, we conclude that the Sirtuin family start functioning at early embryonic stages and is required for various developmental events.     

猪链球菌2型的表面蛋白烯醇化酶在细菌黏附和引发免疫下调中的作用

利用克隆表达获得了具有酶活性的猪链球菌2型(S.suis2)05ZYH33重组烯醇化酶(Enolase)蛋白。流式细胞术FCM的细胞定位发现Enolase可部分存在于S.suis205ZYH33细菌的表面。进而利用Hep2细胞探讨猪链球菌表面Enolase参与细菌对宿主细胞的黏附作用。免疫空斑试验的结果提示,Enolase可能作为一个免疫下调蛋白对于引发猪链球菌相关疾病发挥着重要的作用。     

Development and optimization of a cell‐based assay for the selection of synthetic compounds that potentiate bone morphogenetic protein‐2 activity

The requirement of large amounts of the recombinant human bone morphogenetic protein‐2 (BMP‐2) produces a huge translational barrier for its routine clinical use due to high cost. This leads to an urgent need to develop alternative methods to lower costs and/or increase efficacies for using BMP‐2. In this study, we describe the development and optimization of a cell‐based assay that is sensitive, reproducible, and reliable in identifying reagents that potentiate the effects of BMP‐2 in inducing transdifferentiation of C2C12 myoblasts into the osteoblastic phenotype. The assay is based on a BMP‐responsive Smad1‐driven luciferase reporter gene. LIM mineralization protein‐1 (LMP‐1) is a novel intracellular LIM domain protein that has been shown by our group to enhance cellular responsiveness to BMP‐2. Our previous report elucidated that the binding of LMP‐1 with the WW2 domain in Smad ubiquitin regulatory factor‐1 (Smurf1) rescues the osteogenic Smads from degradation. Here, using the optimized cell‐based assay, we first evaluated the activity of the recombinantly prepared proteins, LMP‐1, and its mutant (LMP‐1ΔSmurf1) that lacks the Smurf1‐WW2 domain‐binding motif. Both the wild type and the mutant proteins were engineered to contain an 11‐amino acid HIV‐TAT protein derived membrane transduction domain to aid the cellular delivery of recombinant proteins. The cell‐based reporter assay confirmed that LMP‐1 potentiates the BMP‐induced stimulation of C2C12 cells towards the osteoblastic phenotype. The potentiating effect of LMP‐1 was significantly reduced when a specific‐motif known to interact with Smurf1 was mutated. We validated the results obtained in the reporter assay by also monitoring the expression of mRNA for osteocalcin and alkaline phosphatase (ALP) which is widely accepted osteoblast differentiation marker genes. Finally, we provide further confirmation of our results by measuring the activity of alkaline phosphatase in support of the accuracy and reliability of our cell‐based assay. Direct delivery of synthesized protein can be limited by high cost, instability or inadequate post‐translational modifications. Thus, there would be a clear benefit for a low cost, cell penetrable chemical compound. We successfully used our gene expression‐based assay to choose an active compound from a select group of compounds that were identified by computational screenings as the most likely candidates for mimicking the function of LMP‐1. Among them, we selected SVAK‐3, a compound that showed a dose‐dependent potentiation of BMP‐2 activity in inducing osteoblastic differentiation of C2C12 cells. We show that either the full length LMP‐1 protein or its potential mimetic compound consistently exhibit similar potentiation of BMP‐2 activity even when multiple markers of the osteoblastic phenotype were parallely monitored. Published in 2009 by John Wiley & Sons, Ltd.     

The proteomic to biology inference,a frequently overlooked concern in the interpretation of proteomic data: A plea for functional validation

Proteomics will celebrate its 20th year in 2014. In this relatively short period of time, it has invaded most areas of biology and its use will probably continue to spread in the future. These two decades have seen a considerable increase in the speed and sensitivity of protein identification and characterization, even from complex samples. Indeed, what was a challenge twenty years ago is now little more than a daily routine. Although not completely over, the technological challenge now makes room to another challenge, which is the best possible appraisal and exploitation of proteomic data to draw the best possible conclusions from a biological point of view. The point developed in this paper is that proteomic data are almost always fragmentary. This means in turn that although better than an mRNA level, a protein level is often insufficient to draw a valid conclusion from a biological point of view, especially in a world where PTMs play such an important role. This means in turn that transformation of proteomic data into biological data requires an important intermediate layer of functional validation, i.e. not merely the confirmation of protein abundance changes by other methods, but a functional appraisal of the biological consequences of the protein level changes highlighted by the proteomic screens.