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Sample records for detect protein complexes

Detection of protein complex from protein-protein interaction network using Markov clustering

NASA Astrophysics Data System (ADS)

Ochieng, P. J.; Kusuma, W. A.; Haryanto, T.

2017-05-01

Detection of complexes, or groups of functionally related proteins, is an important challenge while analysing biological networks. However, existing algorithms to identify protein complexes are insufficient when applied to dense networks of experimentally derived interaction data. Therefore, we introduced a graph clustering method based on Markov clustering algorithm to identify protein complex within highly interconnected protein-protein interaction networks. Protein-protein interaction network was first constructed to develop geometrical network, the network was then partitioned using Markov clustering to detect protein complexes. The interest of the proposed method was illustrated by its application to Human Proteins associated to type II diabetes mellitus. Flow simulation of MCL algorithm was initially performed and topological properties of the resultant network were analysed for detection of the protein complex. The results indicated the proposed method successfully detect an overall of 34 complexes with 11 complexes consisting of overlapping modules and 20 non-overlapping modules. The major complex consisted of 102 proteins and 521 interactions with cluster modularity and density of 0.745 and 0.101 respectively. The comparison analysis revealed MCL out perform AP, MCODE and SCPS algorithms with high clustering coefficient (0.751) network density and modularity index (0.630). This demonstrated MCL was the most reliable and efficient graph clustering algorithm for detection of protein complexes from PPI networks.
A Type-2 fuzzy data fusion approach for building reliable weighted protein interaction networks with application in protein complex detection.

PubMed

Mehranfar, Adele; Ghadiri, Nasser; Kouhsar, Morteza; Golshani, Ashkan

2017-09-01

Detecting the protein complexes is an important task in analyzing the protein interaction networks. Although many algorithms predict protein complexes in different ways, surveys on the interaction networks indicate that about 50% of detected interactions are false positives. Consequently, the accuracy of existing methods needs to be improved. In this paper we propose a novel algorithm to detect the protein complexes in 'noisy' protein interaction data. First, we integrate several biological data sources to determine the reliability of each interaction and determine more accurate weights for the interactions. A data fusion component is used for this step, based on the interval type-2 fuzzy voter that provides an efficient combination of the information sources. This fusion component detects the errors and diminishes their effect on the detection protein complexes. So in the first step, the reliability scores have been assigned for every interaction in the network. In the second step, we have proposed a general protein complex detection algorithm by exploiting and adopting the strong points of other algorithms and existing hypotheses regarding real complexes. Finally, the proposed method has been applied for the yeast interaction datasets for predicting the interactions. The results show that our framework has a better performance regarding precision and F-measure than the existing approaches. Copyright © 2017 Elsevier Ltd. All rights reserved.
Solid-state nanopore detection of protein complexes: applications in healthcare and protein kinetics.

PubMed

Freedman, Kevin J; Bastian, Arangassery R; Chaiken, Irwin; Kim, Min Jun

2013-03-11

Protein conjugation provides a unique look into many biological phenomena and has been used for decades for molecular recognition purposes. In this study, the use of solid-state nanopores for the detection of gp120-associated complexes are investigated. They exhibit monovalent and multivalent binding to anti-gp120 antibody monomer and dimers. In order to investigate the feasibility of many practical applications related to nanopores, detection of specific protein complexes is attempted within a heterogeneous protein sample, and the role of voltage on complexed proteins is researched. It is found that the electric field within the pore can result in unbinding of a freely translocating protein complex within the transient event durations measured experimentally. The strong dependence of the unbinding time with voltage can be used to improve the detection capability of the nanopore system by adding an additional level of specificity that can be probed. These data provide a strong framework for future protein-specific detection schemes, which are shown to be feasible in the realm of a 'real-world' sample and an automated multidimensional method of detecting events. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Multi-Dimensional Scaling based grouping of known complexes and intelligent protein complex detection.

PubMed

Rehman, Zia Ur; Idris, Adnan; Khan, Asifullah

2018-06-01

Protein-Protein Interactions (PPI) play a vital role in cellular processes and are formed because of thousands of interactions among proteins. Advancements in proteomics technologies have resulted in huge PPI datasets that need to be systematically analyzed. Protein complexes are the locally dense regions in PPI networks, which extend important role in metabolic pathways and gene regulation. In this work, a novel two-phase protein complex detection and grouping mechanism is proposed. In the first phase, topological and biological features are extracted for each complex, and prediction performance is investigated using Bagging based Ensemble classifier (PCD-BEns). Performance evaluation through cross validation shows improvement in comparison to CDIP, MCode, CFinder and PLSMC methods Second phase employs Multi-Dimensional Scaling (MDS) for the grouping of known complexes by exploring inter complex relations. It is experimentally observed that the combination of topological and biological features in the proposed approach has greatly enhanced prediction performance for protein complex detection, which may help to understand various biological processes, whereas application of MDS based exploration may assist in grouping potentially similar complexes. Copyright © 2018 Elsevier Ltd. All rights reserved.
[Application of PLA Method for Detection of p53/p63/p73 Complexes in Situ in Tumour Cells and Tumour Tissue].

PubMed

Hrabal, V; Nekulová, M; Nenutil, R; Holčaková, J; Coates, P J; Vojtěšek, B

2017-01-01

PLA (proximity ligation assay) can be used for detection of protein-protein interactions in situ directly in cells and tissues. Due to its high sensitivity and specificity it is useful for detection, localization and quantification of protein complexes with single molecule resolution. One of the mechanisms of mutated p53 gain of function is formation of proten-protein complexes with other members of p53 family - p63 and p73. These interactions influences chemosensitivity and invasivity of cancer cells and this is why these complexes are potential targets of anti-cancer therapy. The aim of this work is to detect p53/p63/p73 interactions in situ in tumour cells and tumour tissue using PLA method. Unique in-house antibodies for specific detection of p63 and p73 isoforms were developed and characterized. Potein complexes were detected using PLA in established cell lines SVK14, HCC1806 and FaDu and in paraffin sections of colorectal carcinoma tissue. Cell lines were also processed to paraffin blocks. p53/T-antigen and ΔNp63/T-antigen protein complexes were detected in SVK14 cells using PLA. Interactions of ΔNp63 and TAp73 isoforms were found in HCC1806 cell line with endogenous expression of these proteins. In FaDu cell line mut-p53/TAp73 complex was localized but not mut-p53/ΔNp63 complex. p53 tetramer was detected directly in colorectal cancer tissue. During development of PLA method for detection of protein complexes between p53 family members we detected interactions of p53 and p63 with T-antigen and mut-p53 and ΔNp63 with TAp73 tumour suppressor in tumour cell lines and p53 tetramers in paraffin sections of colorectal cancer tissue. PLA will be further used for detection of p53/p63, p53/p73 and p63/p73 interactions in tumour tissues and it could be also used for screening of compounds that can block formation of p53/p63/p73 protein complexes.Key words: p53 protein family - protein interaction mapping - immunofluorescence This work was supported by MEYS - NPS I - LO1413. The authors declare they have no potential conflicts of interest concerning drugs, products, or services used in the study. The Editorial Board declares that the manuscript met the ICMJE recommendation for biomedical papers.Submitted: 13. 3. 2017Accepted: 26. 3. 2017.
Predicting protein complexes using a supervised learning method combined with local structural information.

PubMed

Dong, Yadong; Sun, Yongqi; Qin, Chao

2018-01-01

The existing protein complex detection methods can be broadly divided into two categories: unsupervised and supervised learning methods. Most of the unsupervised learning methods assume that protein complexes are in dense regions of protein-protein interaction (PPI) networks even though many true complexes are not dense subgraphs. Supervised learning methods utilize the informative properties of known complexes; they often extract features from existing complexes and then use the features to train a classification model. The trained model is used to guide the search process for new complexes. However, insufficient extracted features, noise in the PPI data and the incompleteness of complex data make the classification model imprecise. Consequently, the classification model is not sufficient for guiding the detection of complexes. Therefore, we propose a new robust score function that combines the classification model with local structural information. Based on the score function, we provide a search method that works both forwards and backwards. The results from experiments on six benchmark PPI datasets and three protein complex datasets show that our approach can achieve better performance compared with the state-of-the-art supervised, semi-supervised and unsupervised methods for protein complex detection, occasionally significantly outperforming such methods.
Global Membrane Protein Interactome Analysis using In vivo Crosslinking and Mass Spectrometry-based Protein Correlation Profiling*

PubMed Central

Larance, Mark; Kirkwood, Kathryn J.; Tinti, Michele; Brenes Murillo, Alejandro; Ferguson, Michael A. J.; Lamond, Angus I.

2016-01-01

We present a methodology using in vivo crosslinking combined with HPLC-MS for the global analysis of endogenous protein complexes by protein correlation profiling. Formaldehyde crosslinked protein complexes were extracted with high yield using denaturing buffers that maintained complex solubility during chromatographic separation. We show this efficiently detects both integral membrane and membrane-associated protein complexes,in addition to soluble complexes, allowing identification and analysis of complexes not accessible in native extracts. We compare the protein complexes detected by HPLC-MS protein correlation profiling in both native and formaldehyde crosslinked U2OS cell extracts. These proteome-wide data sets of both in vivo crosslinked and native protein complexes from U2OS cells are freely available via a searchable online database (www.peptracker.com/epd). Raw data are also available via ProteomeXchange (identifier PXD003754). PMID:27114452
Identifying protein complex by integrating characteristic of core-attachment into dynamic PPI network.

PubMed

Shen, Xianjun; Yi, Li; Jiang, Xingpeng; He, Tingting; Yang, Jincai; Xie, Wei; Hu, Po; Hu, Xiaohua

2017-01-01

How to identify protein complex is an important and challenging task in proteomics. It would make great contribution to our knowledge of molecular mechanism in cell life activities. However, the inherent organization and dynamic characteristic of cell system have rarely been incorporated into the existing algorithms for detecting protein complexes because of the limitation of protein-protein interaction (PPI) data produced by high throughput techniques. The availability of time course gene expression profile enables us to uncover the dynamics of molecular networks and improve the detection of protein complexes. In order to achieve this goal, this paper proposes a novel algorithm DCA (Dynamic Core-Attachment). It detects protein-complex core comprising of continually expressed and highly connected proteins in dynamic PPI network, and then the protein complex is formed by including the attachments with high adhesion into the core. The integration of core-attachment feature into the dynamic PPI network is responsible for the superiority of our algorithm. DCA has been applied on two different yeast dynamic PPI networks and the experimental results show that it performs significantly better than the state-of-the-art techniques in terms of prediction accuracy, hF-measure and statistical significance in biology. In addition, the identified complexes with strong biological significance provide potential candidate complexes for biologists to validate.
A Novel Algorithm for Detecting Protein Complexes with the Breadth First Search

PubMed Central

Tang, Xiwei; Wang, Jianxin; Li, Min; He, Yiming; Pan, Yi

2014-01-01

Most biological processes are carried out by protein complexes. A substantial number of false positives of the protein-protein interaction (PPI) data can compromise the utility of the datasets for complexes reconstruction. In order to reduce the impact of such discrepancies, a number of data integration and affinity scoring schemes have been devised. The methods encode the reliabilities (confidence) of physical interactions between pairs of proteins. The challenge now is to identify novel and meaningful protein complexes from the weighted PPI network. To address this problem, a novel protein complex mining algorithm ClusterBFS (Cluster with Breadth-First Search) is proposed. Based on the weighted density, ClusterBFS detects protein complexes of the weighted network by the breadth first search algorithm, which originates from a given seed protein used as starting-point. The experimental results show that ClusterBFS performs significantly better than the other computational approaches in terms of the identification of protein complexes. PMID:24818139
Detection of Protein Complexes Based on Penalized Matrix Decomposition in a Sparse Protein⁻Protein Interaction Network.

PubMed

Cao, Buwen; Deng, Shuguang; Qin, Hua; Ding, Pingjian; Chen, Shaopeng; Li, Guanghui

2018-06-15

High-throughput technology has generated large-scale protein interaction data, which is crucial in our understanding of biological organisms. Many complex identification algorithms have been developed to determine protein complexes. However, these methods are only suitable for dense protein interaction networks, because their capabilities decrease rapidly when applied to sparse protein⁻protein interaction (PPI) networks. In this study, based on penalized matrix decomposition ( PMD ), a novel method of penalized matrix decomposition for the identification of protein complexes (i.e., PMD pc ) was developed to detect protein complexes in the human protein interaction network. This method mainly consists of three steps. First, the adjacent matrix of the protein interaction network is normalized. Second, the normalized matrix is decomposed into three factor matrices. The PMD pc method can detect protein complexes in sparse PPI networks by imposing appropriate constraints on factor matrices. Finally, the results of our method are compared with those of other methods in human PPI network. Experimental results show that our method can not only outperform classical algorithms, such as CFinder, ClusterONE, RRW, HC-PIN, and PCE-FR, but can also achieve an ideal overall performance in terms of a composite score consisting of F-measure, accuracy (ACC), and the maximum matching ratio (MMR).
Characterization, stoichiometry, and stability of salivary protein-tannin complexes by ESI-MS and ESI-MS/MS.

PubMed

Canon, Francis; Paté, Franck; Meudec, Emmanuelle; Marlin, Thérèse; Cheynier, Véronique; Giuliani, Alexandre; Sarni-Manchado, Pascale

2009-12-01

Numerous protein-polyphenol interactions occur in biological and food domains particularly involving proline-rich proteins, which are representative of the intrinsically unstructured protein group (IUP). Noncovalent protein-ligand complexes are readily detected by electrospray ionization mass spectrometry (ESI-MS), which also gives access to ligand binding stoichiometry. Surprisingly, the study of interactions between polyphenolic molecules and proteins is still an area where ESI-MS has poorly benefited, whereas it has been extensively applied to the detection of noncovalent complexes. Electrospray ionization mass spectrometry has been applied to the detection and the characterization of the complexes formed between tannins and a human salivary proline-rich protein (PRP), namely IB5. The study of the complex stability was achieved by low-energy collision-induced dissociation (CID) measurements, which are commonly implemented using triple quadrupole, hybrid quadrupole time-of-flight, or ion trap instruments. Complexes composed of IB5 bound to a model polyphenol EgCG have been detected by ESI-MS and further analyzed by MS/MS. Mild ESI interface conditions allowed us to observe intact noncovalent PRP-tannin complexes with stoichiometries ranging from 1:1 to 1:5. Thus, ESI-MS shows its efficiency for (1) the study of PRP-tannin interactions, (2) the determination of stoichiometry, and (3) the study of complex stability. We were able to establish unambiguously both their stoichiometries and their overall subunit architecture via tandem mass spectrometry and solution disruption experiments. Our results prove that IB5.EgCG complexes are maintained intact in the gas phase.
Identifying Hierarchical and Overlapping Protein Complexes Based on Essential Protein-Protein Interactions and “Seed-Expanding” Method

PubMed Central

Ren, Jun; Zhou, Wei; Wang, Jianxin

2014-01-01

Many evidences have demonstrated that protein complexes are overlapping and hierarchically organized in PPI networks. Meanwhile, the large size of PPI network wants complex detection methods have low time complexity. Up to now, few methods can identify overlapping and hierarchical protein complexes in a PPI network quickly. In this paper, a novel method, called MCSE, is proposed based on λ-module and “seed-expanding.” First, it chooses seeds as essential PPIs or edges with high edge clustering values. Then, it identifies protein complexes by expanding each seed to a λ-module. MCSE is suitable for large PPI networks because of its low time complexity. MCSE can identify overlapping protein complexes naturally because a protein can be visited by different seeds. MCSE uses the parameter λ_th to control the range of seed expanding and can detect a hierarchical organization of protein complexes by tuning the value of λ_th. Experimental results of S. cerevisiae show that this hierarchical organization is similar to that of known complexes in MIPS database. The experimental results also show that MCSE outperforms other previous competing algorithms, such as CPM, CMC, Core-Attachment, Dpclus, HC-PIN, MCL, and NFC, in terms of the functional enrichment and matching with known protein complexes. PMID:25143945
Filtering Gene Ontology semantic similarity for identifying protein complexes in large protein interaction networks.

PubMed

Wang, Jian; Xie, Dong; Lin, Hongfei; Yang, Zhihao; Zhang, Yijia

2012-06-21

Many biological processes recognize in particular the importance of protein complexes, and various computational approaches have been developed to identify complexes from protein-protein interaction (PPI) networks. However, high false-positive rate of PPIs leads to challenging identification. A protein semantic similarity measure is proposed in this study, based on the ontology structure of Gene Ontology (GO) terms and GO annotations to estimate the reliability of interactions in PPI networks. Interaction pairs with low GO semantic similarity are removed from the network as unreliable interactions. Then, a cluster-expanding algorithm is used to detect complexes with core-attachment structure on filtered network. Our method is applied to three different yeast PPI networks. The effectiveness of our method is examined on two benchmark complex datasets. Experimental results show that our method performed better than other state-of-the-art approaches in most evaluation metrics. The method detects protein complexes from large scale PPI networks by filtering GO semantic similarity. Removing interactions with low GO similarity significantly improves the performance of complex identification. The expanding strategy is also effective to identify attachment proteins of complexes.
Protein Stains to Detect Antigen on Membranes.

PubMed

Dsouza, Anil; Scofield, R Hal

2015-01-01

Western blotting (protein blotting/electroblotting) is the gold standard in the analysis of complex protein mixtures. Electroblotting drives protein molecules from a polyacrylamide (or less commonly, of an agarose) gel to the surface of a binding membrane, thereby facilitating an increased availability of the sites with affinity for both general and specific protein reagents. The analysis of these complex protein mixtures is achieved by the detection of specific protein bands on a membrane, which in turn is made possible by the visualization of protein bands either by chemical staining or by reaction with an antibody of a conjugated ligand. Chemical methods employ staining with organic dyes, metal chelates, autoradiography, fluorescent dyes, complexing with silver, or prelabeling with fluorophores. All of these methods have differing sensitivities and quantitative determinations vary significantly. This review will describe the various protein staining methods applied to membranes after western blotting. "Detection" precedes and is a prerequisite to obtaining qualitative and quantitative data on the proteins in a sample, as much as to comparing the protein composition of different samples. "Detection" is often synonymous to staining, i.e., the reversible or irreversible binding by the proteins of a colored organic or inorganic chemical.
Protein stains to detect antigen on membranes.

PubMed

D'souza, Anil; Scofield, R Hal

2009-01-01

Western blotting (protein blotting/electroblotting) is the gold standard in the analysis of complex protein mixtures. Electroblotting drives protein molecules from a polyacrylamide (or less commonly, of an agarose) gel to the surface of a binding membrane, thereby facilitating an increased availability of the sites with affinity for both general and specific protein reagents. The analysis of these complex protein mixtures is achieved by the detection of specific protein bands on a membrane, which in turn is made possible by the visualization of protein bands either by chemical staining or by reaction with an antibody of a conjugated ligand. Chemical methods employ staining with organic dyes, metal chelates, autoradiography, fluorescent dyes, complexing with silver, or prelabeling with fluorophores. All of these methods have differing sensitivities and quantitative determinations vary significantly. This review will describe the various protein staining methods applied to membranes after electrophoresis. "Detection" precedes and is a prerequisite to obtaining qualitative and quantitative data on the proteins in a sample, as much as to comparing the protein composition of different samples. Detection is often synonymous to staining, i.e., the reversible or irreversible binding by the proteins of a colored organic or inorganic chemical.
A Method for Predicting Protein Complexes from Dynamic Weighted Protein-Protein Interaction Networks.

PubMed

Liu, Lizhen; Sun, Xiaowu; Song, Wei; Du, Chao

2018-06-01

Predicting protein complexes from protein-protein interaction (PPI) network is of great significance to recognize the structure and function of cells. A protein may interact with different proteins under different time or conditions. Existing approaches only utilize static PPI network data that may lose much temporal biological information. First, this article proposed a novel method that combines gene expression data at different time points with traditional static PPI network to construct different dynamic subnetworks. Second, to further filter out the data noise, the semantic similarity based on gene ontology is regarded as the network weight together with the principal component analysis, which is introduced to deal with the weight computing by three traditional methods. Third, after building a dynamic PPI network, a predicting protein complexes algorithm based on "core-attachment" structural feature is applied to detect complexes from each dynamic subnetworks. Finally, it is revealed from the experimental results that our method proposed in this article performs well on detecting protein complexes from dynamic weighted PPI networks.
Discovering protein complexes in protein interaction networks via exploring the weak ties effect

PubMed Central

2012-01-01

Background Studying protein complexes is very important in biological processes since it helps reveal the structure-functionality relationships in biological networks and much attention has been paid to accurately predict protein complexes from the increasing amount of protein-protein interaction (PPI) data. Most of the available algorithms are based on the assumption that dense subgraphs correspond to complexes, failing to take into account the inherence organization within protein complex and the roles of edges. Thus, there is a critical need to investigate the possibility of discovering protein complexes using the topological information hidden in edges. Results To provide an investigation of the roles of edges in PPI networks, we show that the edges connecting less similar vertices in topology are more significant in maintaining the global connectivity, indicating the weak ties phenomenon in PPI networks. We further demonstrate that there is a negative relation between the weak tie strength and the topological similarity. By using the bridges, a reliable virtual network is constructed, in which each maximal clique corresponds to the core of a complex. By this notion, the detection of the protein complexes is transformed into a classic all-clique problem. A novel core-attachment based method is developed, which detects the cores and attachments, respectively. A comprehensive comparison among the existing algorithms and our algorithm has been made by comparing the predicted complexes against benchmark complexes. Conclusions We proved that the weak tie effect exists in the PPI network and demonstrated that the density is insufficient to characterize the topological structure of protein complexes. Furthermore, the experimental results on the yeast PPI network show that the proposed method outperforms the state-of-the-art algorithms. The analysis of detected modules by the present algorithm suggests that most of these modules have well biological significance in context of complexes, suggesting that the roles of edges are critical in discovering protein complexes. PMID:23046740
Detection of in situ protein-protein complexes at the Drosophila larval neuromuscular junction using proximity ligation assay.

PubMed

Wang, Simon; Yoo, SooHyun; Kim, Hae-Yoon; Wang, Mannan; Zheng, Clare; Parkhouse, Wade; Krieger, Charles; Harden, Nicholas

2015-01-20

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development. Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.
Singlet oxygen Triplet Energy Transfer based imaging technology for mapping protein-protein proximity in intact cells

PubMed Central

To, Tsz-Leung; Fadul, Michael J.; Shu, Xiaokun

2014-01-01

Many cellular processes are carried out by large protein complexes that can span several tens of nanometers. Whereas Forster resonance energy transfer has a detection range of <10 nm, here we report the theoretical development and experimental demonstration of a new fluorescence imaging technology with a detection range of up to several tens of nanometers: singlet oxygen triplet energy transfer. We demonstrate that our method confirms the topology of a large protein complex in intact cells, which spans from the endoplasmic reticulum to the outer mitochondrial membrane and the matrix. This new method is thus suited for mapping protein proximity in large protein complexes. PMID:24905026
Ink-native electrophoresis: an alternative to blue-native electrophoresis more suitable for in-gel detection of enzymatic activity.

PubMed

Kaneko, Keisuke; Sueyoshi, Noriyuki; Kameshita, Isamu; Ishida, Atsuhiko

2013-09-15

Blue-native electrophoresis (BNE) is a useful technique for analyzing protein complexes, but the Coomassie brilliant blue (CBB) dye used in BNE often hampers in-gel detection of enzymatic activity. Here we report an improved method, termed ink-native electrophoresis (INE), in which Pelikan 4001 fountain pen ink is used as a charge-shifting agent instead of CBB. INE is more suitable than BNE for in-gel detection of protein kinase activity after polyacrylamide gel electrophoresis (PAGE), and its performance in protein complex separation is comparable to that of conventional BNE. INE may provide a powerful tool to isolate and analyze various protein complexes. Copyright © 2013 Elsevier Inc. All rights reserved.

In-depth proteomic analysis of Varroa destructor: Detection of DWV-complex, ABPV, VdMLV and honeybee proteins in the mite.

PubMed

Erban, Tomas; Harant, Karel; Hubalek, Martin; Vitamvas, Pavel; Kamler, Martin; Poltronieri, Palmiro; Tyl, Jan; Markovic, Martin; Titera, Dalibor

2015-09-11

We investigated pathogens in the parasitic honeybee mite Varroa destructor using nanoLC-MS/MS (TripleTOF) and 2D-E-MS/MS proteomics approaches supplemented with affinity-chromatography to concentrate trace target proteins. Peptides were detected from the currently uncharacterized Varroa destructor Macula-like virus (VdMLV), the deformed wing virus (DWV)-complex and the acute bee paralysis virus (ABPV). Peptide alignments revealed detection of complete structural DWV-complex block VP2-VP1-VP3, VDV-1 helicase and single-amino-acid substitution A/K/Q in VP1, the ABPV structural block VP1-VP4-VP2-VP3 including uncleaved VP4/VP2, and VdMLV coat protein. Isoforms of viral structural proteins of highest abundance were localized via 2D-E. The presence of all types of capsid/coat proteins of a particular virus suggested the presence of virions in Varroa. Also, matches between the MWs of viral structural proteins on 2D-E and their theoretical MWs indicated that viruses were not digested. The absence/scarce detection of non-structural proteins compared with high-abundance structural proteins suggest that the viruses did not replicate in the mite; hence, virions accumulate in the Varroa gut via hemolymph feeding. Hemolymph feeding also resulted in the detection of a variety of honeybee proteins. The advantages of MS-based proteomics for pathogen detection, false-positive pathogen detection, virus replication, posttranslational modifications, and the presence of honeybee proteins in Varroa are discussed.
In-depth proteomic analysis of Varroa destructor: Detection of DWV-complex, ABPV, VdMLV and honeybee proteins in the mite

PubMed Central

Erban, Tomas; Harant, Karel; Hubalek, Martin; Vitamvas, Pavel; Kamler, Martin; Poltronieri, Palmiro; Tyl, Jan; Markovic, Martin; Titera, Dalibor

2015-01-01

We investigated pathogens in the parasitic honeybee mite Varroa destructor using nanoLC-MS/MS (TripleTOF) and 2D-E-MS/MS proteomics approaches supplemented with affinity-chromatography to concentrate trace target proteins. Peptides were detected from the currently uncharacterized Varroa destructor Macula-like virus (VdMLV), the deformed wing virus (DWV)-complex and the acute bee paralysis virus (ABPV). Peptide alignments revealed detection of complete structural DWV-complex block VP2-VP1-VP3, VDV-1 helicase and single-amino-acid substitution A/K/Q in VP1, the ABPV structural block VP1-VP4-VP2-VP3 including uncleaved VP4/VP2, and VdMLV coat protein. Isoforms of viral structural proteins of highest abundance were localized via 2D-E. The presence of all types of capsid/coat proteins of a particular virus suggested the presence of virions in Varroa. Also, matches between the MWs of viral structural proteins on 2D-E and their theoretical MWs indicated that viruses were not digested. The absence/scarce detection of non-structural proteins compared with high-abundance structural proteins suggest that the viruses did not replicate in the mite; hence, virions accumulate in the Varroa gut via hemolymph feeding. Hemolymph feeding also resulted in the detection of a variety of honeybee proteins. The advantages of MS-based proteomics for pathogen detection, false-positive pathogen detection, virus replication, posttranslational modifications, and the presence of honeybee proteins in Varroa are discussed. PMID:26358842
Advances in ultra-high performance liquid chromatography coupled to tandem mass spectrometry for sensitive detection of several food allergens in complex and processed foodstuffs.

PubMed

Planque, M; Arnould, T; Dieu, M; Delahaut, P; Renard, P; Gillard, N

2016-09-16

Sensitive detection of food allergens is affected by food processing and foodstuff complexity. It is therefore a challenge to detect cross-contamination in food production that could endanger an allergic customer's life. Here we used ultra-high performance liquid chromatography coupled to tandem mass spectrometry for simultaneous detection of traces of milk (casein, whey protein), egg (yolk, white), soybean, and peanut allergens in different complex and/or heat-processed foodstuffs. The method is based on a single protocol (extraction, trypsin digestion, and purification) applicable to the different tested foodstuffs: chocolate, ice cream, tomato sauce, and processed cookies. The determined limits of quantitation, expressed in total milk, egg, peanut, or soy proteins (and not soluble proteins) per kilogram of food, are: 0.5mg/kg for milk (detection of caseins), 5mg/kg for milk (detection of whey), 2.5mg/kg for peanut, 5mg/kg for soy, 3.4mg/kg for egg (detection of egg white), and 30.8mg/kg for egg (detection of egg yolk). The main advantage is the ability of the method to detect four major food allergens simultaneously in processed and complex matrices with very high sensitivity and specificity. Copyright © 2016 Elsevier B.V. All rights reserved.
Advantages of Molecular Weight Identification during Native MS Screening.

PubMed

Khan, Ahad; Bresnick, Anne; Cahill, Sean; Girvin, Mark; Almo, Steve; Quinn, Ronald

2018-05-09

Native mass spectrometry detection of ligand-protein complexes allowed rapid detection of natural product binders of apo and calcium-bound S100A4 (a member of the metal binding protein S100 family), T cell/transmembrane, immunoglobulin (Ig), and mucin protein 3, and T cell immunoreceptor with Ig and ITIM (immunoreceptor tyrosine-based inhibitory motif) domains precursor protein from extracts and fractions. Based on molecular weight common hits were detected binding to all four proteins. Seven common hits were identified as apigenin 6- C - β - D -glucoside 8- C - α - L -arabinoside, sweroside, 4',5-dihydroxy-7-methoxyflavanone-6- C -rutinoside, loganin acid, 6- C -glucosylnaringenin, biochanin A 7- O -rutinoside and quercetin 3- O -rutinoside. Mass guided isolation and NMR identification of hits confirmed the mass accuracy of the ligand in the ligand-protein MS complexes. Thus, molecular weight ID from ligand-protein complexes by electrospray ionization Fourier transform mass spectrometry allowed rapid dereplication. Native mass spectrometry using electrospray ionization Fourier transform mass spectrometry is a tool for dereplication and metabolomics analysis. Georg Thieme Verlag KG Stuttgart · New York.
Four-color single-molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes.

PubMed

DeRocco, Vanessa; Anderson, Trevor; Piehler, Jacob; Erie, Dorothy A; Weninger, Keith

2010-11-01

To enable studies of conformational changes within multimolecular complexes, we present a simultaneous, four-color single molecule fluorescence methodology implemented with total internal reflection illumination and camera-based, wide-field detection. We further demonstrate labeling histidine-tagged proteins noncovalently with Tris-nitrilotriacetic acid (Tris-NTA)-conjugated dyes to achieve single molecule detection. We combine these methods to colocalize the mismatch repair protein MutSα on DNA while monitoring MutSα-induced DNA bending using Förster resonance energy transfer (FRET) and to monitor assembly of membrane-tethered SNARE protein complexes.
Four-color single molecule fluorescence with noncovalent dye labeling to monitor dynamic multimolecular complexes

PubMed Central

DeRocco, Vanessa C.; Anderson, Trevor; Piehler, Jacob; Erie, Dorothy A.; Weninger, Keith

2010-01-01

To allow studies of conformational changes within multi-molecular complexes, we present a simultaneous, 4-color single molecule fluorescence methodology implemented with total internal reflection illumination and camera based, wide-field detection. We further demonstrate labeling histidine-tagged proteins non-covalently with tris-Nitrilotriacetic acid (tris-NTA) conjugated dyes to achieve single molecule detection. We combine these methods to co-localize the mismatch repair protein MutSα on DNA while monitoring MutSα-induced DNA bending using Förster resonance energy transfer (FRET) and to monitor assembly of membrane-tethered SNARE protein complexes. PMID:21091445
Predicting Physical Interactions between Protein Complexes*

PubMed Central

Clancy, Trevor; Rødland, Einar Andreas; Nygard, Ståle; Hovig, Eivind

2013-01-01

Protein complexes enact most biochemical functions in the cell. Dynamic interactions between protein complexes are frequent in many cellular processes. As they are often of a transient nature, they may be difficult to detect using current genome-wide screens. Here, we describe a method to computationally predict physical interactions between protein complexes, applied to both humans and yeast. We integrated manually curated protein complexes and physical protein interaction networks, and we designed a statistical method to identify pairs of protein complexes where the number of protein interactions between a complex pair is due to an actual physical interaction between the complexes. An evaluation against manually curated physical complex-complex interactions in yeast revealed that 50% of these interactions could be predicted in this manner. A community network analysis of the highest scoring pairs revealed a biologically sensible organization of physical complex-complex interactions in the cell. Such analyses of proteomes may serve as a guide to the discovery of novel functional cellular relationships. PMID:23438732
Detection and characterization of protein interactions in vivo by a simple live-cell imaging method.

PubMed

Gallego, Oriol; Specht, Tanja; Brach, Thorsten; Kumar, Arun; Gavin, Anne-Claude; Kaksonen, Marko

2013-01-01

Over the last decades there has been an explosion of new methodologies to study protein complexes. However, most of the approaches currently used are based on in vitro assays (e.g. nuclear magnetic resonance, X-ray, electron microscopy, isothermal titration calorimetry etc). The accurate measurement of parameters that define protein complexes in a physiological context has been largely limited due to technical constrains. Here, we present PICT (Protein interactions from Imaging of Complexes after Translocation), a new method that provides a simple fluorescence microscopy readout for the study of protein complexes in living cells. We take advantage of the inducible dimerization of FK506-binding protein (FKBP) and FKBP-rapamycin binding (FRB) domain to translocate protein assemblies to membrane associated anchoring platforms in yeast. In this assay, GFP-tagged prey proteins interacting with the FRB-tagged bait will co-translocate to the FKBP-tagged anchor sites upon addition of rapamycin. The interactions are thus encoded into localization changes and can be detected by fluorescence live-cell imaging under different physiological conditions or upon perturbations. PICT can be automated for high-throughput studies and can be used to quantify dissociation rates of protein complexes in vivo. In this work we have used PICT to analyze protein-protein interactions from three biological pathways in the yeast Saccharomyces cerevisiae: Mitogen-activated protein kinase cascade (Ste5-Ste11-Ste50), exocytosis (exocyst complex) and endocytosis (Ede1-Syp1).
Template-based structure modeling of protein-protein interactions

PubMed Central

Szilagyi, Andras; Zhang, Yang

2014-01-01

The structure of protein-protein complexes can be constructed by using the known structure of other protein complexes as a template. The complex structure templates are generally detected either by homology-based sequence alignments or, given the structure of monomer components, by structure-based comparisons. Critical improvements have been made in recent years by utilizing interface recognition and by recombining monomer and complex template libraries. Encouraging progress has also been witnessed in genome-wide applications of template-based modeling, with modeling accuracy comparable to high-throughput experimental data. Nevertheless, bottlenecks exist due to the incompleteness of the proteinprotein complex structure library and the lack of methods for distant homologous template identification and full-length complex structure refinement. PMID:24721449
Protein-protein interactions among enzymes of starch biosynthesis in high-amylose barley genotypes reveal differential roles of heteromeric enzyme complexes in the synthesis of A and B granules.

PubMed

Ahmed, Zaheer; Tetlow, Ian J; Ahmed, Regina; Morell, Matthew K; Emes, Michael J

2015-04-01

The present study investigated the role of protein phosphorylation, and protein complex formation between key enzymes of amylopectin synthesis, in barley genotypes exhibiting "high amylose" phenotypes. Starch branching enzyme (SBE) down-regulated lines (ΔSBEIIa and ΔSBEIIb), starch synthase (SS)IIa (ssiia(-), sex6) and SSIII (ssiii(-), amo1) mutants were compared to a reference genotype, OAC Baxter. Down-regulation of either SBEIIa or IIb caused pleiotropic effects on SSI and starch phosphorylase (SP) and resulted in formation of novel protein complexes in which the missing SBEII isoform was substituted by SBEI and SP. In the ΔSBEIIb down-regulated line, soluble SP activity was undetectable. Nonetheless, SP was incorporated into a heteromeric protein complex with SBEI and SBEIIa and was readily detected in starch granules. In amo1, unlike other mutants, the data suggest that both SBEIIa and SBEIIb are in a protein complex with SSI and SSIIa. In the sex6 mutant no protein complexes involving SBEIIa or SBEIIb were detected in amyloplasts. Studies with Pro-Q Diamond revealed that GBSS, SSI, SSIIa, SBEIIb and SP are phosphorylated in their granule bound state. Alteration in the granule proteome in ΔSBEIIa and ΔSBEIIb lines, suggests that different protein complexes are involved in the synthesis of A and B granules. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.
RNA Whole-Mount In situ Hybridisation Proximity Ligation Assay (rISH-PLA), an Assay for Detecting RNA-Protein Complexes in Intact Cells.

PubMed

Roussis, Ioannis M; Guille, Matthew; Myers, Fiona A; Scarlett, Garry P

2016-01-01

Techniques for studying RNA-protein interactions have lagged behind those for DNA-protein complexes as a consequence of the complexities associated with working with RNA. Here we present a method for the modification of the existing In Situ Hybridisation-Proximity Ligation Assay (ISH-PLA) protocol to adapt it to the study of RNA regulation (rISH-PLA). As proof of principle we used the well-characterised interaction of the Xenopus laevis Staufen RNA binding protein with Vg1 mRNA, the complex of which co-localises to the vegetal pole of Xenopus oocytes. The applicability of both the Stau1 antibody and the Locked Nucleic Acid probe (LNA) recognising Vg1 mRNA were independently validated by whole-mount Immunohistochemistry and whole-mount in situ hybridisation assays respectively prior to combining them in the rISH-PLA assay. The rISH-PLA assay allows the identification of a given RNA-protein complex at subcellular and single cell resolution, thus avoiding the lack of spatial resolution and sensitivity associated with assaying heterogenous cell populations from which conventional RNA-protein interaction detection techniques suffer. This technique will be particularly usefully for studying the activity of RNA binding proteins (RBPs) in complex mixtures of cells, for example tissue sections or whole embryos.
Highly Reproducible Label Free Quantitative Proteomic Analysis of RNA Polymerase Complexes*

PubMed Central

Mosley, Amber L.; Sardiu, Mihaela E.; Pattenden, Samantha G.; Workman, Jerry L.; Florens, Laurence; Washburn, Michael P.

2011-01-01

The use of quantitative proteomics methods to study protein complexes has the potential to provide in-depth information on the abundance of different protein components as well as their modification state in various cellular conditions. To interrogate protein complex quantitation using shotgun proteomic methods, we have focused on the analysis of protein complexes using label-free multidimensional protein identification technology and studied the reproducibility of biological replicates. For these studies, we focused on three highly related and essential multi-protein enzymes, RNA polymerase I, II, and III from Saccharomyces cerevisiae. We found that label-free quantitation using spectral counting is highly reproducible at the protein and peptide level when analyzing RNA polymerase I, II, and III. In addition, we show that peptide sampling does not follow a random sampling model, and we show the need for advanced computational models to predict peptide detection probabilities. In order to address these issues, we used the APEX protocol to model the expected peptide detectability based on whole cell lysate acquired using the same multidimensional protein identification technology analysis used for the protein complexes. Neither method was able to predict the peptide sampling levels that we observed using replicate multidimensional protein identification technology analyses. In addition to the analysis of the RNA polymerase complexes, our analysis provides quantitative information about several RNAP associated proteins including the RNAPII elongation factor complexes DSIF and TFIIF. Our data shows that DSIF and TFIIF are the most highly enriched RNAP accessory factors in Rpb3-TAP purifications and demonstrate our ability to measure low level associated protein abundance across biological replicates. In addition, our quantitative data supports a model in which DSIF and TFIIF interact with RNAPII in a dynamic fashion in agreement with previously published reports. PMID:21048197
Identifying Dynamic Protein Complexes Based on Gene Expression Profiles and PPI Networks

PubMed Central

Li, Min; Chen, Weijie; Wang, Jianxin; Pan, Yi

2014-01-01

Identification of protein complexes from protein-protein interaction networks has become a key problem for understanding cellular life in postgenomic era. Many computational methods have been proposed for identifying protein complexes. Up to now, the existing computational methods are mostly applied on static PPI networks. However, proteins and their interactions are dynamic in reality. Identifying dynamic protein complexes is more meaningful and challenging. In this paper, a novel algorithm, named DPC, is proposed to identify dynamic protein complexes by integrating PPI data and gene expression profiles. According to Core-Attachment assumption, these proteins which are always active in the molecular cycle are regarded as core proteins. The protein-complex cores are identified from these always active proteins by detecting dense subgraphs. Final protein complexes are extended from the protein-complex cores by adding attachments based on a topological character of “closeness” and dynamic meaning. The protein complexes produced by our algorithm DPC contain two parts: static core expressed in all the molecular cycle and dynamic attachments short-lived. The proposed algorithm DPC was applied on the data of Saccharomyces cerevisiae and the experimental results show that DPC outperforms CMC, MCL, SPICi, HC-PIN, COACH, and Core-Attachment based on the validation of matching with known complexes and hF-measures. PMID:24963481
Evaluation of Multi-tRNA Synthetase Complex by Multiple Reaction Monitoring Mass Spectrometry Coupled with Size Exclusion Chromatography

PubMed Central

Kim, Jun Seok; Lee, Cheolju

2015-01-01

Eight aminoacyl-tRNA synthetases (M, K, Q, D, R, I, EP and LARS) and three auxiliary proteins (AIMP1, 2 and 3) are known to form a multi-tRNA synthetase complex (MSC) in mammalian cells. We combined size exclusion chromatography (SEC) with reversed-phase liquid chromatography multiple reaction monitoring mass spectrometry (RPLC-MRM-MS) to characterize MSC components and free ARS proteins in human embryonic kidney (HEK 293T) cells. Crude cell extract and affinity-purified proteins were fractionated by SEC in non-denaturing state and ARSs were monitored in each fraction by MRM-MS. The eleven MSC components appeared mostly in earlier SEC fractions demonstrating their participation in complex formation. TARSL2 and AIMP2-DX2, despite their low abundance, were co-purified with KARS and detected in the SEC fractions, where MSC appeared. Moreover, other large complex-forming ARS proteins, such as VARS and FARS, were detected in earlier fractions. The MRM-MS results were further confirmed by western blot analysis. Our study demonstrates usefulness of combined SEC-MRM analysis for the characterization of protein complexes and in understanding the behavior of minor isoforms or variant proteins. PMID:26544075
Evaluation of Multi-tRNA Synthetase Complex by Multiple Reaction Monitoring Mass Spectrometry Coupled with Size Exclusion Chromatography.

PubMed

Park, Seong-Jun; Ahn, Hee-Sung; Kim, Jun Seok; Lee, Cheolju

2015-01-01

Eight aminoacyl-tRNA synthetases (M, K, Q, D, R, I, EP and LARS) and three auxiliary proteins (AIMP1, 2 and 3) are known to form a multi-tRNA synthetase complex (MSC) in mammalian cells. We combined size exclusion chromatography (SEC) with reversed-phase liquid chromatography multiple reaction monitoring mass spectrometry (RPLC-MRM-MS) to characterize MSC components and free ARS proteins in human embryonic kidney (HEK 293T) cells. Crude cell extract and affinity-purified proteins were fractionated by SEC in non-denaturing state and ARSs were monitored in each fraction by MRM-MS. The eleven MSC components appeared mostly in earlier SEC fractions demonstrating their participation in complex formation. TARSL2 and AIMP2-DX2, despite their low abundance, were co-purified with KARS and detected in the SEC fractions, where MSC appeared. Moreover, other large complex-forming ARS proteins, such as VARS and FARS, were detected in earlier fractions. The MRM-MS results were further confirmed by western blot analysis. Our study demonstrates usefulness of combined SEC-MRM analysis for the characterization of protein complexes and in understanding the behavior of minor isoforms or variant proteins.
The Network Organization of Cancer-associated Protein Complexes in Human Tissues

PubMed Central

Zhao, Jing; Lee, Sang Hoon; Huss, Mikael; Holme, Petter

2013-01-01

Differential gene expression profiles for detecting disease genes have been studied intensively in systems biology. However, it is known that various biological functions achieved by proteins follow from the ability of the protein to form complexes by physically binding to each other. In other words, the functional units are often protein complexes rather than individual proteins. Thus, we seek to replace the perspective of disease-related genes by disease-related complexes, exemplifying with data on 39 human solid tissue cancers and their original normal tissues. To obtain the differential abundance levels of protein complexes, we apply an optimization algorithm to genome-wide differential expression data. From the differential abundance of complexes, we extract tissue- and cancer-selective complexes, and investigate their relevance to cancer. The method is supported by a clustering tendency of bipartite cancer-complex relationships, as well as a more concrete and realistic approach to disease-related proteomics. PMID:23567845
Thermal proximity coaggregation for system-wide profiling of protein complex dynamics in cells.

PubMed

Tan, Chris Soon Heng; Go, Ka Diam; Bisteau, Xavier; Dai, Lingyun; Yong, Chern Han; Prabhu, Nayana; Ozturk, Mert Burak; Lim, Yan Ting; Sreekumar, Lekshmy; Lengqvist, Johan; Tergaonkar, Vinay; Kaldis, Philipp; Sobota, Radoslaw M; Nordlund, Pär

2018-03-09

Proteins differentially interact with each other across cellular states and conditions, but an efficient proteome-wide strategy to monitor them is lacking. We report the application of thermal proximity coaggregation (TPCA) for high-throughput intracellular monitoring of protein complex dynamics. Significant TPCA signatures observed among well-validated protein-protein interactions correlate positively with interaction stoichiometry and are statistically observable in more than 350 annotated human protein complexes. Using TPCA, we identified many complexes without detectable differential protein expression, including chromatin-associated complexes, modulated in S phase of the cell cycle. Comparison of six cell lines by TPCA revealed cell-specific interactions even in fundamental cellular processes. TPCA constitutes an approach for system-wide studies of protein complexes in nonengineered cells and tissues and might be used to identify protein complexes that are modulated in diseases. Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.
A water-soluble conjugated polymer for protein identification and denaturation detection.

PubMed

Xu, Qingling; Wu, Chunxian; Zhu, Chunlei; Duan, Xinrui; Liu, Libing; Han, Yuchun; Wang, Yilin; Wang, Shu

2010-12-03

Rapid and sensitive methods to detect proteins and protein denaturation have become increasingly needful in the field of proteomics, medical diagnostics, and biology. In this paper, we have reported the synthesis of a new cationic water-soluble conjugated polymer that contains fluorene and diene moieties in the backbone (PFDE) for protein identification by sensing an array of PFDE solutions in different ionic strengths using the linear discriminant analysis technique (LDA). The PFDE can form complexes with proteins by electrostatic and/or hydrophobic interactions and exhibits different fluorescence response. Three main factors contribute to the fluorescence response of PFDE, namely, the net charge density on the protein surface, the hydrophobic nature of the protein, and the metalloprotein characteristics. The denaturation of proteins can also be detected using PFDE as a fluorescent probe. The interactions between PFDE and proteins were also studied by dynamic light scattering (DLS) and isothermal titration microcalorimetry (ITC) techniques. In contrast to other methods based on conjugated polymers, the synthesis of a series of quencher or dye-labeled acceptors or protein substrates has been avoided in our method, which significantly reduces the cost and the synthetic complexity. Our method provides promising applications on protein identification and denaturation detection in a simple, fast, and label-free manner based on non-specific interaction-induced perturbation of PFDE fluorescence response.
Monochromatic multicomponent fluorescence sedimentation velocity for the study of high-affinity protein interactions

PubMed Central

Zhao, Huaying; Fu, Yan; Glasser, Carla; Andrade Alba, Eric J; Mayer, Mark L; Patterson, George; Schuck, Peter

2016-01-01

The dynamic assembly of multi-protein complexes underlies fundamental processes in cell biology. A mechanistic understanding of assemblies requires accurate measurement of their stoichiometry, affinity and cooperativity, and frequently consideration of multiple co-existing complexes. Sedimentation velocity analytical ultracentrifugation equipped with fluorescence detection (FDS-SV) allows the characterization of protein complexes free in solution with high size resolution, at concentrations in the nanomolar and picomolar range. Here, we extend the capabilities of FDS-SV with a single excitation wavelength from single-component to multi-component detection using photoswitchable fluorescent proteins (psFPs). We exploit their characteristic quantum yield of photo-switching to imprint spatio-temporal modulations onto the sedimentation signal that reveal different psFP-tagged protein components in the mixture. This novel approach facilitates studies of heterogeneous multi-protein complexes at orders of magnitude lower concentrations and for higher-affinity systems than previously possible. Using this technique we studied high-affinity interactions between the amino-terminal domains of GluA2 and GluA3 AMPA receptors. DOI: http://dx.doi.org/10.7554/eLife.17812.001 PMID:27436096
High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes.

PubMed

Wittig, Ilka; Karas, Michael; Schägger, Hermann

2007-07-01

Clear native electrophoresis and blue native electrophoresis are microscale techniques for the isolation of membrane protein complexes. The Coomassie Blue G-250 dye, used in blue native electrophoresis, interferes with in-gel fluorescence detection and in-gel catalytic activity assays. This problem can be overcome by omitting the dye in clear native electrophoresis. However, clear native electrophoresis suffers from enhanced protein aggregation and broadening of protein bands during electrophoresis and therefore has been used rarely. To preserve the advantages of both electrophoresis techniques we substituted Coomassie dye in the cathode buffer of blue native electrophoresis by non-colored mixtures of anionic and neutral detergents. Like Coomassie dye, these mixed micelles imposed a charge shift on the membrane proteins to enhance their anodic migration and improved membrane protein solubility during electrophoresis. This improved clear native electrophoresis offers a high resolution of membrane protein complexes comparable to that of blue native electrophoresis. We demonstrate the superiority of high resolution clear native electrophoresis for in-gel catalytic activity assays of mitochondrial complexes I-V. We present the first in-gel histochemical staining protocol for respiratory complex III. Moreover we demonstrate the special advantages of high resolution clear native electrophoresis for in-gel detection of fluorescent labeled proteins labeled by reactive fluorescent dyes and tagged by fluorescent proteins. The advantages of high resolution clear native electrophoresis make this technique superior for functional proteomics analyses.

Two dimensional Blue Native-/SDS-PAGE analysis of SLP family adaptor protein complexes.

PubMed

Swamy, Mahima; Kulathu, Yogesh; Ernst, Sandra; Reth, Michael; Schamel, Wolfgang W A

2006-04-15

SH2 domain containing leukocyte protein (SLP) adaptor proteins serve a central role in the antigen-mediated activation of lymphocytes by organizing multiprotein signaling complexes. Here, we use two dimensional native-/SDS-gel electrophoresis to study the number, size and relative abundance of protein complexes containing SLP family proteins. In non-stimulated T cells all SLP-76 proteins are in a approximately 400 kDa complex with the small adaptor protein Grb2-like adaptor protein downstream of Shc (Gads), whereas half of Gads is monomeric. This constitutive SLP-76/Gads complex could be reconstituted in Drosophila S2 cells expressing both components, suggesting that it might not contain additional subunits. In contrast, in B cells SLP-65 exists in a 180 kDa complex as well as in monomeric form. Since the complex was not found in S2 cells expressing only SLP-65, it was not di/trimeric SLP-65. Upon antigen-stimulation only the complexed SLP-65 was phosphorylated. Surprisingly, stimulation-induced alteration of SLP complexes could not be detected, suggesting that active signaling complexes form only transiently, and are of low abundance.
Structure-Based Characterization of Multiprotein Complexes

PubMed Central

Wiederstein, Markus; Gruber, Markus; Frank, Karl; Melo, Francisco; Sippl, Manfred J.

2014-01-01

Summary Multiprotein complexes govern virtually all cellular processes. Their 3D structures provide important clues to their biological roles, especially through structural correlations among protein molecules and complexes. The detection of such correlations generally requires comprehensive searches in databases of known protein structures by means of appropriate structure-matching techniques. Here, we present a high-speed structure search engine capable of instantly matching large protein oligomers against the complete and up-to-date database of biologically functional assemblies of protein molecules. We use this tool to reveal unseen structural correlations on the level of protein quaternary structure and demonstrate its general usefulness for efficiently exploring complex structural relationships among known protein assemblies. PMID:24954616
Coevolution study of mitochondria respiratory chain proteins: toward the understanding of protein--protein interaction.

PubMed

Yang, Ming; Ge, Yan; Wu, Jiayan; Xiao, Jingfa; Yu, Jun

2011-05-20

Coevolution can be seen as the interdependency between evolutionary histories. In the context of protein evolution, functional correlation proteins are ever-present coordinated evolutionary characters without disruption of organismal integrity. As to complex system, there are two forms of protein--protein interactions in vivo, which refer to inter-complex interaction and intra-complex interaction. In this paper, we studied the difference of coevolution characters between inter-complex interaction and intra-complex interaction using "Mirror tree" method on the respiratory chain (RC) proteins. We divided the correlation coefficients of every pairwise RC proteins into two groups corresponding to the binary protein--protein interaction in intra-complex and the binary protein--protein interaction in inter-complex, respectively. A dramatical discrepancy is detected between the coevolution characters of the two sets of protein interactions (Wilcoxon test, p-value = 4.4 × 10(-6)). Our finding reveals some critical information on coevolutionary study and assists the mechanical investigation of protein--protein interaction. Furthermore, the results also provide some unique clue for supramolecular organization of protein complexes in the mitochondrial inner membrane. More detailed binding sites map and genome information of nuclear encoded RC proteins will be extraordinary valuable for the further mitochondria dynamics study. Copyright © 2011. Published by Elsevier Ltd.
Recording information on protein complexes in an information management system.

PubMed

Savitsky, Marc; Diprose, Jonathan M; Morris, Chris; Griffiths, Susanne L; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S; Blake, Richard; Stuart, David I; Esnouf, Robert M

2011-08-01

The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein-protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described. Copyright © 2011 Elsevier Inc. All rights reserved.
An automated method for finding molecular complexes in large protein interaction networks

PubMed Central

Bader, Gary D; Hogue, Christopher WV

2003-01-01

Background Recent advances in proteomics technologies such as two-hybrid, phage display and mass spectrometry have enabled us to create a detailed map of biomolecular interaction networks. Initial mapping efforts have already produced a wealth of data. As the size of the interaction set increases, databases and computational methods will be required to store, visualize and analyze the information in order to effectively aid in knowledge discovery. Results This paper describes a novel graph theoretic clustering algorithm, "Molecular Complex Detection" (MCODE), that detects densely connected regions in large protein-protein interaction networks that may represent molecular complexes. The method is based on vertex weighting by local neighborhood density and outward traversal from a locally dense seed protein to isolate the dense regions according to given parameters. The algorithm has the advantage over other graph clustering methods of having a directed mode that allows fine-tuning of clusters of interest without considering the rest of the network and allows examination of cluster interconnectivity, which is relevant for protein networks. Protein interaction and complex information from the yeast Saccharomyces cerevisiae was used for evaluation. Conclusion Dense regions of protein interaction networks can be found, based solely on connectivity data, many of which correspond to known protein complexes. The algorithm is not affected by a known high rate of false positives in data from high-throughput interaction techniques. The program is available from . PMID:12525261
MCL-CAw: a refinement of MCL for detecting yeast complexes from weighted PPI networks by incorporating core-attachment structure

PubMed Central

2010-01-01

Background The reconstruction of protein complexes from the physical interactome of organisms serves as a building block towards understanding the higher level organization of the cell. Over the past few years, several independent high-throughput experiments have helped to catalogue enormous amount of physical protein interaction data from organisms such as yeast. However, these individual datasets show lack of correlation with each other and also contain substantial number of false positives (noise). Over these years, several affinity scoring schemes have also been devised to improve the qualities of these datasets. Therefore, the challenge now is to detect meaningful as well as novel complexes from protein interaction (PPI) networks derived by combining datasets from multiple sources and by making use of these affinity scoring schemes. In the attempt towards tackling this challenge, the Markov Clustering algorithm (MCL) has proved to be a popular and reasonably successful method, mainly due to its scalability, robustness, and ability to work on scored (weighted) networks. However, MCL produces many noisy clusters, which either do not match known complexes or have additional proteins that reduce the accuracies of correctly predicted complexes. Results Inspired by recent experimental observations by Gavin and colleagues on the modularity structure in yeast complexes and the distinctive properties of "core" and "attachment" proteins, we develop a core-attachment based refinement method coupled to MCL for reconstruction of yeast complexes from scored (weighted) PPI networks. We combine physical interactions from two recent "pull-down" experiments to generate an unscored PPI network. We then score this network using available affinity scoring schemes to generate multiple scored PPI networks. The evaluation of our method (called MCL-CAw) on these networks shows that: (i) MCL-CAw derives larger number of yeast complexes and with better accuracies than MCL, particularly in the presence of natural noise; (ii) Affinity scoring can effectively reduce the impact of noise on MCL-CAw and thereby improve the quality (precision and recall) of its predicted complexes; (iii) MCL-CAw responds well to most available scoring schemes. We discuss several instances where MCL-CAw was successful in deriving meaningful complexes, and where it missed a few proteins or whole complexes due to affinity scoring of the networks. We compare MCL-CAw with several recent complex detection algorithms on unscored and scored networks, and assess the relative performance of the algorithms on these networks. Further, we study the impact of augmenting physical datasets with computationally inferred interactions for complex detection. Finally, we analyse the essentiality of proteins within predicted complexes to understand a possible correlation between protein essentiality and their ability to form complexes. Conclusions We demonstrate that core-attachment based refinement in MCL-CAw improves the predictions of MCL on yeast PPI networks. We show that affinity scoring improves the performance of MCL-CAw. PMID:20939868
A Sulfhydryl-Reactive Ruthenium (II) Complex and Its Conjugation to Protein G as a Universal Reagent for Fluorescent Immunoassays

PubMed Central

Goud, Thirumani Venkatshwar; Huang, Bor-Rong; Lin, Tzu-Chau; Biellmann, Jean-François; Chen, Chien-Sheng

2012-01-01

To develop a fluorescent ruthenium complex for biosensing, we synthesized a novel sulfhydryl-reactive compound, 4-bromophenanthroline bis-2,2′-dipyridine Ruthenium bis (hexafluorophosphate). The synthesized Ru(II) complex was crosslinked with thiol-modified protein G to form a universal reagent for fluorescent immunoassays. The resulting Ru(II)-protein G conjugates were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The emission peak wavelength of the Ru(II)-protein G conjugate was 602 nm at the excitation of 452 nm which is similar to the spectra of the Ru(II) complex, indicating that Ru(II)-protein G conjugates still remain the same fluorescence after conjugation. To test the usefulness of the conjugate for biosensing, immunoglobulin G (IgG) binding assay was conducted. The result showed that Ru(II)-protein G conjugates were capable of binding IgG and the more cross-linkers to modify protein G, the higher conjugation efficiency. To demonstrate the feasibility of Ru(II)-protein G conjugates for fluorescent immunoassays, the detection of recombinant histidine-tagged protein using the conjugates and anti-histidine antibody was developed. The results showed that the histidine-tagged protein was successfully detected with dose-response, indicating that Ru(II)-protein G conjugate is a useful universal fluorescent reagent for quantitative immunoassays. PMID:22563441
A universal method for detection of amyloidogenic misfolded proteins.

PubMed

Yam, Alice Y; Wang, Xuemei; Gao, Carol Man; Connolly, Michael D; Zuckermann, Ronald N; Bleu, Thieu; Hall, John; Fedynyshyn, Joseph P; Allauzen, Sophie; Peretz, David; Salisbury, Cleo M

2011-05-24

Diseases associated with the misfolding of endogenous proteins, such as Alzheimer's disease and type II diabetes, are becoming increasingly prevalent. The pathophysiology of these diseases is not totally understood, but mounting evidence suggests that the misfolded protein aggregates themselves may be toxic to cells and serve as key mediators of cell death. As such, an assay that can detect aggregates in a sensitive and selective fashion could provide the basis for early detection of disease, before cellular damage occurs. Here we report the evolution of a reagent that can selectively capture diverse misfolded proteins by interacting with a common supramolecular feature of protein aggregates. By coupling this enrichment tool with protein specific immunoassays, diverse misfolded proteins and sub-femtomole amounts of oligomeric aggregates can be detected in complex biological matrices. We anticipate that this near-universal approach for quantitative misfolded protein detection will become a useful research tool for better understanding amyloidogenic protein pathology as well as serve as the basis for early detection of misfolded protein diseases.
Predicting overlapping protein complexes from weighted protein interaction graphs by gradually expanding dense neighborhoods.

PubMed

Dimitrakopoulos, Christos; Theofilatos, Konstantinos; Pegkas, Andreas; Likothanassis, Spiros; Mavroudi, Seferina

2016-07-01

Proteins are vital biological molecules driving many fundamental cellular processes. They rarely act alone, but form interacting groups called protein complexes. The study of protein complexes is a key goal in systems biology. Recently, large protein-protein interaction (PPI) datasets have been published and a plethora of computational methods that provide new ideas for the prediction of protein complexes have been implemented. However, most of the methods suffer from two major limitations: First, they do not account for proteins participating in multiple functions and second, they are unable to handle weighted PPI graphs. Moreover, the problem remains open as existing algorithms and tools are insufficient in terms of predictive metrics. In the present paper, we propose gradually expanding neighborhoods with adjustment (GENA), a new algorithm that gradually expands neighborhoods in a graph starting from highly informative "seed" nodes. GENA considers proteins as multifunctional molecules allowing them to participate in more than one protein complex. In addition, GENA accepts weighted PPI graphs by using a weighted evaluation function for each cluster. In experiments with datasets from Saccharomyces cerevisiae and human, GENA outperformed Markov clustering, restricted neighborhood search and clustering with overlapping neighborhood expansion, three state-of-the-art methods for computationally predicting protein complexes. Seven PPI networks and seven evaluation datasets were used in total. GENA outperformed existing methods in 16 out of 18 experiments achieving an average improvement of 5.5% when the maximum matching ratio metric was used. Our method was able to discover functionally homogeneous protein clusters and uncover important network modules in a Parkinson expression dataset. When used on the human networks, around 47% of the detected clusters were enriched in gene ontology (GO) terms with depth higher than five in the GO hierarchy. In the present manuscript, we introduce a new method for the computational prediction of protein complexes by making the realistic assumption that proteins participate in multiple protein complexes and cellular functions. Our method can detect accurate and functionally homogeneous clusters. Copyright © 2016 Elsevier B.V. All rights reserved.
Structure-based characterization of multiprotein complexes.

PubMed

Wiederstein, Markus; Gruber, Markus; Frank, Karl; Melo, Francisco; Sippl, Manfred J

2014-07-08

Multiprotein complexes govern virtually all cellular processes. Their 3D structures provide important clues to their biological roles, especially through structural correlations among protein molecules and complexes. The detection of such correlations generally requires comprehensive searches in databases of known protein structures by means of appropriate structure-matching techniques. Here, we present a high-speed structure search engine capable of instantly matching large protein oligomers against the complete and up-to-date database of biologically functional assemblies of protein molecules. We use this tool to reveal unseen structural correlations on the level of protein quaternary structure and demonstrate its general usefulness for efficiently exploring complex structural relationships among known protein assemblies. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.
Evaluation of several two-dimensional gel electrophoresis techniques in cardiac proteomics.

PubMed

Li, Zhao Bo; Flint, Paul W; Boluyt, Marvin O

2005-09-01

Two-dimensional gel electrophoresis (2-DE) is currently the best method for separating complex mixtures of proteins, and its use is gradually becoming more common in cardiac proteome analysis. A number of variations in basic 2-DE have emerged, but their usefulness in analyzing cardiac tissue has not been evaluated. The purpose of the present study was to systematically evaluate the capabilities and limitations of several 2-DE techniques for separating proteins from rat heart tissue. Immobilized pH gradient strips of various pH ranges, parameters of protein loading and staining, subcellular fractionation, and detection of phosphorylated proteins were studied. The results provide guidance for proteome analysis of cardiac and other tissues in terms of selection of the isoelectric point separating window for cardiac proteins, accurate quantitation of cardiac protein abundance, stabilization of technical variation, reduction of sample complexity, enrichment of low-abundant proteins, and detection of phosphorylated proteins.
CytoCluster: A Cytoscape Plugin for Cluster Analysis and Visualization of Biological Networks.

PubMed

Li, Min; Li, Dongyan; Tang, Yu; Wu, Fangxiang; Wang, Jianxin

2017-08-31

Nowadays, cluster analysis of biological networks has become one of the most important approaches to identifying functional modules as well as predicting protein complexes and network biomarkers. Furthermore, the visualization of clustering results is crucial to display the structure of biological networks. Here we present CytoCluster, a cytoscape plugin integrating six clustering algorithms, HC-PIN (Hierarchical Clustering algorithm in Protein Interaction Networks), OH-PIN (identifying Overlapping and Hierarchical modules in Protein Interaction Networks), IPCA (Identifying Protein Complex Algorithm), ClusterONE (Clustering with Overlapping Neighborhood Expansion), DCU (Detecting Complexes based on Uncertain graph model), IPC-MCE (Identifying Protein Complexes based on Maximal Complex Extension), and BinGO (the Biological networks Gene Ontology) function. Users can select different clustering algorithms according to their requirements. The main function of these six clustering algorithms is to detect protein complexes or functional modules. In addition, BinGO is used to determine which Gene Ontology (GO) categories are statistically overrepresented in a set of genes or a subgraph of a biological network. CytoCluster can be easily expanded, so that more clustering algorithms and functions can be added to this plugin. Since it was created in July 2013, CytoCluster has been downloaded more than 9700 times in the Cytoscape App store and has already been applied to the analysis of different biological networks. CytoCluster is available from http://apps.cytoscape.org/apps/cytocluster.
CytoCluster: A Cytoscape Plugin for Cluster Analysis and Visualization of Biological Networks

PubMed Central

Li, Min; Li, Dongyan; Tang, Yu; Wang, Jianxin

2017-01-01

Nowadays, cluster analysis of biological networks has become one of the most important approaches to identifying functional modules as well as predicting protein complexes and network biomarkers. Furthermore, the visualization of clustering results is crucial to display the structure of biological networks. Here we present CytoCluster, a cytoscape plugin integrating six clustering algorithms, HC-PIN (Hierarchical Clustering algorithm in Protein Interaction Networks), OH-PIN (identifying Overlapping and Hierarchical modules in Protein Interaction Networks), IPCA (Identifying Protein Complex Algorithm), ClusterONE (Clustering with Overlapping Neighborhood Expansion), DCU (Detecting Complexes based on Uncertain graph model), IPC-MCE (Identifying Protein Complexes based on Maximal Complex Extension), and BinGO (the Biological networks Gene Ontology) function. Users can select different clustering algorithms according to their requirements. The main function of these six clustering algorithms is to detect protein complexes or functional modules. In addition, BinGO is used to determine which Gene Ontology (GO) categories are statistically overrepresented in a set of genes or a subgraph of a biological network. CytoCluster can be easily expanded, so that more clustering algorithms and functions can be added to this plugin. Since it was created in July 2013, CytoCluster has been downloaded more than 9700 times in the Cytoscape App store and has already been applied to the analysis of different biological networks. CytoCluster is available from http://apps.cytoscape.org/apps/cytocluster. PMID:28858211
Recording information on protein complexes in an information management system

PubMed Central

Savitsky, Marc; Diprose, Jonathan M.; Morris, Chris; Griffiths, Susanne L.; Daniel, Edward; Lin, Bill; Daenke, Susan; Bishop, Benjamin; Siebold, Christian; Wilson, Keith S.; Blake, Richard; Stuart, David I.; Esnouf, Robert M.

2011-01-01

The Protein Information Management System (PiMS) is a laboratory information management system (LIMS) designed for use with the production of proteins in a research environment. The software is distributed under the CCP4 licence, and so is available free of charge to academic laboratories. Like most LIMS, the underlying PiMS data model originally had no support for protein–protein complexes. To support the SPINE2-Complexes project the developers have extended PiMS to meet these requirements. The modifications to PiMS, described here, include data model changes, additional protocols, some user interface changes and functionality to detect when an experiment may have formed a complex. Example data are shown for the production of a crystal of a protein complex. Integration with SPINE2-Complexes Target Tracker application is also described. PMID:21605682
Generation and purification of highly-specific antibodies for detecting post-translationally modified proteins in vivo

PubMed Central

Arur, Swathi; Schedl, Tim

2014-01-01

Post-translational modifications alter protein structure, affecting activity, stability, localization and/or binding partners. Antibodies that specifically recognize post-translationally modified proteins have a number of uses including immuno-cytochemistry and immuno-precipitation of the modified protein to purify protein-protein and protein-nucleic acid complexes. However, antibodies directed at modified sites on individual proteins are often non-specific. Here we describe a protocol to purify polyclonal antibodies that specifically detect the modified protein of interest. The approach uses iterative rounds of subtraction and affinity purification, using stringent washes to remove antibodies that recognize the unmodified protein and low sequence complexity epitopes containing the modified amino acid. Dot and western blots assays are employed to assess antibody preparation specificity. The approach is designed to overcome the common occurrence that a single round of subtraction and affinity purification is not sufficient to obtain a modified protein specific antibody preparation. One full round of antibody purification and specificity testing takes 6 days of discontinuous time. PMID:24457330
Anti-RAINBOW dye-specific antibodies as universal tools for the visualization of prestained protein molecular weight markers in Western blot analysis.

PubMed

Schüchner, Stefan; Andorfer, Peter; Mudrak, Ingrid; Ogris, Egon

2016-08-17

Western blotting is one of the most widely used techniques in molecular biology and biochemistry. Prestained proteins are used as molecular weight standards in protein electrophoresis. In the chemiluminescent Western blot analysis, however, these colored protein markers are invisible leaving researchers with the unsatisfying situation that the signal for the protein of interest and the signal for the markers are not captured simultaneously and have to be merged in an error-prone step. To allow the simultaneous detection of marker proteins we generated monoclonal antibodies specific for the protein dyes. To elicit a dye rather than protein specific immune response we immunized mice sequentially with dye-carrier protein complexes, in which a new carrier protein was used for each subsequent immunization. Moreover, by sequentially immunizing with dye-carrier protein complexes, in which different but structurally related dyes were used, we could also generate an antibody, termed anti-RAINBOW, that cross-reacted even with structurally related dyes not used in the immunizations. Our novel antibodies represent convenient tools for the simultaneous Western blot detection of commercially available prestained marker proteins in combination with the detection of any specific protein of interest. These antibodies will render obsolete the anachronistic tradition of manually charting marker bands on film.
Anti-RAINBOW dye-specific antibodies as universal tools for the visualization of prestained protein molecular weight markers in Western blot analysis

PubMed Central

Schüchner, Stefan; Andorfer, Peter; Mudrak, Ingrid; Ogris, Egon

2016-01-01

Western blotting is one of the most widely used techniques in molecular biology and biochemistry. Prestained proteins are used as molecular weight standards in protein electrophoresis. In the chemiluminescent Western blot analysis, however, these colored protein markers are invisible leaving researchers with the unsatisfying situation that the signal for the protein of interest and the signal for the markers are not captured simultaneously and have to be merged in an error-prone step. To allow the simultaneous detection of marker proteins we generated monoclonal antibodies specific for the protein dyes. To elicit a dye rather than protein specific immune response we immunized mice sequentially with dye-carrier protein complexes, in which a new carrier protein was used for each subsequent immunization. Moreover, by sequentially immunizing with dye-carrier protein complexes, in which different but structurally related dyes were used, we could also generate an antibody, termed anti-RAINBOW, that cross-reacted even with structurally related dyes not used in the immunizations. Our novel antibodies represent convenient tools for the simultaneous Western blot detection of commercially available prestained marker proteins in combination with the detection of any specific protein of interest. These antibodies will render obsolete the anachronistic tradition of manually charting marker bands on film. PMID:27531616
Tumor detection and elimination by a targeted gallium corrole

PubMed Central

Agadjanian, Hasmik; Ma, Jun; Rentsendorj, Altan; Valluripalli, Vinod; Hwang, Jae Youn; Mahammed, Atif; Farkas, Daniel L.; Gray, Harry B.; Gross, Zeev; Medina-Kauwe, Lali K.

2009-01-01

Sulfonated gallium(III) corroles are intensely fluorescent macrocyclic compounds that spontaneously assemble with carrier proteins to undergo cell entry. We report in vivo imaging and therapeutic efficacy of a tumor-targeted corrole noncovalently assembled with a heregulin-modified protein directed at the human epidermal growth factor receptor (HER). Systemic delivery of this protein-corrole complex results in tumor accumulation, which can be visualized in vivo owing to intensely red corrole fluorescence. Targeted delivery in vivo leads to tumor cell death while normal tissue is spared. These findings contrast with the effects of doxorubicin, which can elicit cardiac damage during therapy and required direct intratumoral injection to yield similar levels of tumor shrinkage compared with the systemically delivered corrole. The targeted complex ablated tumors at >5 times a lower dose than untargeted systemic doxorubicin, and the corrole did not damage heart tissue. Complexes remained intact in serum and the carrier protein elicited no detectable immunogenicity. The sulfonated gallium(III) corrole functions both for tumor detection and intervention with safety and targeting advantages over standard chemotherapeutic agents. PMID:19342490
A Plant Immune Receptor Detects Pathogen Effectors that Target WRKY Transcription Factors.

PubMed

Sarris, Panagiotis F; Duxbury, Zane; Huh, Sung Un; Ma, Yan; Segonzac, Cécile; Sklenar, Jan; Derbyshire, Paul; Cevik, Volkan; Rallapalli, Ghanasyam; Saucet, Simon B; Wirthmueller, Lennart; Menke, Frank L H; Sohn, Kee Hoon; Jones, Jonathan D G

2015-05-21

Defense against pathogens in multicellular eukaryotes depends on intracellular immune receptors, yet surveillance by these receptors is poorly understood. Several plant nucleotide-binding, leucine-rich repeat (NB-LRR) immune receptors carry fusions with other protein domains. The Arabidopsis RRS1-R NB-LRR protein carries a C-terminal WRKY DNA binding domain and forms a receptor complex with RPS4, another NB-LRR protein. This complex detects the bacterial effectors AvrRps4 or PopP2 and then activates defense. Both bacterial proteins interact with the RRS1 WRKY domain, and PopP2 acetylates lysines to block DNA binding. PopP2 and AvrRps4 interact with other WRKY domain-containing proteins, suggesting these effectors interfere with WRKY transcription factor-dependent defense, and RPS4/RRS1 has integrated a "decoy" domain that enables detection of effectors that target WRKY proteins. We propose that NB-LRR receptor pairs, one member of which carries an additional protein domain, enable perception of pathogen effectors whose function is to target that domain. Copyright © 2015 Elsevier Inc. All rights reserved.
Optical Lock-In Detection of FRET Using Synthetic and Genetically Encoded Optical Switches

PubMed Central

Mao, Shu; Benninger, Richard K. P.; Yan, Yuling; Petchprayoon, Chutima; Jackson, David; Easley, Christopher J.; Piston, David W.; Marriott, Gerard

2008-01-01

The Förster resonance energy transfer (FRET) technique is widely used for studying protein interactions within live cells. The effectiveness and sensitivity of determining FRET, however, can be reduced by photobleaching, cross talk, autofluorescence, and unlabeled, endogenous proteins. We present a FRET imaging method using an optical switch probe, Nitrobenzospiropyran (NitroBIPS), which substantially improves the sensitivity of detection to <1% FRET efficiency. Through orthogonal optical control of the colorful merocyanine and colorless spiro states of the NitroBIPS acceptor, donor fluorescence can be measured both in the absence and presence of FRET in the same FRET pair in the same cell. A SNAP-tag approach is used to generate a green fluorescent protein-alkylguaninetransferase fusion protein (GFP-AGT) that is labeled with benzylguanine-NitroBIPS. In vivo imaging studies on this green fluorescent protein-alkylguaninetransferase (GFP-AGT) (NitroBIPS) complex, employing optical lock-in detection of FRET, allow unambiguous resolution of FRET efficiencies below 1%, equivalent to a few percent of donor-tagged proteins in complexes with acceptor-tagged proteins. PMID:18281383

Live Cell Visualization of Multiple Protein-Protein Interactions with BiFC Rainbow.

PubMed

Wang, Sheng; Ding, Miao; Xue, Boxin; Hou, Yingping; Sun, Yujie

2018-05-18

As one of the most powerful tools to visualize PPIs in living cells, bimolecular fluorescence complementation (BiFC) has gained great advancement during recent years, including deep tissue imaging with far-red or near-infrared fluorescent proteins or super-resolution imaging with photochromic fluorescent proteins. However, little progress has been made toward simultaneous detection and visualization of multiple PPIs in the same cell, mainly due to the spectral crosstalk. In this report, we developed novel BiFC assays based on large-Stokes-shift fluorescent proteins (LSS-FPs) to detect and visualize multiple PPIs in living cells. With the large excitation/emission spectral separation, LSS-FPs can be imaged together with normal Stokes shift fluorescent proteins to realize multicolor BiFC imaging using a simple illumination scheme. We also further demonstrated BiFC rainbow combining newly developed BiFC assays with previously established mCerulean/mVenus-based BiFC assays to achieve detection and visualization of four PPI pairs in the same cell. Additionally, we prove that with the complete spectral separation of mT-Sapphire and CyOFP1, LSS-FP-based BiFC assays can be readily combined with intensity-based FRET measurement to detect ternary protein complex formation with minimal spectral crosstalk. Thus, our newly developed LSS-FP-based BiFC assays not only expand the fluorescent protein toolbox available for BiFC but also facilitate the detection and visualization of multiple protein complex interactions in living cells.
Electrophoretic Mobility Shift Assay (EMSA) for Detecting Protein-Nucleic Acid Interactions

PubMed Central

Hellman, Lance M.; Fried, Michael G.

2009-01-01

The gel electrophoresis mobility shift assay (EMSA) is used to detect protein complexes with nucleic acids. It is the core technology underlying a wide range of qualitative and quantitative analyses for the characterization of interacting systems. In the classical assay, solutions of protein and nucleic acid are combined and the resulting mixtures are subjected to electrophoresis under native conditions through polyacrylamide or agarose gel. After electrophoresis, the distribution of species containing nucleic acid is determined, usually by autoradiography of 32P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more slowly than the corresponding free nucleic acid. In this article, we identify the most important factors that determine the stabilities and electrophoretic mobilities of complexes under assay conditions. A representative protocol is provided and commonly used variants are discussed. Expected outcomes are briefly described. References to extensions of the method and a troubleshooting guide are provided. PMID:17703195
Identifying protein complexes in PPI network using non-cooperative sequential game.

PubMed

Maulik, Ujjwal; Basu, Srinka; Ray, Sumanta

2017-08-21

Identifying protein complexes from protein-protein interaction (PPI) network is an important and challenging task in computational biology as it helps in better understanding of cellular mechanisms in various organisms. In this paper we propose a noncooperative sequential game based model for protein complex detection from PPI network. The key hypothesis is that protein complex formation is driven by mechanism that eventually optimizes the number of interactions within the complex leading to dense subgraph. The hypothesis is drawn from the observed network property named small world. The proposed multi-player game model translates the hypothesis into the game strategies. The Nash equilibrium of the game corresponds to a network partition where each protein either belong to a complex or form a singleton cluster. We further propose an algorithm to find the Nash equilibrium of the sequential game. The exhaustive experiment on synthetic benchmark and real life yeast networks evaluates the structural as well as biological significance of the network partitions.
Visual Reading Method for Detection of Bacterial Tannase

PubMed Central

Osawa, R.; Walsh, T. P.

1993-01-01

Tannase activity of bacteria capable of degrading tannin-protein complexes was determined by a newly developed visual reading method. The method is based on two phenomena: (i) the ability of tannase to hydrolyze methyl gallate to release free gallic acid and (ii) the green to brown coloration of gallic acid after prolonged exposure to oxygen in an alkaline condition. The method has been successfully used to detect the presence of tannase in the cultures of bacteria capable of degrading tannin-protein complexes. PMID:16348918
Benchtop Detection of Proteins

NASA Technical Reports Server (NTRS)

Scardelletti, Maximilian C.; Varaljay, Vanessa

2007-01-01

A process, and a benchtop-scale apparatus for implementing the process, have been developed to detect proteins associated with specific microbes in water. The process and apparatus may also be useful for detection of proteins in other, more complex liquids. There may be numerous potential applications, including monitoring lakes and streams for contamination, testing of blood and other bodily fluids in medical laboratories, and testing for microbial contamination of liquids in restaurants and industrial food-processing facilities. A sample can be prepared and analyzed by use of this process and apparatus within minutes, whereas an equivalent analysis performed by use of other processes and equipment can often take hours to days. The process begins with the conjugation of near-infrared-fluorescent dyes to antibodies that are specific to a particular protein. Initially, the research has focused on using near-infrared dyes to detect antigens or associated proteins in solution, which has proven successful vs. microbial cells, and streamlining the technique in use for surface protein detection on microbes would theoretically render similar results. However, it is noted that additional work is needed to transition protein-based techniques to microbial cell detection. Consequently, multiple such dye/antibody pairs could be prepared to enable detection of multiple selected microbial species, using a different dye for each species. When excited by near-infrared light of a suitable wavelength, each dye fluoresces at a unique longer wavelength that differs from those of the other dyes, enabling discrimination among the various species. In initial tests, the dye/antibody pairs are mixed into a solution suspected of containing the selected proteins, causing the binding of the dye/antibody pairs to such suspect proteins that may be present. The solution is then run through a microcentrifuge that includes a membrane that acts as a filter in that it retains the dye/antibody/protein complexes while allowing any remaining unbound dye/antibody pairs to flow away. The retained dye/antibody/protein complexes are transferred to a cuvette, wherein they are irradiated with light from a miniature near-infrared laser delivered via a fiber-optic cable. The resulting fluorescence from the dye(s) is measured by use of a miniature spectrometer, the output of which is digitized, then analyzed by laptop computer. The software running in the computer identifies the protein species by the wavelengths of their spectral peaks and determines the amounts of the proteins, and thus, one day, microbes of the various species from the intensities of the peaks. The abovementioned removal of the unbound dye/antibody pairs during centrifugation prevents false positive readings. The process proves successful in detecting proteins in solution and thus can now be employed for use in microbe detection.
From pull-down data to protein interaction networks and complexes with biological relevance.

PubMed

Zhang, Bing; Park, Byung-Hoon; Karpinets, Tatiana; Samatova, Nagiza F

2008-04-01

Recent improvements in high-throughput Mass Spectrometry (MS) technology have expedited genome-wide discovery of protein-protein interactions by providing a capability of detecting protein complexes in a physiological setting. Computational inference of protein interaction networks and protein complexes from MS data are challenging. Advances are required in developing robust and seamlessly integrated procedures for assessment of protein-protein interaction affinities, mathematical representation of protein interaction networks, discovery of protein complexes and evaluation of their biological relevance. A multi-step but easy-to-follow framework for identifying protein complexes from MS pull-down data is introduced. It assesses interaction affinity between two proteins based on similarity of their co-purification patterns derived from MS data. It constructs a protein interaction network by adopting a knowledge-guided threshold selection method. Based on the network, it identifies protein complexes and infers their core components using a graph-theoretical approach. It deploys a statistical evaluation procedure to assess biological relevance of each found complex. On Saccharomyces cerevisiae pull-down data, the framework outperformed other more complicated schemes by at least 10% in F(1)-measure and identified 610 protein complexes with high-functional homogeneity based on the enrichment in Gene Ontology (GO) annotation. Manual examination of the complexes brought forward the hypotheses on cause of false identifications. Namely, co-purification of different protein complexes as mediated by a common non-protein molecule, such as DNA, might be a source of false positives. Protein identification bias in pull-down technology, such as the hydrophilic bias could result in false negatives.
Bioluminescence methodology for the detection of protein-protein interactions within the voltage-gated sodium channel macromolecular complex.

PubMed

Shavkunov, Alexander; Panova, Neli; Prasai, Anesh; Veselenak, Ron; Bourne, Nigel; Stoilova-McPhie, Svetla; Laezza, Fernanda

2012-04-01

Protein-protein interactions are critical molecular determinants of ion channel function and emerging targets for pharmacological interventions. Yet, current methodologies for the rapid detection of ion channel macromolecular complexes are still lacking. In this study we have adapted a split-luciferase complementation assay (LCA) for detecting the assembly of the voltage-gated Na+ (Nav) channel C-tail and the intracellular fibroblast growth factor 14 (FGF14), a functionally relevant component of the Nav channelosome that controls gating and targeting of Nav channels through direct interaction with the channel C-tail. In the LCA, two complementary N-terminus and C-terminus fragments of the firefly luciferase were fused, respectively, to a chimera of the CD4 transmembrane segment and the C-tail of Nav1.6 channel (CD4-Nav1.6-NLuc) or FGF14 (CLuc-FGF14). Co-expression of CLuc-FGF14 and CD4-Nav1.6-NLuc in live cells led to a robust assembly of the FGF14:Nav1.6 C-tail complex, which was attenuated by introducing single-point mutations at the predicted FGF14:Nav channel interface. To evaluate the dynamic regulation of the FGF14:Nav1.6 C-tail complex by signaling pathways, we investigated the effect of kinase inhibitors on the complex formation. Through a platform of counter screenings, we show that the p38/MAPK inhibitor, PD169316, and the IκB kinase inhibitor, BAY 11-7082, reduce the FGF14:Nav1.6 C-tail complementation, highlighting a potential role of the p38MAPK and the IκB/NFκB pathways in controlling neuronal excitability through protein-protein interactions. We envision the methodology presented here as a new valuable tool to allow functional evaluations of protein-channel complexes toward probe development and drug discovery targeting ion channels implicated in human disorders.
Chemical Methods for the Direct Detection and Labeling of S-Nitrosothiols

PubMed Central

Bechtold, Erika

2012-01-01

Abstract Significance: Posttranslational modification of proteins through phosphorylation, glycosylation, and oxidation adds complexity to the proteome by reversibly altering the structure and function of target proteins in a highly controlled fashion. Recent Advances: The study of reversible cysteine oxidation highlights a role for this oxidative modification in complex signal transduction pathways. Nitric oxide (NO), and its respective metabolites (including reactive nitrogen species), participates in a variety of these cellular redox processes, including the reversible oxidation of cysteine to S-nitrosothiols (RSNOs). RSNOs act as endogenous transporters of NO, but also possess beneficial effects independent of NO-related signaling, which suggests a complex and versatile biological role. In this review, we highlight the importance of RSNOs as a required posttranslational modification and summarize the current methods available for detecting S-nitrosation. Critical Issues: Given the limitations of these indirect detection methods, the review covers recent developments toward the direct detection of RSNOs by phosphine-based chemical probes. The intrinsic properties that dictate this phosphine/RSNO reactivity are summarized. In general, RSNOs (both small molecule and protein) react with phosphines to yield reactive S-substituted aza-ylides that undergo further reactions leading to stable RSNO-based adducts. Future Directions: This newly explored chemical reactivity forms the basis of a number of exciting potential chemical methods for protein RSNO detection in biological systems. Antioxid. Redox Signal. 17, 981–991. PMID:22356122
xTract: software for characterizing conformational changes of protein complexes by quantitative cross-linking mass spectrometry.

PubMed

Walzthoeni, Thomas; Joachimiak, Lukasz A; Rosenberger, George; Röst, Hannes L; Malmström, Lars; Leitner, Alexander; Frydman, Judith; Aebersold, Ruedi

2015-12-01

Chemical cross-linking in combination with mass spectrometry generates distance restraints of amino acid pairs in close proximity on the surface of native proteins and protein complexes. In this study we used quantitative mass spectrometry and chemical cross-linking to quantify differences in cross-linked peptides obtained from complexes in spatially discrete states. We describe a generic computational pipeline for quantitative cross-linking mass spectrometry consisting of modules for quantitative data extraction and statistical assessment of the obtained results. We used the method to detect conformational changes in two model systems: firefly luciferase and the bovine TRiC complex. Our method discovers and explains the structural heterogeneity of protein complexes using only sparse structural information.
Detecting transitions in protein dynamics using a recurrence quantification analysis based bootstrap method.

PubMed

Karain, Wael I

2017-11-28

Proteins undergo conformational transitions over different time scales. These transitions are closely intertwined with the protein's function. Numerous standard techniques such as principal component analysis are used to detect these transitions in molecular dynamics simulations. In this work, we add a new method that has the ability to detect transitions in dynamics based on the recurrences in the dynamical system. It combines bootstrapping and recurrence quantification analysis. We start from the assumption that a protein has a "baseline" recurrence structure over a given period of time. Any statistically significant deviation from this recurrence structure, as inferred from complexity measures provided by recurrence quantification analysis, is considered a transition in the dynamics of the protein. We apply this technique to a 132 ns long molecular dynamics simulation of the β-Lactamase Inhibitory Protein BLIP. We are able to detect conformational transitions in the nanosecond range in the recurrence dynamics of the BLIP protein during the simulation. The results compare favorably to those extracted using the principal component analysis technique. The recurrence quantification analysis based bootstrap technique is able to detect transitions between different dynamics states for a protein over different time scales. It is not limited to linear dynamics regimes, and can be generalized to any time scale. It also has the potential to be used to cluster frames in molecular dynamics trajectories according to the nature of their recurrence dynamics. One shortcoming for this method is the need to have large enough time windows to insure good statistical quality for the recurrence complexity measures needed to detect the transitions.
Measles virus attachment proteins with impaired ability to bind CD46 interact more efficiently with the homologous fusion protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Corey, Elizabeth A.; Iorio, Ronald M.; Program in Immunology and Virology, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, MA 01655

2009-01-05

Fusion promotion by measles virus (MV) depends on an interaction between the hemagglutinin (H) and fusion (F) glycoproteins. Amino acid substitutions in MV H that drastically reduce hemagglutinating activity result in an increase in the amount of H (primarily the 74 kDa isoform) detectable in a complex with F at the cell surface. This is in direct contrast to the loss of the ability to detect a complex between the fusion protein of Newcastle disease virus and most attachment proteins that lack receptor binding activity. These opposing results provide support for the existence of different mechanisms for the regulation ofmore » fusion by these two paramyxoviruses.« less
Ribosomal RNA maturation in Schizosaccharomyces pombe is dependent on a large ribonucleoprotein complex of the internal transcribed spacer 1.

PubMed

Lalev, A I; Abeyrathne, P D; Nazar, R N

2000-09-08

The interdependency of steps in the processing of pre-rRNA in Schizosaccharomyces pombe suggests that RNA processing, at least in part, acts as a quality control mechanism which helps assure that only functional RNA is incorporated into mature ribosomes. To determine further the role of the transcribed spacer regions in rRNA processing and to detect interactions which underlie the interdependencies, the ITS1 sequence was examined for its ability to form ribonucleoprotein complexes with cellular proteins. When incubated with protein extract, the spacer formed a specific large RNP. This complex was stable to fractionation by agarose or polyacrylamide gel electrophoresis. Modification exclusion analyses indicated that the proteins interact with a helical domain which is conserved in the internal transcribed spacers. Mutagenic analyses confirmed an interaction with this sequence and indicated that this domain is critical to the efficient maturation of the precursor RNA. The protein constituents, purified by affinity chromatography using the ITS1 sequence, retained an ability to form stable RNP. Protein analyses of gel purified complex, prepared with affinity-purified proteins, indicated at least 20 protein components ranging in size from 20-200 kDa. Peptide mapping by Maldi-Toff mass spectroscopy identified eight hypothetical RNA binding proteins which included four different RNA-binding motifs. Another protein was putatively identified as a pseudouridylate synthase. Additional RNA constituents were not detected. The significance of this complex with respect to rRNA maturation and interdependence in rRNA processing is discussed. Copyright 2000 Academic Press.
Development of bimolecular fluorescence complementation using rsEGFP2 for detection and super-resolution imaging of protein-protein interactions in live cells

PubMed Central

Wang, Sheng; Ding, Miao; Chen, Xuanze; Chang, Lei; Sun, Yujie

2017-01-01

Direct visualization of protein-protein interactions (PPIs) at high spatial and temporal resolution in live cells is crucial for understanding the intricate and dynamic behaviors of signaling protein complexes. Recently, bimolecular fluorescence complementation (BiFC) assays have been combined with super-resolution imaging techniques including PALM and SOFI to visualize PPIs at the nanometer spatial resolution. RESOLFT nanoscopy has been proven as a powerful live-cell super-resolution imaging technique. With regard to the detection and visualization of PPIs in live cells with high temporal and spatial resolution, here we developed a BiFC assay using split rsEGFP2, a highly photostable and reversibly photoswitchable fluorescent protein previously developed for RESOLFT nanoscopy. Combined with parallelized RESOLFT microscopy, we demonstrated the high spatiotemporal resolving capability of a rsEGFP2-based BiFC assay by detecting and visualizing specifically the heterodimerization interactions between Bcl-xL and Bak as well as the dynamics of the complex on mitochondria membrane in live cells. PMID:28663931
Evaluating the efficacy of a structure-derived amino acid substitution matrix in detecting protein homologs by BLAST and PSI-BLAST.

PubMed

Goonesekere, Nalin Cw

2009-01-01

The large numbers of protein sequences generated by whole genome sequencing projects require rapid and accurate methods of annotation. The detection of homology through computational sequence analysis is a powerful tool in determining the complex evolutionary and functional relationships that exist between proteins. Homology search algorithms employ amino acid substitution matrices to detect similarity between proteins sequences. The substitution matrices in common use today are constructed using sequences aligned without reference to protein structure. Here we present amino acid substitution matrices constructed from the alignment of a large number of protein domain structures from the structural classification of proteins (SCOP) database. We show that when incorporated into the homology search algorithms BLAST and PSI-blast, the structure-based substitution matrices enhance the efficacy of detecting remote homologs.
Polyethylenimine-coated Fe3O4 nanoparticles effectively quench fluorescent DNA, which can be developed as a novel platform for protein detection.

PubMed

Ma, Long; Sun, Nana; Zhang, Jinyan; Tu, Chunhao; Cao, Xiuqi; Duan, Demin; Diao, Aipo; Man, Shuli

2017-11-23

We report a novel assembly of polyethyleneimine (PEI)-coated Fe 3 O 4 nanoparticles (NPs) with single-stranded DNA (ssDNA), and the fluorescence of the dye labeled in the DNA is remarkably quenched. In the presence of a target protein, the protein-DNA aptamer mutual interaction releases the ssDNA from this assembly and hence restores the fluorescence. This feature could be adopted to develop an aptasensor for protein detection. As a proof-of-concept, for the first time, we have used this proposed sensing strategy to detect thrombin selectively and sensitively. Furthermore, simultaneous multiple detection of thrombin and lysozyme in a complex protein mixture has been proven to be possible.
Proteomic technology for biomarker profiling in cancer: an update*

PubMed Central

Alaoui-Jamali, Moulay A.; Xu, Ying-jie

2006-01-01

The progress in the understanding of cancer progression and early detection has been slow and frustrating due to the complex multifactorial nature and heterogeneity of the cancer syndrome. To date, no effective treatment is available for advanced cancers, which remain a major cause of morbidity and mortality. Clearly, there is urgent need to unravel novel biomarkers for early detection. Most of the functional information of the cancer-associated genes resides in the proteome. The later is an exceptionally complex biological system involving several proteins that function through posttranslational modifications and dynamic intermolecular collisions with partners. These protein complexes can be regulated by signals emanating from cancer cells, their surrounding tissue microenvironment, and/or from the host. Some proteins are secreted and/or cleaved into the extracellular milieu and may represent valuable serum biomarkers for diagnosis purpose. It is estimated that the cancer proteome may include over 1.5 million proteins as a result of posttranslational processing and modifications. Such complexity clearly highlights the need for ultra-high resolution proteomic technology for robust quantitative protein measurements and data acquisition. This review is to update the current research efforts in high-resolution proteomic technology for discovery and monitoring cancer biomarkers. PMID:16625706
Lys48 ubiquitination during the intraerythrocytic cycle of the rodent malaria parasite, Plasmodium chabaudi.

PubMed

González-López, Lorena; Carballar-Lejarazú, Rebeca; Arrevillaga Boni, Gerardo; Cortés-Martínez, Leticia; Cázares-Raga, Febe Elena; Trujillo-Ocampo, Abel; Rodríguez, Mario H; James, Anthony A; Hernández-Hernández, Fidel de la Cruz

2017-01-01

Ubiquitination tags proteins for different functions within the cell. One of the most abundant and studied ubiquitin modification is the Lys48 polyubiquitin chain that modifies proteins for their destruction by proteasome. In Plasmodium is proposed that post-translational regulation is fundamental for parasite development during its complex life-cycle; thus, the objective of this work was to analyze the ubiquitination during Plasmodium chabaudi intraerythrocytic stages. Ubiquitinated proteins were detected during intraerythrocytic stages of Plasmodium chabaudi by immunofluorescent microscopy, bidimensional electrophoresis (2-DE) combined with immunoblotting and mass spectrometry. All the studied stages presented protein ubiquitination and Lys48 polyubiquitination with more abundance during the schizont stage. Three ubiquitinated proteins were identified for rings, five for trophozoites and twenty for schizonts. Only proteins detected with a specific anti- Lys48 polyubiquitin antibody were selected for Mass Spectrometry analysis and two of these identified proteins were selected in order to detect the specific amino acid residues where ubiquitin is placed. Ubiquitinated proteins during the ring and trophozoite stages were related with the invasion process and in schizont proteins were related with nucleic acid metabolism, glycolysis and protein biosynthesis. Most of the ubiquitin detection was during the schizont stage and the Lys48 polyubiquitination during this stage was related to proteins that are expected to be abundant during the trophozoite stage. The evidence that these Lys48 polyubiquitinated proteins are tagged for destruction by the proteasome complex suggests that this type of post-translational modification is important in the regulation of protein abundance during the life-cycle and may also contribute to the parasite cell-cycle progression.
Characterization of Native Protein Complexes and Protein Isoform Variation Using Size-fractionation-based Quantitative Proteomics*

PubMed Central

Kirkwood, Kathryn J.; Ahmad, Yasmeen; Larance, Mark; Lamond, Angus I.

2013-01-01

Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach, we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1-MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific Gene Ontology terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on the native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data-sharing resource available to the community. PMID:24043423
Characterization of native protein complexes and protein isoform variation using size-fractionation-based quantitative proteomics.

PubMed

Kirkwood, Kathryn J; Ahmad, Yasmeen; Larance, Mark; Lamond, Angus I

2013-12-01

Proteins form a diverse array of complexes that mediate cellular function and regulation. A largely unexplored feature of such protein complexes is the selective participation of specific protein isoforms and/or post-translationally modified forms. In this study, we combined native size-exclusion chromatography (SEC) with high-throughput proteomic analysis to characterize soluble protein complexes isolated from human osteosarcoma (U2OS) cells. Using this approach, we have identified over 71,500 peptides and 1,600 phosphosites, corresponding to over 8,000 proteins, distributed across 40 SEC fractions. This represents >50% of the predicted U2OS cell proteome, identified with a mean peptide sequence coverage of 27% per protein. Three biological replicates were performed, allowing statistical evaluation of the data and demonstrating a high degree of reproducibility in the SEC fractionation procedure. Specific proteins were detected interacting with multiple independent complexes, as typified by the separation of distinct complexes for the MRFAP1-MORF4L1-MRGBP interaction network. The data also revealed protein isoforms and post-translational modifications that selectively associated with distinct subsets of protein complexes. Surprisingly, there was clear enrichment for specific Gene Ontology terms associated with differential size classes of protein complexes. This study demonstrates that combined SEC/MS analysis can be used for the system-wide annotation of protein complexes and to predict potential isoform-specific interactions. All of these SEC data on the native separation of protein complexes have been integrated within the Encyclopedia of Proteome Dynamics, an online, multidimensional data-sharing resource available to the community.
The Detection of Protein via ZnO Resonant Raman Scattering Signal

NASA Astrophysics Data System (ADS)

Shan, Guiye; Yang, Guoliang; Wang, Shuang; Liu, Yichun

2008-03-01

Detecting protein with high sensitivity and specificity is essential for disease diagnostics, drug screening and other application. Semiconductor nanoparticles show better properties than organic dye molecules when used as markers for optical measurements. We used ZnO nanoparticles as markers for detecting protein in resonant Raman scattering measurements. The highly sensitive detection of proteins was achieved by an antibody-based sandwich assay. A probe for the target protein was constructed by binding the ZnO/Au nanoparticles to a primary antibody by eletrostatic interaction between Au and the antibody. A secondary antibody, which could be specifically recognized by target protein, was attached to a solid surface. The ZnO/Au-antibody probe could specifically recognize and bind to the complex of the target protein and secondary antibody. Our measurements using the resonant Raman scattering signal of ZnO nanoparticles showed good selectivity and sensitivity for the target protein.

Searching for microbial protein over-expression in a complex matrix using automated high throughput MS-based proteomics tools.

PubMed

Akeroyd, Michiel; Olsthoorn, Maurien; Gerritsma, Jort; Gutker-Vermaas, Diana; Ekkelkamp, Laurens; van Rij, Tjeerd; Klaassen, Paul; Plugge, Wim; Smit, Ed; Strupat, Kerstin; Wenzel, Thibaut; van Tilborg, Marcel; van der Hoeven, Rob

2013-03-10

In the discovery of new enzymes genomic and cDNA expression libraries containing thousands of differential clones are generated to obtain biodiversity. These libraries need to be screened for the activity of interest. Removing so-called empty and redundant clones significantly reduces the size of these expression libraries and therefore speeds up new enzyme discovery. Here, we present a sensitive, generic workflow for high throughput screening of successful microbial protein over-expression in microtiter plates containing a complex matrix based on mass spectrometry techniques. MALDI-LTQ-Orbitrap screening followed by principal component analysis and peptide mass fingerprinting was developed to obtain a throughput of ∼12,000 samples per week. Alternatively, a UHPLC-MS(2) approach including MS(2) protein identification was developed for microorganisms with a complex protein secretome with a throughput of ∼2000 samples per week. TCA-induced protein precipitation enhanced by addition of bovine serum albumin is used for protein purification prior to MS detection. We show that this generic workflow can effectively reduce large expression libraries from fungi and bacteria to their minimal size by detection of successful protein over-expression using MS. Copyright © 2012 Elsevier B.V. All rights reserved.
Bimolecular fluorescence complementation: visualization of molecular interactions in living cells.

PubMed

Kerppola, Tom K

2008-01-01

A variety of experimental methods have been developed for the analysis of protein interactions. The majority of these methods either require disruption of the cells to detect molecular interactions or rely on indirect detection of the protein interaction. The bimolecular fluorescence complementation (BiFC) assay provides a direct approach for the visualization of molecular interactions in living cells and organisms. The BiFC approach is based on the facilitated association between two fragments of a fluorescent protein when the fragments are brought together by an interaction between proteins fused to the fragments. The BiFC approach has been used for visualization of interactions among a variety of structurally diverse interaction partners in many different cell types. It enables detection of transient complexes as well as complexes formed by a subpopulation of the interaction partners. It is essential to include negative controls in each experiment in which the interface between the interaction partners has been mutated or deleted. The BiFC assay has been adapted for simultaneous visualization of multiple protein complexes in the same cell and the competition for shared interaction partners. A ubiquitin-mediated fluorescence complementation assay has also been developed for visualization of the covalent modification of proteins by ubiquitin family peptides. These fluorescence complementation assays have a great potential to illuminate a variety of biological interactions in the future.
Cardiac mitochondrial matrix and respiratory complex protein phosphorylation

PubMed Central

Covian, Raul

2012-01-01

It has become appreciated over the last several years that protein phosphorylation within the cardiac mitochondrial matrix and respiratory complexes is extensive. Given the importance of oxidative phosphorylation and the balance of energy metabolism in the heart, the potential regulatory effect of these classical signaling events on mitochondrial function is of interest. However, the functional impact of protein phosphorylation and the kinase/phosphatase system responsible for it are relatively unknown. Exceptions include the well-characterized pyruvate dehydrogenase and branched chain α-ketoacid dehydrogenase regulatory system. The first task of this review is to update the current status of protein phosphorylation detection primarily in the matrix and evaluate evidence linking these events with enzymatic function or protein processing. To manage the scope of this effort, we have focused on the pathways involved in energy metabolism. The high sensitivity of modern methods of detecting protein phosphorylation and the low specificity of many kinases suggests that detection of protein phosphorylation sites without information on the mole fraction of phosphorylation is difficult to interpret, especially in metabolic enzymes, and is likely irrelevant to function. However, several systems including protein translocation, adenine nucleotide translocase, cytochrome c, and complex IV protein phosphorylation have been well correlated with enzymatic function along with the classical dehydrogenase systems. The second task is to review the current understanding of the kinase/phosphatase system within the matrix. Though it is clear that protein phosphorylation occurs within the matrix, based on 32P incorporation and quantitative mass spectrometry measures, the kinase/phosphatase system responsible for this process is ill-defined. An argument is presented that remnants of the much more labile bacterial protein phosphoryl transfer system may be present in the matrix and that the evaluation of this possibility will require the application of approaches developed for bacterial cell signaling to the mitochondria. PMID:22886415
Label-free proteomic analysis of intestinal mucosa proteins in common carp (Cyprinus carpio) infected with Aeromonas hydrophila.

PubMed

Di, Guilan; Li, Hui; Zhang, Chao; Zhao, Yanjing; Zhou, Chuanjiang; Naeem, Sajid; Li, Li; Kong, Xianghui

2017-07-01

Outbreaks of infectious diseases in common carp Cyprinus carpio, a major cultured fish in northern regions of China, constantly result in significant economic losses. Until now, information proteomic on immune defence remains limited. In the present study, a profile of intestinal mucosa immune response in Cyprinus carpio was investigated after 0, 12, 36 and 84 h after challenging tissues with Aeromonas hydrophila at a concentration of 1.4 × 10 8 CFU/mL. Proteomic profiles in different samples were compared using label-free quantitative proteomic approach. Based on MASCOT database search, 1149 proteins were identified in samples after normalisation of proteins. Treated groups 1 (T1) and 2 (T2) were first clustered together and then clustered with control (C group). The distance between C and treated group 3 (T3) represented the maxima according to hierarchical cluster analysis. Therefore, comparative analysis between C and T3 was selected in the following analysis. A total of 115 proteins with differential abundance were detected to show conspicuous expressing variances. A total of 52 up-regulated proteins and 63 down-regulated proteins were detected in T3. Gene ontology analysis showed that identified up-regulated differentially expressed proteins in T3 were mainly localised in the hemoglobin complex, and down-regulated proteins in T3 were mainly localised in the major histocompatibility complex II protein complex. Forty-six proteins of differential abundance (40% of 115) were involved in immune response, with 17 up-regulated and 29 down-regulated proteins detected in T3. This study is the first to report proteome response of carp intestinal mucosa against A. hydrophila infection; information obtained contribute to understanding defence mechanisms of carp intestinal mucosa. Copyright © 2017 Elsevier Ltd. All rights reserved.
Exploring the interactome: microfluidic isolation of proteins and interacting partners for quantitative analysis by electron microscopy.

PubMed

Giss, Dominic; Kemmerling, Simon; Dandey, Venkata; Stahlberg, Henning; Braun, Thomas

2014-05-20

Multimolecular protein complexes are important for many cellular processes. However, the stochastic nature of the cellular interactome makes the experimental detection of complex protein assemblies difficult and quantitative analysis at the single molecule level essential. Here, we present a fast and simple microfluidic method for (i) the quantitative isolation of endogenous levels of untagged protein complexes from minute volumes of cell lysates under close to physiological conditions and (ii) the labeling of specific components constituting these complexes. The method presented uses specific antibodies that are conjugated via a photocleavable linker to magnetic beads that are trapped in microcapillaries to immobilize the target proteins. Proteins are released by photocleavage, eluted, and subsequently analyzed by quantitative transmission electron microscopy at the single molecule level. Additionally, before photocleavage, immunogold can be employed to label proteins that interact with the primary target protein. Thus, the presented method provides a new way to study the interactome and, in combination with single molecule transmission electron microscopy, to structurally characterize the large, dynamic, heterogeneous multimolecular protein complexes formed.
Detection of biomolecules in complex media using surface plasmon resonance sensors

NASA Astrophysics Data System (ADS)

Malone, Michael R.; Masson, Jean-Francois; Barhnart, Margaret; Beaudoin, Stephen; Booksh, Karl S.

2005-11-01

Detection of multiple biologically relevant molecules was accomplished at sub-ng/mL levels in highly fouling media using fiber- optic based surface plasmon resonance sensors. Myocardial infarction markers, myoglobin and cTnI, were quantified in full serum with limits of detection below 1 ng/mL. Biologically relevant levels are between 15-30 ng/mL and 1-5 ng/mL for myoglobin and cTnI respectively. Cytokines involved in chronic wound healing, Interleukin 1, Interleukin 6, and tumor necrosis factor α, were detected at around 1 ng/mL in cell culture media. Preliminary results in monitoring these cytokines in cell cultures expressing the cytokines were obtained. The protein diagnostic of spinal muscular atrophy, survival motor neuron protein, was quantified from cell lysate. To obtain such results in complex media, the sensor's stability to non-specific protein adsorption had to be optimized. A layer of the N-hydroxysuccinimide ester of 16-mercaptohexadecanoic acid is attached to the sensor. This layer optimizes the antibody attachment to the sensor while minimizing the non-specific signal from serum proteins.
High-throughput Isolation and Characterization of Untagged Membrane Protein Complexes: Outer Membrane Complexes of Desulfovibrio vulgaris

PubMed Central

2012-01-01

Cell membranes represent the “front line” of cellular defense and the interface between a cell and its environment. To determine the range of proteins and protein complexes that are present in the cell membranes of a target organism, we have utilized a “tagless” process for the system-wide isolation and identification of native membrane protein complexes. As an initial subject for study, we have chosen the Gram-negative sulfate-reducing bacterium Desulfovibrio vulgaris. With this tagless methodology, we have identified about two-thirds of the outer membrane- associated proteins anticipated. Approximately three-fourths of these appear to form homomeric complexes. Statistical and machine-learning methods used to analyze data compiled over multiple experiments revealed networks of additional protein–protein interactions providing insight into heteromeric contacts made between proteins across this region of the cell. Taken together, these results establish a D. vulgaris outer membrane protein data set that will be essential for the detection and characterization of environment-driven changes in the outer membrane proteome and in the modeling of stress response pathways. The workflow utilized here should be effective for the global characterization of membrane protein complexes in a wide range of organisms. PMID:23098413
Protein-Protein Interactions in the Complex between the Enhancer Binding Protein NIFA and the Sensor NIFL from Azotobacter vinelandii

PubMed Central

Money, Tracy; Barrett, Jason; Dixon, Ray; Austin, Sara

2001-01-01

The enhancer binding protein NIFA and the sensor protein NIFL from Azotobacter vinelandii comprise an atypical two-component regulatory system in which signal transduction occurs via complex formation between the two proteins rather than by the phosphotransfer mechanism, which is characteristic of orthodox systems. The inhibitory activity of NIFL towards NIFA is stimulated by ADP binding to the C-terminal domain of NIFL, which bears significant homology to the histidine protein kinase transmitter domains. Adenosine nucleotides, particularly MgADP, also stimulate complex formation between NIFL and NIFA in vitro, allowing isolation of the complex by cochromatography. Using limited proteolysis of the purified proteins, we show here that changes in protease sensitivity of the Q linker regions of both NIFA and NIFL occurred when the complex was formed in the presence of MgADP. The N-terminal domain of NIFA adjacent to the Q linker was also protected by NIFL. Experiments with truncated versions of NIFA demonstrate that the central domain of NIFA is sufficient to cause protection of the Q linker of NIFL, although in this case, stable protein complexes are not detectable by cochromatography. PMID:11157949
CALCOM: a software for calculating the center of mass of proteins.

PubMed

Costantini, Susan; Paladino, Antonella; Facchiano, Angelo M

2008-02-09

The center of mass of a protein is an artificial point useful for detecting important and simple features of proteins structure, shape and association.CALCOM is a software which calculates the center of mass of a protein, starting from PDB protein structure files. In the case of protein complexes and of protein-small ligand complexes, the position of protein residues or of ligand atoms respect to each protein subunit can be evaluated, as well as the distance among the center of mass of the protein subunits, in order to compare different conformations and evaluate the relative motion of subunits. THE SERVICE IS AVAILABLE AT THE URL: http://bioinformatica.isa.cnr.it/CALCOM/.
Efficient and accurate Greedy Search Methods for mining functional modules in protein interaction networks.

PubMed

He, Jieyue; Li, Chaojun; Ye, Baoliu; Zhong, Wei

2012-06-25

Most computational algorithms mainly focus on detecting highly connected subgraphs in PPI networks as protein complexes but ignore their inherent organization. Furthermore, many of these algorithms are computationally expensive. However, recent analysis indicates that experimentally detected protein complexes generally contain Core/attachment structures. In this paper, a Greedy Search Method based on Core-Attachment structure (GSM-CA) is proposed. The GSM-CA method detects densely connected regions in large protein-protein interaction networks based on the edge weight and two criteria for determining core nodes and attachment nodes. The GSM-CA method improves the prediction accuracy compared to other similar module detection approaches, however it is computationally expensive. Many module detection approaches are based on the traditional hierarchical methods, which is also computationally inefficient because the hierarchical tree structure produced by these approaches cannot provide adequate information to identify whether a network belongs to a module structure or not. In order to speed up the computational process, the Greedy Search Method based on Fast Clustering (GSM-FC) is proposed in this work. The edge weight based GSM-FC method uses a greedy procedure to traverse all edges just once to separate the network into the suitable set of modules. The proposed methods are applied to the protein interaction network of S. cerevisiae. Experimental results indicate that many significant functional modules are detected, most of which match the known complexes. Results also demonstrate that the GSM-FC algorithm is faster and more accurate as compared to other competing algorithms. Based on the new edge weight definition, the proposed algorithm takes advantages of the greedy search procedure to separate the network into the suitable set of modules. Experimental analysis shows that the identified modules are statistically significant. The algorithm can reduce the computational time significantly while keeping high prediction accuracy.
Structure Prediction of Protein Complexes

NASA Astrophysics Data System (ADS)

Pierce, Brian; Weng, Zhiping

Protein-protein interactions are critical for biological function. They directly and indirectly influence the biological systems of which they are a part. Antibodies bind with antigens to detect and stop viruses and other infectious agents. Cell signaling is performed in many cases through the interactions between proteins. Many diseases involve protein-protein interactions on some level, including cancer and prion diseases.
Categorizing Biases in High-Confidence High-Throughput Protein-Protein Interaction Data Sets*

PubMed Central

Yu, Xueping; Ivanic, Joseph; Memišević, Vesna; Wallqvist, Anders; Reifman, Jaques

2011-01-01

We characterized and evaluated the functional attributes of three yeast high-confidence protein-protein interaction data sets derived from affinity purification/mass spectrometry, protein-fragment complementation assay, and yeast two-hybrid experiments. The interacting proteins retrieved from these data sets formed distinct, partially overlapping sets with different protein-protein interaction characteristics. These differences were primarily a function of the deployed experimental technologies used to recover these interactions. This affected the total coverage of interactions and was especially evident in the recovery of interactions among different functional classes of proteins. We found that the interaction data obtained by the yeast two-hybrid method was the least biased toward any particular functional characterization. In contrast, interacting proteins in the affinity purification/mass spectrometry and protein-fragment complementation assay data sets were over- and under-represented among distinct and different functional categories. We delineated how these differences affected protein complex organization in the network of interactions, in particular for strongly interacting complexes (e.g. RNA and protein synthesis) versus weak and transient interacting complexes (e.g. protein transport). We quantified methodological differences in detecting protein interactions from larger protein complexes, in the correlation of protein abundance among interacting proteins, and in their connectivity of essential proteins. In the latter case, we showed that minimizing inherent methodology biases removed many of the ambiguous conclusions about protein essentiality and protein connectivity. We used these findings to rationalize how biological insights obtained by analyzing data sets originating from different sources sometimes do not agree or may even contradict each other. An important corollary of this work was that discrepancies in biological insights did not necessarily imply that one detection methodology was better or worse, but rather that, to a large extent, the insights reflected the methodological biases themselves. Consequently, interpreting the protein interaction data within their experimental or cellular context provided the best avenue for overcoming biases and inferring biological knowledge. PMID:21876202
Centralities in simplicial complexes. Applications to protein interaction networks.

PubMed

Estrada, Ernesto; Ross, Grant J

2018-02-07

Complex networks can be used to represent complex systems which originate in the real world. Here we study a transformation of these complex networks into simplicial complexes, where cliques represent the simplices of the complex. We extend the concept of node centrality to that of simplicial centrality and study several mathematical properties of degree, closeness, betweenness, eigenvector, Katz, and subgraph centrality for simplicial complexes. We study the degree distributions of these centralities at the different levels. We also compare and describe the differences between the centralities at the different levels. Using these centralities we study a method for detecting essential proteins in PPI networks of cells and explain the varying abilities of the centrality measures at the different levels in identifying these essential proteins. Copyright © 2017 Elsevier Ltd. All rights reserved.
Two-dimensional blue native/SDS-PAGE analysis of whole cell lysate protein complexes of rice in response to salt stress.

PubMed

Hashemi, Amenehsadat; Gharechahi, Javad; Nematzadeh, Ghorbanali; Shekari, Faezeh; Hosseini, Seyed Abdollah; Salekdeh, Ghasem Hosseini

2016-08-01

To understand the biology of a plant in response to stress, insight into protein-protein interactions, which almost define cell behavior, is thought to be crucial. Here, we provide a comparative complexomics analysis of leaf whole cell lysate of two rice genotypes with contrasting responses to salt using two-dimensional blue native/SDS-PAGE (2D-BN/SDS-PAGE). We aimed to identify changes in subunit composition and stoichiometry of protein complexes elicited by salt. Using mild detergent for protein complex solubilization, we were able to identify 9 protein assemblies as hetero-oligomeric and 30 as homo-oligomeric complexes. A total of 20 proteins were identified as monomers in the 2D-BN/SDS-PAGE gels. In addition to identifying known protein complexes that confirm the technical validity of our analysis, we were also able to discover novel protein-protein interactions. Interestingly, an interaction was detected for glycolytic enzymes enolase (ENO1) and triosephosphate isomerase (TPI) and also for a chlorophyll a-b binding protein and RuBisCo small subunit. To show changes in subunit composition and stoichiometry of protein assemblies during salt stress, the differential abundance of interacting proteins was compared between salt-treated and control plants. A detailed exploration of some of the protein complexes provided novel insight into the function, composition, stoichiometry and dynamics of known and previously uncharacterized protein complexes in response to salt stress. Copyright © 2016 Elsevier GmbH. All rights reserved.
Evaluation of three simple direct or indirect carbonyl detection methods for characterization of oxidative modifications of proteins.

PubMed

Vásquez-Garzón, Verónica R; Rouimi, Patrick; Jouanin, Isabelle; Waeg, Georg; Zarkovic, Neven; Villa-Treviño, Saul; Guéraud, Françoise

2012-05-01

Among disruptions induced by oxidative stress, modifications of proteins, particularly irreversible carbonylation, are associated with the development of several diseases, including cardiovascular diseases, neurodegenerative diseases, and cancer. Carbonylation of proteins can occur directly or indirectly through the adduction of lipid oxidation products. In this study, three classical and easy-to-perform techniques to detect direct or indirect carbonylation of proteins were compared. A model protein apomyoglobin and a complex mixture of rat liver cytosolic proteins were exposed to cumene hydroperoxide oxidation or adduction to the lipid peroxidation product 4-hydroxynonenal in order to test direct or indirect carbonylation, respectively. The technique using a specific anti-4-hydroxynonenal-histidine adduct antibody was effective to detect in vitro modification of model apomyoglobin and cytosolic proteins by 4-hydroxynonenal but not by direct carbonylation which was achieved by techniques using biotin-coupled hydrazide or dinitrophenylhydrazine derivatization of carbonyls. Sequential use of these methods enabled the detection of both direct and indirect carbonyl modification in proteins, although constitutively biotinylated proteins were detected by biotin-hydrazide. Although rather classical and efficient, methods for carbonyl detection on proteins in oxidative stress studies may be biased by some artifactual detections and complicated by proteins multimerizations. The use of more and more specific available antibodies is recommended to complete detection of lipid peroxidation product adducts on proteins.
Analysis of Protein Interactions with Picomolar Binding Affinity by Fluorescence-Detected Sedimentation Velocity

PubMed Central

2014-01-01

The study of high-affinity protein interactions with equilibrium dissociation constants (KD) in the picomolar range is of significant interest in many fields, but the characterization of stoichiometry and free energy of such high-affinity binding can be far from trivial. Analytical ultracentrifugation has long been considered a gold standard in the study of protein interactions but is typically applied to systems with micromolar KD. Here we present a new approach for the study of high-affinity interactions using fluorescence detected sedimentation velocity analytical ultracentrifugation (FDS-SV). Taking full advantage of the large data sets in FDS-SV by direct boundary modeling with sedimentation coefficient distributions c(s), we demonstrate detection and hydrodynamic resolution of protein complexes at low picomolar concentrations. We show how this permits the characterization of the antibody–antigen interactions with low picomolar binding constants, 2 orders of magnitude lower than previously achieved. The strongly size-dependent separation and quantitation by concentration, size, and shape of free and complex species in free solution by FDS-SV has significant potential for studying high-affinity multistep and multicomponent protein assemblies. PMID:24552356
Identifying protein complexes based on brainstorming strategy.

PubMed

Shen, Xianjun; Zhou, Jin; Yi, Li; Hu, Xiaohua; He, Tingting; Yang, Jincai

2016-11-01

Protein complexes comprising of interacting proteins in protein-protein interaction network (PPI network) play a central role in driving biological processes within cells. Recently, more and more swarm intelligence based algorithms to detect protein complexes have been emerging, which have become the research hotspot in proteomics field. In this paper, we propose a novel algorithm for identifying protein complexes based on brainstorming strategy (IPC-BSS), which is integrated into the main idea of swarm intelligence optimization and the improved K-means algorithm. Distance between the nodes in PPI network is defined by combining the network topology and gene ontology (GO) information. Inspired by human brainstorming process, IPC-BSS algorithm firstly selects the clustering center nodes, and then they are separately consolidated with the other nodes with short distance to form initial clusters. Finally, we put forward two ways of updating the initial clusters to search optimal results. Experimental results show that our IPC-BSS algorithm outperforms the other classic algorithms on yeast and human PPI networks, and it obtains many predicted protein complexes with biological significance. Copyright © 2016 Elsevier Inc. All rights reserved.
Exploiting three kinds of interface propensities to identify protein binding sites.

PubMed

Liu, Bin; Wang, Xiaolong; Lin, Lei; Dong, Qiwen; Wang, Xuan

2009-08-01

Predicting the binding sites between two interacting proteins provides important clues to the function of a protein. In this study, we present a building block of proteins called order profiles to use the evolutionary information of the protein sequence frequency profiles and apply this building block to produce a class of propensities called order profile interface propensities. For comparisons, we revisit the usage of residue interface propensities and binary profile interface propensities for protein binding site prediction. Each kind of propensities combined with sequence profiles and accessible surface areas are inputted into SVM. When tested on four types of complexes (hetero-permanent complexes, hetero-transient complexes, homo-permanent complexes and homo-transient complexes), experimental results show that the order profile interface propensities are better than residue interface propensities and binary profile interface propensities. Therefore, order profile is a suitable profile-level building block of the protein sequences and can be widely used in many tasks of computational biology, such as the sequence alignment, the prediction of domain boundary, the designation of knowledge-based potentials and the protein remote homology detection.
Detection and isolation of cell-derived microparticles are compromised by protein complexes resulting from shared biophysical parameters.

PubMed

György, Bence; Módos, Károly; Pállinger, Eva; Pálóczi, Krisztina; Pásztói, Mária; Misják, Petra; Deli, Mária A; Sipos, Aron; Szalai, Anikó; Voszka, István; Polgár, Anna; Tóth, Kálmán; Csete, Mária; Nagy, György; Gay, Steffen; Falus, András; Kittel, Agnes; Buzás, Edit I

2011-01-27

Numerous diseases, recently reported to associate with elevated microvesicle/microparticle (MP) counts, have also long been known to be characterized by accelerated immune complex (IC) formation. The goal of this study was to investigate the potential overlap between parameters of protein complexes (eg, ICs or avidin-biotin complexes) and MPs, which might perturb detection and/or isolation of MPs. In this work, after comprehensive characterization of MPs by electron microscopy, atomic force microscopy, dynamic light-scattering analysis, and flow cytometry, for the first time, we drive attention to the fact that protein complexes, especially insoluble ICs, overlap in biophysical properties (size, light scattering, and sedimentation) with MPs. This, in turn, affects MP quantification by flow cytometry and purification by differential centrifugation, especially in diseases in which IC formation is common, including not only autoimmune diseases, but also hematologic disorders, infections, and cancer. These data may necessitate reevaluation of certain published data on patient-derived MPs and contribute to correct the clinical laboratory assessment of the presence and biologic functions of MPs in health and disease.
Improved Peak Detection and Deconvolution of Native Electrospray Mass Spectra from Large Protein Complexes.

PubMed

Lu, Jonathan; Trnka, Michael J; Roh, Soung-Hun; Robinson, Philip J J; Shiau, Carrie; Fujimori, Danica Galonic; Chiu, Wah; Burlingame, Alma L; Guan, Shenheng

2015-12-01

Native electrospray-ionization mass spectrometry (native MS) measures biomolecules under conditions that preserve most aspects of protein tertiary and quaternary structure, enabling direct characterization of large intact protein assemblies. However, native spectra derived from these assemblies are often partially obscured by low signal-to-noise as well as broad peak shapes because of residual solvation and adduction after the electrospray process. The wide peak widths together with the fact that sequential charge state series from highly charged ions are closely spaced means that native spectra containing multiple species often suffer from high degrees of peak overlap or else contain highly interleaved charge envelopes. This situation presents a challenge for peak detection, correct charge state and charge envelope assignment, and ultimately extraction of the relevant underlying mass values of the noncovalent assemblages being investigated. In this report, we describe a comprehensive algorithm developed for addressing peak detection, peak overlap, and charge state assignment in native mass spectra, called PeakSeeker. Overlapped peaks are detected by examination of the second derivative of the raw mass spectrum. Charge state distributions of the molecular species are determined by fitting linear combinations of charge envelopes to the overall experimental mass spectrum. This software is capable of deconvoluting heterogeneous, complex, and noisy native mass spectra of large protein assemblies as demonstrated by analysis of (1) synthetic mononucleosomes containing severely overlapping peaks, (2) an RNA polymerase II/α-amanitin complex with many closely interleaved ion signals, and (3) human TriC complex containing high levels of background noise. Graphical Abstract ᅟ.

Synaptic activity induces input-specific rearrangements in a targeted synaptic protein interaction network.

PubMed

Lautz, Jonathan D; Brown, Emily A; VanSchoiack, Alison A Williams; Smith, Stephen E P

2018-05-27

Cells utilize dynamic, network level rearrangements in highly interconnected protein interaction networks to transmit and integrate information from distinct signaling inputs. Despite the importance of protein interaction network dynamics, the organizational logic underlying information flow through these networks is not well understood. Previously, we developed the quantitative multiplex co-immunoprecipitation platform, which allows for the simultaneous and quantitative measurement of the amount of co-association between large numbers of proteins in shared complexes. Here, we adapt quantitative multiplex co-immunoprecipitation to define the activity dependent dynamics of an 18-member protein interaction network in order to better understand the underlying principles governing glutamatergic signal transduction. We first establish that immunoprecipitation detected by flow cytometry can detect activity dependent changes in two known protein-protein interactions (Homer1-mGluR5 and PSD-95-SynGAP). We next demonstrate that neuronal stimulation elicits a coordinated change in our targeted protein interaction network, characterized by the initial dissociation of Homer1 and SynGAP-containing complexes followed by increased associations among glutamate receptors and PSD-95. Finally, we show that stimulation of distinct glutamate receptor types results in different modular sets of protein interaction network rearrangements, and that cells activate both modules in order to integrate complex inputs. This analysis demonstrates that cells respond to distinct types of glutamatergic input by modulating different combinations of protein co-associations among a targeted network of proteins. Our data support a model of synaptic plasticity in which synaptic stimulation elicits dissociation of preexisting multiprotein complexes, opening binding slots in scaffold proteins and allowing for the recruitment of additional glutamatergic receptors. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
The detection of hepatitis c virus core antigen using afm chips with immobolized aptamers.

PubMed

Pleshakova, T O; Kaysheva, A L; Bayzyanova, J М; Anashkina, А S; Uchaikin, V F; Ziborov, V S; Konev, V A; Archakov, A I; Ivanov, Y D

2018-01-01

In the present study, the possibility of hepatitis C virus core antigen (HCVcoreAg) detection in buffer solution, using atomic force microscope chip (AFM-chip) with immobilized aptamers, has been demonstrated. The target protein was detected in 1mL of solution at concentrations from 10 -10 М to 10 -13 М. The registration of aptamer/antigen complexes on the chip surface was carried out by atomic force microscopy (AFM). The further mass-spectrometric (MS) identification of AFM-registered objects on the chip surface allowed reliable identification of HCVcoreAg target protein in the complexes. Aptamers, which were designed for therapeutic purposes, have been shown to be effective in HCVcoreAg detection as probe molecules. Copyright © 2017 Elsevier B.V. All rights reserved.
Novel 1:1 labeling and purification process for C-terminal thioester and single cysteine recombinant proteins using generic peptidic toolbox reagents.

PubMed

Portal, Christophe F; Seifert, Jan-Marcus; Buehler, Christof; Meisner-Kober, Nicole-Claudia; Auer, Manfred

2014-07-16

We developed a versatile set of chemical labeling reagents which allow dye ligation to the C-terminus of a protein or a single internal cysteine and target purification in a simple two-step process. This simple process results in a fully 1:1 labeled conjugate suitable for all quantitative fluorescence spectroscopy and imaging experiments. We refer to a "generic labeling toolbox" because of the flexibility to choose one of many available dyes, spacers of different lengths and compositions which increase the target solubility, a variety of affinity purification tags, and different cleavage chemistries to release the 1:1 labeled proteins. Studying protein function in vitro or in the context of live cells and organisms is of vital importance in biological research. Although label free detection technologies gain increasing interest in molecular recognition science, fluorescence spectroscopy is still the most often used detection technique for assays and screens both in academic as well as in industrial groups. For generations, fluorescence spectroscopists have labeled their proteins of interest with small fluorescent dyes by random chemical linking on the proteins' exposed lysines and cysteines. Chemical reactions with a certain excess of activated esters or maleimides of longer wavelength dyes hardly ever result in quantitative labeling of the target protein. Most of the time, more than one exposed amino acid side chain reacts. This results in a mixture of dye-protein complexes of different labeling stoichiometries and labeling sites. Only mass spectrometry allows resolving the precise chemical composition of the conjugates. In "classical" ensemble averaging fluorescent experiments, these labeled proteins are still useful, and quantification of, e.g., ligand binding experiments, is achieved via knowledge of the overall protein concentration and a fluorescent signal change which is proportional to the amount of complex formed. With the development of fluorescence fluctuation analysis techniques working at single molecule resolution, like fluorescence correlation spectroscopy (FCS), fluorescence cross correlation spectroscopy (FCCS), fluorescence intensity diffusion analysis (FIDA), etc., it became important to work with homogeneously labeled target proteins. Each molecule participating in a binding equilibrium should be detectable when it freely fluctuates through the confocal focus of a microscope. The measured photon burst for each transition contains information about the size and the stoichiometry of a protein complex. Therefore, it is important to work with reagents that contain an exact number of tracers per protein at identical positions. The ideal fluorescent tracer-protein complex stoichiometry is 1:1. While genetic tags such as fluorescent proteins (FPs) are widely used to detect proteins, FPs have several limitations compared to chemical tags. For example, FPs cannot easily compete with organic dyes in the flexibility of modification and spectral range; moreover, FPs have disadvantages in brightness and photostability and are therefore not ideal for most biochemical single molecule studies. We present the synthesis of a series of exemplaric toolbox reagents and labeling results on three target proteins which were needed for high throughput screening experiments using fluorescence fluctuation analysis at single molecule resolution. On one target, Hu-antigen R (HuR), we demonstrated the activity of the 1:1 labeled protein in ribonucleic acid (RNA) binding, and the ease of resolving the stoichiometry of an RNA-HuR complex using the same dye on protein and RNA by Fluorescence Intensity Multiple Distribution Analysis (FIMDA) detection.
Generation of GFP Native Protein for Detection of Its Intracellular Uptake by Cell-Penetrating Peptides.

PubMed

Kadkhodayan, S; Sadat, S M; Irani, S; Fotouhi, F; Bolhassani, A

2016-01-01

Different types of lipid- and polymer-based vectors have been developed to deliver proteins into cells, but these methods showed relatively poor efficiency. Recently, a group of short, highly basic peptides known as cell-penetrating peptides (CPPs) were used to carry polypeptides and proteins into cells. In this study, expression and purification of GFP protein was performed using the prokaryotic pET expression system. We used two amphipathic CPPs (Pep-1 and CADY-2) as a novel delivery system to transfer the GFP protein into cells. The morphological features of the CPP/GFP complexes were studied by scanning electron microscopy (SEM), Zetasizer, and SDS-PAGE. The efficiency of GFP transfection using Pep-1 and CADY-2 peptides and TurboFect reagent was compared with FITC-antibody protein control delivered by these transfection vehicles in the HEK-293T cell line. SEM data confirmed formation of discrete nanoparticles with a diameter of below 300 nm. Moreover, formation of the complexes was detected using SDS-PAGE as two individual bands, indicating non-covalent interaction. The size and homogeneity of Pep-1/GFP and CADY-2/GFP complexes were dependent on the ratio of peptide/cargo formulations, and responsible for their biological efficiency. The cells transfected by Pep-1/GFP and CADY-2/GFP complexes at a molar ratio of 20 : 1 demonstrated spreading green regions using fluorescent microscopy. Flow cytometry results showed that the transfection efficiency of Pep-based nanoparticles was similar to CADY-based nanoparticles and comparable with TurboFect-protein complexes. These data open an efficient way for future therapeutic purposes.
Enhanced Detection of Low-Abundance Human Plasma Proteins by Integrating Polyethylene Glycol Fractionation and Immunoaffinity Depletion

PubMed Central

Liu, Haipeng; Yu, Jia; Qiao, Rui; Zhou, Mi; Yang, Yongtao; Zhou, Jian; Xie, Peng

2016-01-01

The enormous depth complexity of the human plasma proteome poses a significant challenge for current mass spectrometry-based proteomic technologies in terms of detecting low-level proteins in plasma, which is essential for successful biomarker discovery efforts. Typically, a single-step analytical approach cannot reduce this intrinsic complexity. Current simplex immunodepletion techniques offer limited capacity for detecting low-abundance proteins, and integrated strategies are thus desirable. In this respect, we developed an improved strategy for analyzing the human plasma proteome by integrating polyethylene glycol (PEG) fractionation with immunoaffinity depletion. PEG fractionation of plasma proteins is simple, rapid, efficient, and compatible with a downstream immunodepletion step. Compared with immunodepletion alone, our integrated strategy substantially improved the proteome coverage afforded by PEG fractionation. Coupling this new protocol with liquid chromatography-tandem mass spectrometry, 135 proteins with reported normal concentrations below 100 ng/mL were confidently identified as common low-abundance proteins. A side-by-side comparison indicated that our integrated strategy was increased by average 43.0% in the identification rate of low-abundance proteins, relying on an average 65.8% increase of the corresponding unique peptides. Further investigation demonstrated that this combined strategy could effectively alleviate the signal-suppressive effects of the major high-abundance proteins by affinity depletion, especially with moderate-abundance proteins after incorporating PEG fractionation, thereby greatly enhancing the detection of low-abundance proteins. In sum, the newly developed strategy of incorporating PEG fractionation to immunodepletion methods can potentially aid in the discovery of plasma biomarkers of therapeutic and clinical interest. PMID:27832179
GeLC-MRM quantitation of mutant KRAS oncoprotein in complex biological samples.

PubMed

Halvey, Patrick J; Ferrone, Cristina R; Liebler, Daniel C

2012-07-06

Tumor-derived mutant KRAS (v-Ki-ras-2 Kirsten rat sarcoma viral oncogene) oncoprotein is a critical driver of cancer phenotypes and a potential biomarker for many epithelial cancers. Targeted mass spectrometry analysis by multiple reaction monitoring (MRM) enables selective detection and quantitation of wild-type and mutant KRAS proteins in complex biological samples. A recently described immunoprecipitation approach (Proc. Nat. Acad. Sci.2011, 108, 2444-2449) can be used to enrich KRAS for MRM analysis, but requires large protein inputs (2-4 mg). Here, we describe sodium dodecyl sulfate-polyacrylamide gel electrophoresis-based enrichment of KRAS in a low molecular weight (20-25 kDa) protein fraction prior to MRM analysis (GeLC-MRM). This approach reduces background proteome complexity, thus, allowing mutant KRAS to be reliably quantified in low protein inputs (5-50 μg). GeLC-MRM detected KRAS mutant variants (G12D, G13D, G12V, G12S) in a panel of cancer cell lines. GeLC-MRM analysis of wild-type and mutant was linear with respect to protein input and showed low variability across process replicates (CV = 14%). Concomitant analysis of a peptide from the highly similar HRAS and NRAS proteins enabled correction of KRAS-targeted measurements for contributions from these other proteins. KRAS peptides were also quantified in fluid from benign pancreatic cysts and pancreatic cancers at concentrations from 0.08 to 1.1 fmol/μg protein. GeLC-MRM provides a robust, sensitive approach to quantitation of mutant proteins in complex biological samples.
Chemiluminescence enzyme immunoassay using ProteinA-bacterial magnetite complex

NASA Astrophysics Data System (ADS)

Matsunaga, Tadashi; Sato, Rika; Kamiya, Shinji; Tanaka, Tsuyosi; Takeyama, Haruko

1999-04-01

Bacterial magnetic particles (BMPs) which have ProteinA expressed on their surface were constructed using magA which is a key gene in BMP biosynthesis in the magnetic bacterium Magnetospirillum sp. AMB-1. Homogenous chemiluminescence enzyme immunoassay using antibody bound ProteinA-BMP complexes was developed for detection of human IgG. A good correlation between the luminescence yield and the concentration of human IgG was obtained in the range of 1-10 3 ng/ml.
Staining Method for Protein Analysis by Capillary Gel Electrophoresis

PubMed Central

Wu, Shuqing; Lu, Joann J; Wang, Shili; Peck, Kristy L.; Li, Guigen; Liu, Shaorong

2009-01-01

A novel staining method and the associated fluorescent dye were developed for protein analysis by capillary SDS-PAGE. The method strategy is to synthesize a pseudo-SDS dye and use it to replace some of the SDS in SDS–protein complexes so that the protein can be fluorescently detected. The pseudo-SDS dye consists of a long, straight alkyl chain connected to a negative charged fluorescent head and binds to proteins just as SDS. The number of dye molecules incorporated with a protein depends on the dye concentration relative to SDS in the sample solution, since SDS and dye bind to proteins competitively. In this work, we synthesized a series of pseudo-SDS dyes, and tested their performances for capillary SDS-PAGE. FT-16 (a fluorescein molecule linked with a hexadodecyl group) seemed to be the best among all the dyes tested. Although the numbers of dye molecules bound to proteins (and the fluorescence signals from these protein complexes) were maximized in the absence of SDS, high-quality separations were obtained when co-complexes of SDS–protein–dye were formed. The migration time correlates well with protein size even after some of the SDS in the SDS–protein complexes was replaced by the pseudo-SDS dye. Under optimized experimental conditions and using a laser-induced fluorescence detector, limits of detection of as low as 0.13 ng/mL (bovine serum albumin) and dynamic ranges over 5 orders of magnitude in which fluorescence response is proportional to the square root of analyte concentration were obtained. The method and dye were also tested for separations of real-world samples from E. coli. PMID:17874848
EzyAmp signal amplification cascade enables isothermal detection of nucleic acid and protein targets.

PubMed

Linardy, Evelyn M; Erskine, Simon M; Lima, Nicole E; Lonergan, Tina; Mokany, Elisa; Todd, Alison V

2016-01-15

Advancements in molecular biology have improved the ability to characterize disease-related nucleic acids and proteins. Recently, there has been an increasing desire for tests that can be performed outside of centralised laboratories. This study describes a novel isothermal signal amplification cascade called EzyAmp (enzymatic signal amplification) that is being developed for detection of targets at the point of care. EzyAmp exploits the ability of some restriction endonucleases to cleave substrates containing nicks within their recognition sites. EzyAmp uses two oligonucleotide duplexes (partial complexes 1 and 2) which are initially cleavage-resistant as they lack a complete recognition site. The recognition site of partial complex 1 can be completed by hybridization of a triggering oligonucleotide (Driver Fragment 1) that is generated by a target-specific initiation event. Binding of Driver Fragment 1 generates a completed complex 1, which upon cleavage, releases Driver Fragment 2. In turn, binding of Driver Fragment 2 to partial complex 2 creates completed complex 2 which when cleaved releases additional Driver Fragment 1. Each cleavage event separates fluorophore quencher pairs resulting in an increase in fluorescence. At this stage a cascade of signal production becomes independent of further target-specific initiation events. This study demonstrated that the EzyAmp cascade can facilitate detection and quantification of nucleic acid targets with sensitivity down to aM concentration. Further, the same cascade detected VEGF protein with a sensitivity of 20nM showing that this universal method for amplifying signal may be linked to the detection of different types of analytes in an isothermal format. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.
Proteomic methods for analysis of S-nitrosation⋄

PubMed Central

Kettenhofen, Nicholas; Broniowska, Katarzyna; Keszler, Agnes; Zhang, Yanhong; Hogg, Neil

2007-01-01

This review discusses proteomic methods to detect and identify S-nitrosated proteins. Protein S-nitrosation, the post-translational modification of thiol residues to form S-nitrosothiols, has been suggested to be a mechanism of cellular redox signaling by which nitric oxide can alter cellular function through modification of protein thiol residues. It has become apparent that methods that will detect and identify low levels of S-nitrosated protein in complex protein mixtures are required in order to fully appreciate the range, extent and selectivity of this modification in both physiological and pathological conditions. While many advances have been made in the detection of either total cellular S-nitrosation or individual S-nitrosothiols, proteomic methods for the detection of S-nitrosation are in relative infancy. This review will discuss the major methods that have been used for the proteomic analysis of protein S-nitrosation and discuss the pros and cons of this methodology. PMID:17360249
Detection of Diverse and High Molecular Weight Nesprin-1 and Nesprin-2 Isoforms Using Western Blotting.

PubMed

Carthew, James; Karakesisoglou, Iakowos

2016-01-01

Heavily utilized in cell and molecular biology, western blotting is considered a crucial technique for the detection and quantification of proteins within complex mixtures. In particular, the detection of members of the nesprin (nuclear envelope spectrin repeat protein) family has proven difficult to analyze due to their substantial isoform diversity, molecular weight variation, and the sheer size of both nesprin-1 and nesprin-2 giant protein variants (>800 kDa). Nesprin isoforms contain distinct domain signatures, perform differential cytoskeletal associations, occupy different subcellular compartments, and vary in their tissue expression profiles. This structural and functional variance highlights the need to distinguish between the full range of proteins within the nesprin protein family, allowing for greater understanding of their specific roles in cell biology and disease. Herein, we describe a western blotting protocol modified for the detection of low to high molecular weight (50-1000 kDa) nesprin proteins.
Slow histidine H/D exchange protocol for thermodynamic analysis of protein folding and stability using mass spectrometry.

PubMed

Tran, Duc T; Banerjee, Sambuddha; Alayash, Abdu I; Crumbliss, Alvin L; Fitzgerald, Michael C

2012-02-07

Described here is a mass spectrometry-based protocol to study the thermodynamic stability of proteins and protein-ligand complexes using the chemical denaturant dependence of the slow H/D exchange reaction of the imidazole C(2) proton in histidine side chains. The protocol is developed using several model protein systems including: ribonuclease (Rnase) A, myoglobin, bovine carbonic anhydrase (BCA) II, hemoglobin (Hb), and the hemoglobin-haptoglobin (Hb-Hp) protein complex. Folding free energies consistent with those previously determined by other more conventional techniques were obtained for the two-state folding proteins, Rnase A and myoglobin. The protocol successfully detected a previously observed partially unfolded intermediate stabilized in the BCA II folding/unfolding reaction, and it could be used to generate a K(d) value of 0.24 nM for the Hb-Hp complex. The compatibility of the protocol with conventional mass spectrometry-based proteomic sample preparation and analysis methods was also demonstrated in an experiment in which the protocol was used to detect the binding of zinc to superoxide dismutase in the yeast cell lysate sample. The yeast cell sample analyses also helped define the scope of the technique, which requires the presence of globally protected histidine residues in a protein's three-dimensional structure for successful application. © 2011 American Chemical Society
Excitation-Energy Transfer Paths from Tryptophans to Coordinated Copper Ions in Engineered Azurins: a Source of Observables for Monitoring Protein Structural Changes

NASA Astrophysics Data System (ADS)

Di Rocco, Giulia; Bernini, Fabrizio; Borsari, Marco; Martinelli, Ilaria; Bortolotti, Carlo Augusto; Battistuzzi, Gianantonio; Ranieri, Antonio; Caselli, Monica; Sola, Marco; Ponterini, Glauco

2016-09-01

The intrinsic fluorescence of recombinant proteins offers a powerful tool to detect and characterize structural changes induced by chemical or biological stimuli. We show that metal-ion binding to a hexahistidine tail can significantly broaden the range of such structurally sensitive fluorescence observables. Bipositive metal-ions as Cu2+, Ni2+ and Zn2+ bind 6xHis-tag azurin and its 6xHis-tagged R129W and W48A-R129W mutants with good efficiency and, thereby, quench their intrinsic fluorescence. Due to a much more favourable spectral overlap, the 6xHis-tag/Cu2+ complex(es) are the most efficient quenchers of both W48 and W129 emissions. Based on simple Förster-type dependence of energy-transfer efficiency on donor/acceptor distance, we can trace several excitation-energy transfer paths across the protein structure. Unexpected lifetime components in the azurin 6xHis-tag/Cu2+ complex emission decays reveal underneath complexity in the conformational landscape of these systems. The new tryptophan emission quenching paths provide additional signals for detecting and identifying protein structural changes.
Characterizing Protein Complexes with UV absorption, Light Scattering, and Refractive Index Detection.

NASA Astrophysics Data System (ADS)

Trainoff, Steven

2009-03-01

Many modern pharmaceuticals and naturally occurring biomolecules consist of complexes of proteins and polyethylene glycol or carbohydrates. In the case of vaccine development, these complexes are often used to induce or amplify immune responses. For protein therapeutics they are used to modify solubility and function, or to control the rate of degradation and elimination of a drug from the body. Characterizing the stoichiometry of these complexes is an important industrial problem that presents a formidable challenge to analytical instrument designers. Traditional analytical methods, such as using florescent tagging, chemical assays, and mass spectrometry perturb the system so dramatically that the complexes are often destroyed or uncontrollably modified by the measurement. A solution to this problem consists of fractionating the samples and then measuring the fractions using sequential non-invasive detectors that are sensitive to different components of the complex. We present results using UV absorption, which is primarily sensitive to the protein fraction, Light Scattering, which measures the total weight average molar mass, and Refractive Index detection, which measures the net concentration. We also present a solution of the problem inter-detector band-broadening problem that has heretofore made this approach impractical. Presented will be instrumentation and an analysis method that overcome these obstacles and make this technique a reliable and robust way of non-invasively characterizing these industrially important compounds.
Bovine Pancreatic Trypsin Inhibitor-Trypsin Complex as a Detection System for Recombinant Proteins

NASA Astrophysics Data System (ADS)

Borjigin, Jimo; Nathans, Jeremy

1993-01-01

Bovine pancreatic trypsin inhibitor (BPTI) binds to trypsin and anhydrotrypsin (an enzymatically inactive derivative of trypsin) with affinities of 6 x 10-14 and 1.1 x 10-13 M, respectively. We have taken advantage of the high affinity and specificity of this binding reaction to develop a protein tagging system in which biotinylated trypsin or biotinylated anhydrotrypsin is used as the reagent to detect recombinant fusion proteins into which BPTI has been inserted. Two proteins, opsin and growth hormone, were used as targets for insertional mutagenesis with BPTI. In each case, both domains of the fusion protein appear to be correctly folded. The fusion proteins can be specifically and efficiently detected by biotinylated trypsin or biotinylated anhydrotrypsin, as demonstrated by staining of transfected cells, protein blotting, affinity purification, and a mobility shift assay in SDS/polyacrylamide gels.
Protein-nucleotide contacts in motor proteins detected by DNP-enhanced solid-state NMR.

PubMed

Wiegand, Thomas; Liao, Wei-Chih; Ong, Ta Chung; Däpp, Alexander; Cadalbert, Riccardo; Copéret, Christophe; Böckmann, Anja; Meier, Beat H

2017-11-01

DNP (dynamic nuclear polarization)-enhanced solid-state NMR is employed to directly detect protein-DNA and protein-ATP interactions and identify the residue type establishing the intermolecular contacts. While conventional solid-state NMR can detect protein-DNA interactions in large oligomeric protein assemblies in favorable cases, it typically suffers from low signal-to-noise ratios. We show here, for the oligomeric DnaB helicase from Helicobacter pylori complexed with ADP and single-stranded DNA, that this limitation can be overcome by using DNP-enhanced spectroscopy. Interactions are established by DNP-enhanced 31 P- 13 C polarization-transfer experiments followed by the recording of a 2D 13 C- 13 C correlation experiment. The NMR spectra were obtained in less than 2 days and allowed the identification of residues of the motor protein involved in nucleotide binding.
The effects of serial skin testing with purified protein derivative on the level and quality of antibodies to complex and defined antigens in Mycobacterium bovis-infected cattle

USDA-ARS?s Scientific Manuscript database

Several serologic tests designed to detect antibodies to immunodominant Mycobacterium bovis antigens have recently emerged as ancillary tests for the detection of bovine tuberculosis in cattle, particularly when applied after injection of purified protein derivative (PPD) for skin test that signific...
In situ hybridization at the electron microscope level: localization of transcripts on ultrathin sections of Lowicryl K4M-embedded tissue using biotinylated probes and protein A-gold complexes

PubMed Central

1986-01-01

A technique has been developed for localizing hybrids formed in situ on semi-thin and ultrathin sections of Lowicryl K4M-embedded tissue. Biotinylated dUTP (Bio-11-dUTP and/or Bio-16-dUTP) was incorporated into mitochondrial rDNA and small nuclear U1 probes by nick- translation. The probes were hybridized to sections of Drosophila ovaries and subsequently detected with an anti-biotin antibody and protein A-gold complex. On semi-thin sections, probe detection was achieved by amplification steps with anti-protein A antibody and protein A-gold with subsequent silver enhancement. At the electron microscope level, specific labeling was obtained over structures known to be the site of expression of the appropriate genes (i.e., either over mitochondria or over nuclei). The labeling pattern at the light microscope level (semi-thin sections) was consistent with that obtained at the electron microscope level. The described nonradioactive procedures for hybrid detection on Lowicryl K4M-embedded tissue sections offer several advantages: rapid signal detection: superior morphological preservation and spatial resolution; and signal-to-noise ratios equivalent to radiolabeling. PMID:3084498
Methods for protein complex prediction and their contributions towards understanding the organisation, function and dynamics of complexes.

PubMed

Srihari, Sriganesh; Yong, Chern Han; Patil, Ashwini; Wong, Limsoon

2015-09-14

Complexes of physically interacting proteins constitute fundamental functional units responsible for driving biological processes within cells. A faithful reconstruction of the entire set of complexes is therefore essential to understand the functional organisation of cells. In this review, we discuss the key contributions of computational methods developed till date (approximately between 2003 and 2015) for identifying complexes from the network of interacting proteins (PPI network). We evaluate in depth the performance of these methods on PPI datasets from yeast, and highlight their limitations and challenges, in particular at detecting sparse and small or sub-complexes and discerning overlapping complexes. We describe methods for integrating diverse information including expression profiles and 3D structures of proteins with PPI networks to understand the dynamics of complex formation, for instance, of time-based assembly of complex subunits and formation of fuzzy complexes from intrinsically disordered proteins. Finally, we discuss methods for identifying dysfunctional complexes in human diseases, an application that is proving invaluable to understand disease mechanisms and to discover novel therapeutic targets. We hope this review aptly commemorates a decade of research on computational prediction of complexes and constitutes a valuable reference for further advancements in this exciting area. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
Nicotine affects protein complex rearrangement in Caenorhabditis elegans cells.

PubMed

Sobkowiak, Robert; Zielezinski, Andrzej; Karlowski, Wojciech M; Lesicki, Andrzej

2017-10-01

Nicotine may affect cell function by rearranging protein complexes. We aimed to determine nicotine-induced alterations of protein complexes in Caenorhabditis elegans (C. elegans) cells, thereby revealing links between nicotine exposure and protein complex modulation. We compared the proteomic alterations induced by low and high nicotine concentrations (0.01 mM and 1 mM) with the control (no nicotine) in vivo by using mass spectrometry (MS)-based techniques, specifically the cetyltrimethylammonium bromide (CTAB) discontinuous gel electrophoresis coupled with liquid chromatography (LC)-MS/MS and spectral counting. As a result, we identified dozens of C. elegans proteins that are present exclusively or in higher abundance in either nicotine-treated or untreated worms. Based on these results, we report a possible network that captures the key protein components of nicotine-induced protein complexes and speculate how the different protein modules relate to their distinct physiological roles. Using functional annotation of detected proteins, we hypothesize that the identified complexes can modulate the energy metabolism and level of oxidative stress. These proteins can also be involved in modulation of gene expression and may be crucial in Alzheimer's disease. The findings reported in our study reveal putative intracellular interactions of many proteins with the cytoskeleton and may contribute to the understanding of the mechanisms of nicotinic acetylcholine receptor (nAChR) signaling and trafficking in cells.

Mitochondrial NADH Fluorescence is Enhanced by Complex I Binding

PubMed Central

Blinova, Ksenia; Levine, Rodney L.; Boja, Emily S.; Griffiths, Gary L.; Shi, Zhen-Dan; Ruddy, Brian; Balaban, Robert S.

2012-01-01

Mitochondrial NADH fluorescence has been a useful tool in evaluating mitochondrial energetics both in vitro and in vivo. Mitochondrial NADH fluorescence is enhanced several fold in the matrix through extended fluorescence lifetimes (EFL). However, the actual binding sites responsible for NADH EFL are unknown. We tested the hypothesis that NADH binding to Complex I is a significant source of mitochondrial NADH fluorescence enhancement. To test this hypothesis, the effect of Complex I binding on NADH fluorescence efficiency was evaluated in purified protein, and in native gels of the entire porcine heart mitochondria proteome. To avoid the oxidation of NADH in these preparations, we conducted the binding experiments under anoxic conditions in a specially designed apparatus. Purified intact Complex I enhanced NADH fluorescence in native gels approximately 10 fold. However, no enhancement was detected in denatured individual Complex I subunit proteins. In the Clear and Ghost native gels of the entire mitochondrial proteome, NADH fluorescence enhancement was localized to regions where NADH oxidation occurred in the presence of oxygen. Inhibitor and mass spectroscopy studies revealed that the fluorescence enhancement was specific to Complex I proteins. No fluorescence enhancement was detected for MDH or other dehydrogenases in this assay system, at physiological mole fractions of the matrix proteins. These data suggest that NADH associated with Complex I significantly contributes to the overall mitochondrial NADH fluorescence signal and provides an explanation for the well established close correlation of mitochondrial NADH fluorescence and the metabolic state. PMID:18702505
mRNA 3' of the A site bound codon is located close to protein S3 on the human 80S ribosome.

PubMed

Molotkov, Maxim V; Graifer, Dmitri M; Popugaeva, Elena A; Bulygin, Konstantin N; Meschaninova, Maria I; Ven'yaminova, Aliya G; Karpova, Galina G

2006-07-01

Ribosomal proteins neighboring the mRNA downstream of the codon bound at the decoding site of human 80S ribosomes were identified using three sets of mRNA analogues that contained a UUU triplet at the 5' terminus and a perfluorophenylazide cross-linker at guanosine, adenosine or uridine residues placed at various locations 3' of this triplet. The positions of modified mRNA nucleotides on the ribosome were governed by tRNA(Phe) cognate to the UUU triplet targeted to the P site. Upon mild UV-irradiation, the mRNA analogues cross-linked preferentially to the 40S subunit, to the proteins and to a lesser extent to the 18S rRNA. Cross-linked nucleotides of 18S rRNA were identified previously. In the present study, it is shown that among the proteins the main target for cross-linking with all the mRNA analogues tested was protein S3 (homologous to prokaryotic S3, S3p); minor cross-linking to protein S2 (S5p) was also detected. Both proteins cross-linked to mRNA analogues in the ternary complexes as well as in the binary complexes (without tRNA). In the ternary complexes protein S15 (S19p) also cross-linked, the yield of the cross-link decreased significantly when the modified nucleotide moved from position +5 to position +12 with respect to the first nucleotide of the P site bound codon. In several ternary complexes minor cross-linking to protein S30 was likewise detected. The results of this study indicate that S3 is a key protein at the mRNA binding site neighboring mRNA downstream of the codon at the decoding site in the human ribosome.
Nanometer-Sized Diamond Particle as a Probe for Biolabeling

PubMed Central

Chao, Jui-I.; Perevedentseva, Elena; Chung, Pei-Hua; Liu, Kuang-Kai; Cheng, Chih-Yuan; Chang, Chia-Ching; Cheng, Chia-Liang

2007-01-01

A novel method is proposed using nanometer-sized diamond particles as detection probes for biolabeling. The advantages of nanodiamond's unique properties were demonstrated in its biocompatibility, nontoxicity, easily detected Raman signal, and intrinsic fluorescence from its natural defects without complicated pretreatments. Carboxylated nanodiamond's (cND's) penetration ability, noncytotoxicity, and visualization of cND-cell interactions are demonstrated on A549 human lung epithelial cells. Protein-targeted cell interaction visualization was demonstrated with cND-lysozyme complex interaction with bacteria Escherichia coli. It is shown that the developed biomolecule-cND complex preserves the original functions of the test protein. The easily detected natural fluorescent and Raman intrinsic signals, penetration ability, and low cytotoxicity of cNDs render them promising agents in multiple medical applications. PMID:17513352
Mapping a Noncovalent Protein-Peptide Interface by Top-Down FTICR Mass Spectrometry Using Electron Capture Dissociation

NASA Astrophysics Data System (ADS)

Clarke, David J.; Murray, Euan; Hupp, Ted; Mackay, C. Logan; Langridge-Smith, Pat R. R.

2011-08-01

Noncovalent protein-ligand and protein-protein complexes are readily detected using electrospray ionization mass spectrometry (ESI MS). Furthermore, recent reports have demonstrated that careful use of electron capture dissociation (ECD) fragmentation allows covalent backbone bonds of protein complexes to be dissociated without disruption of noncovalent protein-ligand interactions. In this way the site of protein-ligand interfaces can be identified. To date, protein-ligand complexes, which have proven tractable to this technique, have been mediated by ionic electrostatic interactions, i.e., ion pair interactions or salt bridging. Here we extend this methodology by applying ECD to study a protein-peptide complex that contains no electrostatics interactions. We analyzed the complex between the 21 kDa p53-inhibitor protein anterior gradient-2 and its hexapeptide binding ligand (PTTIYY). ECD fragmentation of the 1:1 complex occurs with retention of protein-peptide binding and analysis of the resulting fragments allows the binding interface to be localized to a C-terminal region between residues 109 and 175. These finding are supported by a solution-phase competition assay, which implicates the region between residues 108 and 122 within AGR2 as the PTTIYY binding interface. Our study expands previous findings by demonstrating that top-down ECD mass spectrometry can be used to determine directly the sites of peptide-protein interfaces. This highlights the growing potential of using ECD and related top-down fragmentation techniques for interrogation of protein-protein interfaces.
A rapid and accurate approach for prediction of interactomes from co-elution data (PrInCE).

PubMed

Stacey, R Greg; Skinnider, Michael A; Scott, Nichollas E; Foster, Leonard J

2017-10-23

An organism's protein interactome, or complete network of protein-protein interactions, defines the protein complexes that drive cellular processes. Techniques for studying protein complexes have traditionally applied targeted strategies such as yeast two-hybrid or affinity purification-mass spectrometry to assess protein interactions. However, given the vast number of protein complexes, more scalable methods are necessary to accelerate interaction discovery and to construct whole interactomes. We recently developed a complementary technique based on the use of protein correlation profiling (PCP) and stable isotope labeling in amino acids in cell culture (SILAC) to assess chromatographic co-elution as evidence of interacting proteins. Importantly, PCP-SILAC is also capable of measuring protein interactions simultaneously under multiple biological conditions, allowing the detection of treatment-specific changes to an interactome. Given the uniqueness and high dimensionality of co-elution data, new tools are needed to compare protein elution profiles, control false discovery rates, and construct an accurate interactome. Here we describe a freely available bioinformatics pipeline, PrInCE, for the analysis of co-elution data. PrInCE is a modular, open-source library that is computationally inexpensive, able to use label and label-free data, and capable of detecting tens of thousands of protein-protein interactions. Using a machine learning approach, PrInCE offers greatly reduced run time, more predicted interactions at the same stringency, prediction of protein complexes, and greater ease of use over previous bioinformatics tools for co-elution data. PrInCE is implemented in Matlab (version R2017a). Source code and standalone executable programs for Windows and Mac OSX are available at https://github.com/fosterlab/PrInCE , where usage instructions can be found. An example dataset and output are also provided for testing purposes. PrInCE is the first fast and easy-to-use data analysis pipeline that predicts interactomes and protein complexes from co-elution data. PrInCE allows researchers without bioinformatics expertise to analyze high-throughput co-elution datasets.
Surface plasmon resonance application for herbicide detection

NASA Astrophysics Data System (ADS)

Chegel, Vladimir I.; Shirshov, Yuri M.; Piletskaya, Elena V.; Piletsky, Sergey A.

1998-01-01

The optoelectronic biosensor, based on Surface Plasmon Resonance (SPR) for detection of photosynthesis-inhibiting herbicides in aqueous solutions is presented. The pesticide capability to replace plastoquinone from its complex with D1 protein is used for the detection. This replacement reaction results in the changes of the optical characteristics of protein layer, immobilized on the gold surface. Monitoring of these changes with SPR-technique permit to determine 0.1 - 5.0 mkg/ml herbicide in solution within one hour.
Surface plasmon resonance application for herbicide detection

NASA Astrophysics Data System (ADS)

Chegel, Vladimir I.; Shirshov, Yuri M.; Piletskaya, Elena V.; Piletsky, Sergey A.

1997-12-01

The optoelectronic biosensor, based on Surface Plasmon Resonance (SPR) for detection of photosynthesis-inhibiting herbicides in aqueous solutions is presented. The pesticide capability to replace plastoquinone from its complex with D1 protein is used for the detection. This replacement reaction results in the changes of the optical characteristics of protein layer, immobilized on the gold surface. Monitoring of these changes with SPR-technique permit to determine 0.1 - 5.0 mkg/ml herbicide in solution within one hour.
Prediction of physical protein protein interactions

NASA Astrophysics Data System (ADS)

Szilágyi, András; Grimm, Vera; Arakaki, Adrián K.; Skolnick, Jeffrey

2005-06-01

Many essential cellular processes such as signal transduction, transport, cellular motion and most regulatory mechanisms are mediated by protein-protein interactions. In recent years, new experimental techniques have been developed to discover the protein-protein interaction networks of several organisms. However, the accuracy and coverage of these techniques have proven to be limited, and computational approaches remain essential both to assist in the design and validation of experimental studies and for the prediction of interaction partners and detailed structures of protein complexes. Here, we provide a critical overview of existing structure-independent and structure-based computational methods. Although these techniques have significantly advanced in the past few years, we find that most of them are still in their infancy. We also provide an overview of experimental techniques for the detection of protein-protein interactions. Although the developments are promising, false positive and false negative results are common, and reliable detection is possible only by taking a consensus of different experimental approaches. The shortcomings of experimental techniques affect both the further development and the fair evaluation of computational prediction methods. For an adequate comparative evaluation of prediction and high-throughput experimental methods, an appropriately large benchmark set of biophysically characterized protein complexes would be needed, but is sorely lacking.
Detection of retromer assembly in Plasmodium falciparum by immunosensing coupled to Surface Plasmon Resonance.

PubMed

Iqbal, Mohd Shameel; Siddiqui, Asim Azhar; Banerjee, Chinmoy; Nag, Shiladitya; Mazumder, Somnath; De, Rudranil; Saha, Shubhra Jyoti; Karri, Suresh Kumar; Bandyopadhyay, Uday

Retromer complex plays a crucial role in intracellular protein trafficking and is conserved throughout the eukaryotes including malaria parasite, Plasmodium falciparum, where it is partially conserved. The assembly of retromer complex in RBC stages of malarial parasite is extremely difficult to explore because of its complicated physiology, small size, and intra-erythrocytic location. Nonetheless, understanding of retromer assembly may pave new ways for the development of novel antimalarials targeting parasite-specific protein trafficking pathways. Here, we investigated the assembly of retromer complex in P. falciparum, by an immunosensing method through highly sensitive Surface Plasmon Resonance (SPR) technique. After taking leads from the bioinformatics search and literature, different interacting proteins were identified and specific antibodies were raised against them. The sensor chip was prepared by covalently linking antibody specific to one component and the whole cell lysate was passed through it in order to trap the interacting complex. Antibodies raised against other interacting components were used to detect them in the trapped complex on the SPR chip. We were able to detect three different components in the retromer complex trapped by the immobilized antibody specific against a different component on a sensor chip. The assay was reproduced and validated in a different two-component CD74-MIF system in mammalian cells. We, thus, illustrate the assembly of retromer complex in P. falciparum through a bio-sensing approach that combines SPR with immunosensing requiring a very small amount of sample from the native source. Copyright © 2018 Elsevier B.V. All rights reserved.
Detection of changes in gene regulatory patterns, elicited by perturbations of the Hsp90 molecular chaperone complex, by visualizing multiple experiments with an animation

PubMed Central

2011-01-01

Background To make sense out of gene expression profiles, such analyses must be pushed beyond the mere listing of affected genes. For example, if a group of genes persistently display similar changes in expression levels under particular experimental conditions, and the proteins encoded by these genes interact and function in the same cellular compartments, this could be taken as very strong indicators for co-regulated protein complexes. One of the key requirements is having appropriate tools to detect such regulatory patterns. Results We have analyzed the global adaptations in gene expression patterns in the budding yeast when the Hsp90 molecular chaperone complex is perturbed either pharmacologically or genetically. We integrated these results with publicly accessible expression, protein-protein interaction and intracellular localization data. But most importantly, all experimental conditions were simultaneously and dynamically visualized with an animation. This critically facilitated the detection of patterns of gene expression changes that suggested underlying regulatory networks that a standard analysis by pairwise comparison and clustering could not have revealed. Conclusions The results of the animation-assisted detection of changes in gene regulatory patterns make predictions about the potential roles of Hsp90 and its co-chaperone p23 in regulating whole sets of genes. The simultaneous dynamic visualization of microarray experiments, represented in networks built by integrating one's own experimental with publicly accessible data, represents a powerful discovery tool that allows the generation of new interpretations and hypotheses. PMID:21672238
Comparative Network-Based Recovery Analysis and Proteomic Profiling of Neurological Changes in Valproic Acid-Treated Mice

PubMed Central

2013-01-01

Despite its prominence for characterization of complex mixtures, LC–MS/MS frequently fails to identify many proteins. Network-based analysis methods, based on protein–protein interaction networks (PPINs), biological pathways, and protein complexes, are useful for recovering non-detected proteins, thereby enhancing analytical resolution. However, network-based analysis methods do come in varied flavors for which the respective efficacies are largely unknown. We compare the recovery performance and functional insights from three distinct instances of PPIN-based approaches, viz., Proteomics Expansion Pipeline (PEP), Functional Class Scoring (FCS), and Maxlink, in a test scenario of valproic acid (VPA)-treated mice. We find that the most comprehensive functional insights, as well as best non-detected protein recovery performance, are derived from FCS utilizing real biological complexes. This outstrips other network-based methods such as Maxlink or Proteomics Expansion Pipeline (PEP). From FCS, we identified known biological complexes involved in epigenetic modifications, neuronal system development, and cytoskeletal rearrangements. This is congruent with the observed phenotype where adult mice showed an increase in dendritic branching to allow the rewiring of visual cortical circuitry and an improvement in their visual acuity when tested behaviorally. In addition, PEP also identified a novel complex, comprising YWHAB, NR1, NR2B, ACTB, and TJP1, which is functionally related to the observed phenotype. Although our results suggest different network analysis methods can produce different results, on the whole, the findings are mutually supportive. More critically, the non-overlapping information each provides can provide greater holistic understanding of complex phenotypes. PMID:23557376
Non-perturbative Quantification of Ionic Charge Transfer through Nm-Scale Protein Pores Using Graphene Microelectrodes

NASA Astrophysics Data System (ADS)

Ping, Jinglei; Johnson, A. T. Charlie; A. T. Charlie Johnson Team

Conventional electrical methods for detecting charge transfer through protein pores perturb the electrostatic condition of the solution and chemical reactivity of the pore, and are not suitable to be used for complex biofluids. We developed a non-perturbative methodology ( fW input power) for quantifying trans-pore electrical current and detecting the pore status (i.e., open vs. closes) via graphene microelectrodes. Ferritin was used as a model protein featuring a large interior compartment, well-separated from the exterior solution with discrete pores as charge commuting channels. The charge flowing through the ferritin pores transfers into the graphene microelectrode and is recorded by an electrometer. In this example, our methodology enables the quantification of an inorganic nanoparticle-protein nanopore interaction in complex biofluids. The authors acknowledge the support from the Defense Advanced Research Projects Agency (DARPA) and the U.S. Army Research Office under Grant Number W911NF1010093.
PAPERS DEVOTED TO THE MEMORY OF ACADEMICIAN A M PROKHOROV: Immunosensor systems with the Langmuir-film-based fluorescence detection

NASA Astrophysics Data System (ADS)

Chudinova, G. K.; Nagovitsyn, I. A.; Karpov, R. E.; Savranskii, V. V.

2003-09-01

A method is developed for detecting protein antigens for fluorescent immunoassay using a model system based on the technique for preparation of Langmuir films. Fluorescein isothiocyanate and donor-acceptor energy-transfer pairs of markers (the Yb complex of tetraphenyl porphyrin — benzoyl trifluoroacetoneisothiocyanate and derivatives of tetra(carboxyphenyl) porphyrin — cyanine dye containing a five-membered polyene chain), which were nor studied earlier, were used as markers for detecting the binding of an antigen on the surface of Langmuir films of antibodies. Fluorescence was detected in the near-IR region (for the first pair) and in the visible spectral range (for the second pair). To reduce the nonspecific sorption of a protein (antigen), a method was proposed for the preparation of a nonpolar surface by applying an even number of layers of stearic acid as a substrate for the Langmuir — Blodgett film. A high sensitivity of model systems to a protein antigen in solution was achieved (~10-11 M), the assay time being 6 — 8 min. The model system with the first donor — acceptor pair was tested in analysis of the blood plasma. The fluorescence of the Dy3+, Tm3+, and Yb3+ complexes of tetraphenyl porphyrin sensitised by diketonate complexes of lanthanides was studied for the first time and the enhancement of the IR fluorescence of these complexes in a Langmuir film was demonstrated.
Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Klopffleisch, Karsten; Phan, Nguyen; Chen, Jay

2011-01-01

The heterotrimeric G-protein complex is minimally composed of G{alpha}, G{beta}, and G{gamma} subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, wemore » detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification.« less
Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis.

PubMed

Klopffleisch, Karsten; Phan, Nguyen; Augustin, Kelsey; Bayne, Robert S; Booker, Katherine S; Botella, Jose R; Carpita, Nicholas C; Carr, Tyrell; Chen, Jin-Gui; Cooke, Thomas Ryan; Frick-Cheng, Arwen; Friedman, Erin J; Fulk, Brandon; Hahn, Michael G; Jiang, Kun; Jorda, Lucia; Kruppe, Lydia; Liu, Chenggang; Lorek, Justine; McCann, Maureen C; Molina, Antonio; Moriyama, Etsuko N; Mukhtar, M Shahid; Mudgil, Yashwanti; Pattathil, Sivakumar; Schwarz, John; Seta, Steven; Tan, Matthew; Temp, Ulrike; Trusov, Yuri; Urano, Daisuke; Welter, Bastian; Yang, Jing; Panstruga, Ralph; Uhrig, Joachim F; Jones, Alan M

2011-09-27

The heterotrimeric G-protein complex is minimally composed of Gα, Gβ, and Gγ subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification.
Arabidopsis G-protein interactome reveals connections to cell wall carbohydrates and morphogenesis

PubMed Central

Klopffleisch, Karsten; Phan, Nguyen; Augustin, Kelsey; Bayne, Robert S; Booker, Katherine S; Botella, Jose R; Carpita, Nicholas C; Carr, Tyrell; Chen, Jin-Gui; Cooke, Thomas Ryan; Frick-Cheng, Arwen; Friedman, Erin J; Fulk, Brandon; Hahn, Michael G; Jiang, Kun; Jorda, Lucia; Kruppe, Lydia; Liu, Chenggang; Lorek, Justine; McCann, Maureen C; Molina, Antonio; Moriyama, Etsuko N; Mukhtar, M Shahid; Mudgil, Yashwanti; Pattathil, Sivakumar; Schwarz, John; Seta, Steven; Tan, Matthew; Temp, Ulrike; Trusov, Yuri; Urano, Daisuke; Welter, Bastian; Yang, Jing; Panstruga, Ralph; Uhrig, Joachim F; Jones, Alan M

2011-01-01

The heterotrimeric G-protein complex is minimally composed of Gα, Gβ, and Gγ subunits. In the classic scenario, the G-protein complex is the nexus in signaling from the plasma membrane, where the heterotrimeric G-protein associates with heptahelical G-protein-coupled receptors (GPCRs), to cytoplasmic target proteins called effectors. Although a number of effectors are known in metazoans and fungi, none of these are predicted to exist in their canonical forms in plants. To identify ab initio plant G-protein effectors and scaffold proteins, we screened a set of proteins from the G-protein complex using two-hybrid complementation in yeast. After deep and exhaustive interrogation, we detected 544 interactions between 434 proteins, of which 68 highly interconnected proteins form the core G-protein interactome. Within this core, over half of the interactions comprising two-thirds of the nodes were retested and validated as genuine in planta. Co-expression analysis in combination with phenotyping of loss-of-function mutations in a set of core interactome genes revealed a novel role for G-proteins in regulating cell wall modification. PMID:21952135
Coevolution at protein complex interfaces can be detected by the complementarity trace with important impact for predictive docking

PubMed Central

Madaoui, Hocine; Guerois, Raphaël

2008-01-01

Protein surfaces are under significant selection pressure to maintain interactions with their partners throughout evolution. Capturing how selection pressure acts at the interfaces of protein–protein complexes is a fundamental issue with high interest for the structural prediction of macromolecular assemblies. We tackled this issue under the assumption that, throughout evolution, mutations should minimally disrupt the physicochemical compatibility between specific clusters of interacting residues. This constraint drove the development of the so-called Surface COmplementarity Trace in Complex History score (SCOTCH), which was found to discriminate with high efficiency the structure of biological complexes. SCOTCH performances were assessed not only with respect to other evolution-based approaches, such as conservation and coevolution analyses, but also with respect to statistically based scoring methods. Validated on a set of 129 complexes of known structure exhibiting both permanent and transient intermolecular interactions, SCOTCH appears as a robust strategy to guide the prediction of protein–protein complex structures. Of particular interest, it also provides a basic framework to efficiently track how protein surfaces could evolve while keeping their partners in contact. PMID:18511568
Dilution of protein-surfactant complexes: a fluorescence study.

PubMed

Azadi, Glareh; Chauhan, Anuj; Tripathi, Anubhav

2013-09-01

Dilution of protein-surfactant complexes is an integrated step in microfluidic protein sizing, where the contribution of free micelles to the overall fluorescence is reduced by dilution. This process can be further improved by establishing an optimum surfactant concentration and quantifying the amount of protein based on the fluorescence intensity. To this end, we study the interaction of proteins with anionic sodium dodecyl sulfate (SDS) and cationic hexadecyl trimethyl ammonium bromide (CTAB) using a hydrophobic fluorescent dye (sypro orange). We analyze these interactions fluourometrically with bovine serum albumin, carbonic anhydrase, and beta-galactosidase as model proteins. The fluorescent signature of protein-surfactant complexes at various dilution points shows three distinct regions, surfactant dominant, breakdown, and protein dominant region. Based on the dilution behavior of protein-surfactant complexes, we propose a fluorescence model to explain the contribution of free and bound micelles to the overall fluorescence. Our results show that protein peak is observed at 3 mM SDS as the optimum dilution concentration. Furthermore, we study the effect of protein concentration on fluorescence intensity. In a single protein model with a constant dye quantum yield, the peak height increases with protein concentration. Finally, addition of CTAB to the protein-SDS complex at mole fractions above 0.1 shifts the protein peak from 3 mM to 4 mM SDS. The knowledge of protein-surfactant interactions obtained from these studies provides significant insights for novel detection and quantification techniques in microfluidics. © 2013 The Protein Society.
Bio-nano interactions detected by nanochannel electrophoresis.

PubMed

Luan, Binquan

2016-08-01

Engineered nanoparticles have been widely used in industry and are present in many consumer products. However, their bio-safeties especially in a long term are largely unknown. Here, a nanochannel-electrophoresis-based method is proposed for detecting the potential bio-nano interactions that may further lead to damages to human health and/or biological environment. Through proof-of-concept molecular dynamics simulations, it was demonstrated that the transport of a protein-nanoparticle complex is very different from that of a protein along. By monitoring the change of ionic currents induced by a transported analyte as well as the transport velocities of the analyte, the complex (with bio-nano interaction) can be clearly distinguished from the protein alone (with no interaction with tested nanoparticles). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
An integrated native mass spectrometry and top-down proteomics method that connects sequence to structure and function of macromolecular complexes

NASA Astrophysics Data System (ADS)

Li, Huilin; Nguyen, Hong Hanh; Ogorzalek Loo, Rachel R.; Campuzano, Iain D. G.; Loo, Joseph A.

2018-02-01

Mass spectrometry (MS) has become a crucial technique for the analysis of protein complexes. Native MS has traditionally examined protein subunit arrangements, while proteomics MS has focused on sequence identification. These two techniques are usually performed separately without taking advantage of the synergies between them. Here we describe the development of an integrated native MS and top-down proteomics method using Fourier-transform ion cyclotron resonance (FTICR) to analyse macromolecular protein complexes in a single experiment. We address previous concerns of employing FTICR MS to measure large macromolecular complexes by demonstrating the detection of complexes up to 1.8 MDa, and we demonstrate the efficacy of this technique for direct acquirement of sequence to higher-order structural information with several large complexes. We then summarize the unique functionalities of different activation/dissociation techniques. The platform expands the ability of MS to integrate proteomics and structural biology to provide insights into protein structure, function and regulation.

Detection of protein-protein interactions by ribosome display and protein in situ immobilisation.

PubMed

He, Mingyue; Liu, Hong; Turner, Martin; Taussig, Michael J

2009-12-31

We describe a method for identification of protein-protein interactions by combining two cell-free protein technologies, namely ribosome display and protein in situ immobilisation. The method requires only PCR fragments as the starting material, the target proteins being made through cell-free protein synthesis, either associated with their encoding mRNA as ribosome complexes or immobilised on a solid surface. The use of ribosome complexes allows identification of interacting protein partners from their attached coding mRNA. To demonstrate the procedures, we have employed the lymphocyte signalling proteins Vav1 and Grb2 and confirmed the interaction between Grb2 and the N-terminal SH3 domain of Vav1. The method has promise for library screening of pairwise protein interactions, down to the analytical level of individual domain or motif mapping.
Location of RAD51-like protein during meiotic prophase in Eimeria tenella.

PubMed

Del Cacho, Emilio; Gallego, Margarita; Pagés, Marc; Barbero, José Luís; Monteagudo, Luís; Sánchez-Acedo, Caridad

2011-05-31

This study focuses on reporting events in Eimeria tenella oocysts from early to late prophase I in terms of RAD51 protein in association with the synaptonemal complex formed between homologous chromosomes. The aim of the study was the sequential localization of RAD51 protein, which is involved in the repair of double-strand breaks (DSBs) on the eimerian chromosomes as they synapse and desynapse. Structural Maintenance of Chromosome protein SMC3, which plays a role in synaptonemal complex formation, was labeled to identify initiation and progress of chromosome synapsis and desynapsis in parallel with the appearance and disappearance of RAD51 foci. Antibodies directed against RAD51 and cohesin subunit SMC3 proteins were labeled with either fluorescence or colloidal gold to visualize RAD51 protein foci and synaptonemal complexes. RAD51 protein localization during prophase I was studied on meiotic chromosomes spreads obtained from oocysts at different points in time after the start of sporulation. The present findings showed that foci detected with the antibody directed against RAD51 protein first appeared at the pre-leptotene stage before homologous chromosomes began pairing. Subsequently, the foci were detected in association with the lateral elements at the precise sites where synapsis were in progress. These findings lead us to suggest that in E. tenella, homologous chromosome pairing was a DSB-dependent mechanism and reinforced the participation of RAD51 protein in meiotic homology search, alignment and pairing of chromosomes. Copyright © 2010 Elsevier B.V. All rights reserved.
Identification of Crosslinked Peptides after Click-based Enrichment Using Sequential CID and ETD Tandem Mass Spectrometry

PubMed Central

Chowdhury, Saiful M.; Du, Xiuxia; Tolić, Nikola; Wu, Si; Moore, Ronald J.; Mayer, M. Uljana; Smith, Richard D.; Adkins, Joshua N.

2010-01-01

Chemical crosslinking combined with mass spectrometry can be a powerful approach for the identification of protein-protein interactions and for providing constraints on protein structures. However, enrichment of crosslinked peptides is crucial to reduce sample complexity before mass spectrometric analysis. In addition compact crosslinkers are often preferred to provide short spacer lengths, surface accessibility to the protein complexes, and must have reasonable solubility under condition where the native complex structure is stable. In this study, we present a novel compact crosslinker that contains two distinct features: 1) an alkyne tag and 2) a small molecule detection tag (NO2-) to maintain reasonable solubility in water. The alkyne tag enables enrichment of the crosslinked peptide after proteolytic cleavage after coupling of an affinity tag using alkyne-azido click chemistry. Neutral loss of the small NO2- moiety provides a secondary means of detecting crosslinked peptides in MS/MS analyses, providing additional confidence in peptide identifications. We show the labeling efficiency of this crosslinker, which we termed CLIP (Click-enabled Linker for Interacting Proteins) using ubiquitin. The enrichment capability of CLIP is demonstrated for crosslinked ubiquitin in highly complex E. coli cell lysates. Sequential CID-MS/MS and ETD-MS/MS of inter-crosslinked peptides (two peptides connected with a crosslinker) are also demonstrated for improved automated identification of crosslinked peptides. PMID:19496583
The Design of a Quantitative Western Blot Experiment

PubMed Central

Taylor, Sean C.; Posch, Anton

2014-01-01

Western blotting is a technique that has been in practice for more than three decades that began as a means of detecting a protein target in a complex sample. Although there have been significant advances in both the imaging and reagent technologies to improve sensitivity, dynamic range of detection, and the applicability of multiplexed target detection, the basic technique has remained essentially unchanged. In the past, western blotting was used simply to detect a specific target protein in a complex mixture, but now journal editors and reviewers are requesting the quantitative interpretation of western blot data in terms of fold changes in protein expression between samples. The calculations are based on the differential densitometry of the associated chemiluminescent and/or fluorescent signals from the blots and this now requires a fundamental shift in the experimental methodology, acquisition, and interpretation of the data. We have recently published an updated approach to produce quantitative densitometric data from western blots (Taylor et al., 2013) and here we summarize the complete western blot workflow with a focus on sample preparation and data analysis for quantitative western blotting. PMID:24738055
Bloch Surface Waves Biosensors for High Sensitivity Detection of Soluble ERBB2 in a Complex Biological Environment.

PubMed

Sinibaldi, Alberto; Sampaoli, Camilla; Danz, Norbert; Munzert, Peter; Sonntag, Frank; Centola, Fabio; Occhicone, Agostino; Tremante, Elisa; Giacomini, Patrizio; Michelotti, Francesco

2017-08-17

We report on the use of one-dimensional photonic crystals to detect clinically relevant concentrations of the cancer biomarker ERBB2 in cell lysates. Overexpression of the ERBB2 protein is associated with aggressive breast cancer subtypes. To detect soluble ERBB2, we developed an optical set-up which operates in both label-free and fluorescence modes. The detection approach makes use of a sandwich assay, in which the one-dimensional photonic crystals sustaining Bloch surface waves are modified with monoclonal antibodies, in order to guarantee high specificity during the biological recognition. We present the results of exemplary protein G based label-free assays in complex biological matrices, reaching an estimated limit of detection of 0.5 ng/mL. On-chip and chip-to-chip variability of the results is addressed too, providing repeatability rates. Moreover, results on fluorescence operation demonstrate the capability to perform high sensitive cancer biomarker assays reaching a resolution of 0.6 ng/mL, without protein G assistance. The resolution obtained in both modes meets international guidelines and recommendations (15 ng/mL) for ERBB2 quantification assays, providing an alternative tool to phenotype and diagnose molecular cancer subtypes.
Non-covalent pomegranate (Punica granatum) hydrolyzable tannin-protein complexes modulate antigen uptake, processing and presentation by a T-cell hybridoma line co-cultured with murine peritoneal macrophages.

PubMed

Madrigal-Carballo, Sergio; Haas, Linda; Vestling, Martha; Krueger, Christian G; Reed, Jess D

2016-12-01

In this work we characterize the interaction of pomegranate hydrolyzable tannins (HT) with hen egg-white lysozyme (HEL) and determine the effects of non-covalent tannin-protein complexes on macrophage endocytosis, processing and presentation of antigen. We isolated HT from pomegranate and complex to HEL, the resulting non-covalent tannin-protein complex was characterized by gel electrophoresis and MALDI-TOF MS. Finally, cell culture studies and confocal microscopy imaging were conducted on the non-covalent pomegranate HT-HEL protein complexes to evaluate its effect on macrophage antigen uptake, processing and presentation to T-cell hybridomas. Our results indicate that non-covalent pomegranate HT-HEL protein complexes modulate uptake, processing and antigen presentation by mouse peritoneal macrophages. After 4 h of pre-incubation, only trace amounts of IL-2 were detected in the co-cultures treated with HEL alone, whereas a non-covalent pomegranate HT-HEL complex had already reached maximum IL-2 expression. Pomegranate HT may increase rate of endocytose of HEL and subsequent expression of IL-2 by the T-cell hybridomas.
Sensitive detection of C-reactive protein using optical fiber Bragg gratings.

PubMed

Sridevi, S; Vasu, K S; Asokan, S; Sood, A K

2015-03-15

An accurate and highly sensitive sensor platform has been demonstrated for the detection of C-reactive protein (CRP) using optical fiber Bragg gratings (FBGs). The CRP detection has been carried out by monitoring the shift in Bragg wavelength (ΔλB) of an etched FBG (eFBG) coated with an anti-CRP antibody (aCRP)-graphene oxide (GO) complex. The complex is characterized by Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy and atomic force microscopy. A limit of detection of 0.01mg/L has been achieved with a linear range of detection from 0.01mg/L to 100mg/L which includes clinical range of CRP. The eFBG sensor coated with only aCRP (without GO) show much less sensitivity than that of aCRP-GO complex coated eFBG. The eFBG sensors show high specificity to CRP even in the presence of other interfering factors such as urea, creatinine and glucose. The affinity constant of ∼1.1×10(10)M(-1) has been extracted from the data of normalized shift (ΔλB/λB) as a function of CRP concentration. Copyright © 2014 Elsevier B.V. All rights reserved.
A Proteomics Analysis of the Effects of Chronic Hemiparetic Stroke on Troponin T Expression in Human Vastus Lateralis

PubMed Central

Rabek, Jeffrey P.; Hafer-Macko, Charlene E.; Amaning, James K.; DeFord, James H.; Dimayuga, Vincent L.; Madsen, Mark A.; Macko, Richard F.

2009-01-01

Stroke disability is attributed to upper motor neuron deficits resulting from ischemic brain injury. We have developed proteome maps of the Vastus lateralis to examine the effects of ischemic brain injury on paretic skeletal muscle myofilament proteins. Proteomics analyses from seven hemiparetic stroke patients have detected a decrease of three troponin T isoforms in the paretic muscle suggesting that myosin–actin interactions may be attenuated. We propose that ischemic brain injury may prevent troponin T participation in complex formation thereby affecting the protein interactions associated with excitation–contraction coupling. We have also detected a novel skeletal troponin T isoform that has a C-terminal variation. Our data suggest that the decreased slow troponin T isoform pools in the paretic limb may contribute to the gait deficit after stroke. The complexity of the neurological deficit on Vastus lateralis is suggested by the multiple changes in proteins detected by our proteomics mapping. PMID:19447848
Specific and Non-Specific Protein Association in Solution: Computation of Solvent Effects and Prediction of First-Encounter Modes for Efficient Configurational Bias Monte Carlo Simulations

PubMed Central

Cardone, Antonio; Pant, Harish; Hassan, Sergio A.

2013-01-01

Weak and ultra-weak protein-protein association play a role in molecular recognition, and can drive spontaneous self-assembly and aggregation. Such interactions are difficult to detect experimentally, and are a challenge to the force field and sampling technique. A method is proposed to identify low-population protein-protein binding modes in aqueous solution. The method is designed to identify preferential first-encounter complexes from which the final complex(es) at equilibrium evolves. A continuum model is used to represent the effects of the solvent, which accounts for short- and long-range effects of water exclusion and for liquid-structure forces at protein/liquid interfaces. These effects control the behavior of proteins in close proximity and are optimized based on binding enthalpy data and simulations. An algorithm is described to construct a biasing function for self-adaptive configurational-bias Monte Carlo of a set of interacting proteins. The function allows mixing large and local changes in the spatial distribution of proteins, thereby enhancing sampling of relevant microstates. The method is applied to three binary systems. Generalization to multiprotein complexes is discussed. PMID:24044772
Molecular counting by photobleaching in protein complexes with many subunits: best practices and application to the cellulose synthesis complex

PubMed Central

Chen, Yalei; Deffenbaugh, Nathan C.; Anderson, Charles T.; Hancock, William O.

2014-01-01

The constituents of large, multisubunit protein complexes dictate their functions in cells, but determining their precise molecular makeup in vivo is challenging. One example of such a complex is the cellulose synthesis complex (CSC), which in plants synthesizes cellulose, the most abundant biopolymer on Earth. In growing plant cells, CSCs exist in the plasma membrane as six-lobed rosettes that contain at least three different cellulose synthase (CESA) isoforms, but the number and stoichiometry of CESAs in each CSC are unknown. To begin to address this question, we performed quantitative photobleaching of GFP-tagged AtCESA3-containing particles in living Arabidopsis thaliana cells using variable-angle epifluorescence microscopy and developed a set of information-based step detection procedures to estimate the number of GFP molecules in each particle. The step detection algorithms account for changes in signal variance due to changing numbers of fluorophores, and the subsequent analysis avoids common problems associated with fitting multiple Gaussian functions to binned histogram data. The analysis indicates that at least 10 GFP-AtCESA3 molecules can exist in each particle. These procedures can be applied to photobleaching data for any protein complex with large numbers of fluorescently tagged subunits, providing a new analytical tool with which to probe complex composition and stoichiometry. PMID:25232006
Molecular counting by photobleaching in protein complexes with many subunits: best practices and application to the cellulose synthesis complex

DOE PAGES

Chen, Yalei; Deffenbaugh, Nathan C.; Anderson, Charles T.; ...

2014-09-17

The constituents of large, multisubunit protein complexes dictate their functions in cells, but determining their precise molecular makeup in vivo is challenging. One example of such a complex is the cellulose synthesis complex (CSC), which in plants synthesizes cellulose, the most abundant biopolymer on Earth. In growing plant cells, CSCs exist in the plasma membrane as six-lobed rosettes that contain at least three different cellulose synthase (CESA) isoforms, but the number and stoichiometry of CESAs in each CSC are unknown. To begin to address this question, we performed quantitative photobleaching of GFP-tagged AtCESA3-containing particles in living Arabidopsis thaliana cells usingmore » variable-angle epifluorescence microscopy and developed a set of information-based step detection procedures to estimate the number of GFP molecules in each particle. The step detection algorithms account for changes in signal variance due to changing numbers of fluorophores, and the subsequent analysis avoids common problems associated with fitting multiple Gaussian functions to binned histogram data. The analysis indicates that at least 10 GFP-AtCESA3 molecules can exist in each particle. In conclusion, these procedures can be applied to photobleaching data for any protein complex with large numbers of fluorescently tagged subunits, providing a new analytical tool with which to probe complex composition and stoichiometry.« less
Myocilin, a Component of a Membrane-Associated Protein Complex Driven by a Homologous Q-SNARE Domain

PubMed Central

Dismuke, W. Michael; McKay, Brian S.; Stamer, W. Daniel

2012-01-01

Myocilin is a widely expressed protein with no known function, however, mutations in myocilin appear to manifest uniquely as ocular hypertension and the blinding disease glaucoma. Using the protein homology/analogy recognition engine (PHYRE) we find that the olfactomedin domain of myocilin is similar in sequence motif and structure to a six-bladed, kelch repeat motif based on the known crystal structures of such proteins. Additionally, using sequence analysis we identify a coiled-coil segment of myocilin with homology to human Q-SNARE proteins. Using COS-7 cells expressing full length human myocilin and a version lacking the C-terminal olfactomedin domain, we identified a membrane-associated protein complex containing myocilin by hydrodynamic analysis. The myocilin construct that included the coiled-coil but lacked the olfactomedin domain formed complexes similar to the full-length protein, indicating that the coiled-coil domain of myocilin is sufficient for myocilin to bind to the large detergent resistant complex. In human retina and retinal pigment epithelium, which express myocilin, we detected the protein in a large, SDS-resistant, membrane-associated complex. We characterized the hydrodynamic properties of myocilin in human tissues as either a 15s complex with an Mr=405,000–440,000 yielding a slightly elongated globular shape similar to known SNARE complexes or a dimer of 6.4s and Mr=108,000. By identifying the Q-SNARE homology within the second coil of myocilin and documenting its participation in a SNARE-like complex, we provide evidence of a SNARE domain containing protein associated with a human disease. PMID:22463803
Dissortativity and duplications in oral cancer

NASA Astrophysics Data System (ADS)

Shinde, Pramod; Yadav, Alok; Rai, Aparna; Jalan, Sarika

2015-08-01

More than 300 000 new cases worldwide are being diagnosed with oral cancer annually. Complexity of oral cancer renders designing drug targets very difficult. We analyse protein-protein interaction network for the normal and oral cancer tissue and detect crucial changes in the structural properties of the networks in terms of the interactions of the hub proteins and the degree-degree correlations. Further analysis of the spectra of both the networks, while exhibiting universal statistical behaviour, manifest distinction in terms of the zero degeneracy, providing insight to the complexity of the underlying system.
Carcinoembryonic antigen promotes colorectal cancer progression by targeting adherens junction complexes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bajenova, Olga, E-mail: o.bazhenova@spbu.ru; Department of Genetics and Biotechnology, St. Petersburg State University, St. Petersburg 199034; Department of Surgery and Biomedical Sciences, Creighton University, Omaha, NE 68178

2014-06-10

Oncomarkers play important roles in the detection and management of human malignancies. Carcinoembryonic antigen (CEA, CEACAM5) and epithelial cadherin (E-cadherin) are considered as independent tumor markers in monitoring metastatic colorectal cancer. They are both expressed by cancer cells and can be detected in the blood serum. We investigated the effect of CEA production by MIP101 colorectal carcinoma cell lines on E-cadherin adherens junction (AJ) protein complexes. No direct interaction between E-cadherin and CEA was detected; however, the functional relationships between E-cadherin and its AJ partners: α-, β- and p120 catenins were impaired. We discovered a novel interaction between CEA andmore » beta-catenin protein in the CEA producing cells. It is shown in the current study that CEA overexpression alters the splicing of p120 catenin and triggers the release of soluble E-cadherin. The influence of CEA production by colorectal cancer cells on the function of E-cadherin junction complexes may explain the link between the elevated levels of CEA and the increase in soluble E-cadherin during the progression of colorectal cancer. - Highlights: • Elevated level of CEA increases the release of soluble E-cadherin during the progression of colorectal cancer. • CEA over-expression alters the binding preferences between E-cadherin and its partners: α-, β- and p120 catenins in adherens junction complexes. • CEA produced by colorectal cancer cells interacts with beta-catenin protein. • CEA over-expression triggers the increase in nuclear beta-catenin. • CEA over-expression alters the splicing of p120 catenin protein.« less
Aptamer-based SERRS Sensor for Thrombin Detection

PubMed Central

Cho, Hansang; Baker, Brian R.; Wachsmann-Hogiu, Sebastian; Pagba, Cynthia V.; Laurence, Ted A.; Lane, Stephen M.; Lee, Luke P.; Tok, Jeffrey B.-H.

2012-01-01

We describe an aptamer-based Surface Enhanced Resonance Raman Scattering (SERRS) sensor with high sensitivity, specificity, and stability for the detection of a coagulation protein, human α-thrombin. The sensor achieves high sensitivity and a limit of detection of 100 pM by monitoring the SERRS signal change upon the single step of thrombin binding to immobilized thrombin binding aptamer. The selectivity of the sensor is demonstrated by the specific discrimination of thrombin from other protein analytes. The specific recognition and binding of thrombin by the thrombin binding aptamer is essential to the mechanism of the aptamer-based sensor, as shown through measurements using negative control oligonucleotides. In addition, the sensor can detect 1 nM thrombin in the presence of complex biofluids, such as 10% fetal calf serum, demonstrating that the immobilized, 5'-capped, 3'-capped aptamer is sufficiently robust for clinical diagnostic applications. Furthermore, the proposed sensor may be implemented for multiplexed detection using different aptamer-Raman probe complexes. PMID:19367849
[Comparative characteristics of microbial proteases by the level of hydrolysis of protein substrates].

PubMed

Rimareva, L V; Overchenko, M B; Serba, E M; Trifonova, V V

1997-01-01

Screening of enzyme preparations displaying a maximum proteolytic activity at pH 4.0-5.5 and effecting deep proteolysis of plant proteins was performed. Amyloprotooryzin prepared from Aspergillus oryzae 387 containing a complex of proteolytic enzymes was the most effective. The amino acid composition of the hydrolysates obtained was studied. Amyloprotooryzin increased the contents of amino acids by 108-227%, depending on the substrate used. The enzymatic complex of amyloprotooryzin was studied; in addition, proteases, alpha-amylase, exo-beta-glucanase, and xylanase were detected in the complex.
Incorporation of Deoxyribonucleic Acid Precursors by T4 Deoxyribonucleic Acid-Protein Complexes Retained on Glass Fiber Filters

PubMed Central

Miller, Robert C.; Kozinski, Andrzej W.

1970-01-01

Bacteriophage T4 deoxyribonucleic acid (DNA)-protein complexes were retained preferentially on glass fiber filters. DNA polymerase activity in the complex was detected through the incorporation of 3H-labeled DNA precursors. The primer-product DNA hybridized with both phage and Escherichia coli DNA. Density labeling experiments showed that about 30% of incorporated 3H-deoxyadenosine triphosphate was found in DNA which hybridized with phage DNA; this DNA was found to be covalently attached to the primer DNA. PMID:5497903
Complex of simian virus 40 large-T antigen and host 53,000-molecular-weight protein in monkey cells.

PubMed Central

Harlow, E; Pim, D C; Crawford, L V

1981-01-01

Mouse cells transformed by simian virus 40 (SV40) have been shown to contain a complex of the virus-coded large-T antigen with a host 53,000-molecular-weight (53K) protein. Initial attempts to detect a similar complex in lytically infected cells were unsuccessful, and it therefore seemed that the complex might be peculiar to transformed or abortively transformed nonpermissive cells. Immunoprecipitation of [32P]phosphate-labeled extracts of SV40-infected CV-1 African green monkey kidney cells with antibodies specific for large-T or the 53K protein revealed that the large-T-53K protein complex was formed during lytic infections. Only a minor fraction of the large-T present was associated with 53K protein, and large-T and the 53K host protein cosedimented during centrifugation through sucrose gradients. We used monospecific sera and monoclonal antibodies to study the rate of synthesis and phosphorylation of the 53K protein during lytic infections. Infection of CV-1 cells with SV40 increased the rate of synthesis of the 53K protein fivefold over that in mock-infected cells. At the same time, the rate of phosphorylation of the 53K protein increased more than 30-fold compared with control cultures. Monkey cells transformed by UV-irradiated SV40 (Gluzman et al., J. Virol. 22:256-266, 1977) also contained the large-T-53K protein complex. The formation of the complex is therefore not a peculiarity of SV40-transformed rodent cells but is a common feature of SV40 infections. Images PMID:6163871
Detection of the HA-33 protein in botulinum neurotoxin type G complex by mass spectrometry.

PubMed

Kalb, Suzanne R; Baudys, Jakub; Barr, John R

2015-10-23

The disease botulism is caused by intoxication with botulinum neurotoxins (BoNTs), extremely toxic proteins which cause paralysis. This neurotoxin is produced by some members of the Clostridium botulinum and closely related species, and is produced as a protein complex consisting of the neurotoxin and neurotoxin-associated proteins (NAPs). There are seven known serotypes of BoNT, A-G, and the composition of the NAPs can differ between these serotypes. It was previously published that the BoNT/G complex consisted of BoNT/G, nontoxic-nonhemagglutinin (NTNH), Hemagglutinin 70 (HA-70), and HA-17, but that HA-33, a component of the protein complex of other serotypes of BoNT, was not found. Components of the BoNT/G complex were first separated by SDS-PAGE, and bands corresponding to components of the complex were digested and analyzed by LC-MS/MS. Gel bands were identified with sequence coverages of 91% for BoNT/G, 91% for NTNH, 89% for HA-70, and 88% for HA-17. Notably, one gel band was also clearly identified as HA-33 with 93% sequence coverage. The BoNT/G complex consists of BoNT/G, NTNH, HA-70, HA-17, and HA-33. These proteins form the progenitor form of BoNT/G, similar to all other HA positive progenitor toxin complexes.
Label-Free Discovery Array Platform for the Characterization of Glycan Binding Proteins and Glycoproteins.

PubMed

Gray, Christopher J; Sánchez-Ruíz, Antonio; Šardzíková, Ivana; Ahmed, Yassir A; Miller, Rebecca L; Reyes Martinez, Juana E; Pallister, Edward; Huang, Kun; Both, Peter; Hartmann, Mirja; Roberts, Hannah N; Šardzík, Robert; Mandal, Santanu; Turnbull, Jerry E; Eyers, Claire E; Flitsch, Sabine L

2017-04-18

The identification of carbohydrate-protein interactions is central to our understanding of the roles of cell-surface carbohydrates (the glycocalyx), fundamental for cell-recognition events. Therefore, there is a need for fast high-throughput biochemical tools to capture the complexity of these biological interactions. Here, we describe a rapid method for qualitative label-free detection of carbohydrate-protein interactions on arrays of simple synthetic glycans, more complex natural glycosaminoglycans (GAG), and lectins/carbohydrate binding proteins using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The platform can unequivocally identify proteins that are captured from either purified or complex sample mixtures, including biofluids. Identification of proteins bound to the functionalized array is achieved by analyzing either the intact protein mass or, after on-chip proteolytic digestion, the peptide mass fingerprint and/or tandem mass spectrometry of selected peptides, which can yield highly diagnostic sequence information. The platform described here should be a valuable addition to the limited analytical toolbox that is currently available for glycomics.

PLIP: fully automated protein-ligand interaction profiler.

PubMed

Salentin, Sebastian; Schreiber, Sven; Haupt, V Joachim; Adasme, Melissa F; Schroeder, Michael

2015-07-01

The characterization of interactions in protein-ligand complexes is essential for research in structural bioinformatics, drug discovery and biology. However, comprehensive tools are not freely available to the research community. Here, we present the protein-ligand interaction profiler (PLIP), a novel web service for fully automated detection and visualization of relevant non-covalent protein-ligand contacts in 3D structures, freely available at projects.biotec.tu-dresden.de/plip-web. The input is either a Protein Data Bank structure, a protein or ligand name, or a custom protein-ligand complex (e.g. from docking). In contrast to other tools, the rule-based PLIP algorithm does not require any structure preparation. It returns a list of detected interactions on single atom level, covering seven interaction types (hydrogen bonds, hydrophobic contacts, pi-stacking, pi-cation interactions, salt bridges, water bridges and halogen bonds). PLIP stands out by offering publication-ready images, PyMOL session files to generate custom images and parsable result files to facilitate successive data processing. The full python source code is available for download on the website. PLIP's command-line mode allows for high-throughput interaction profiling. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
Definition and characterization of a "trypsinosome" from specific peptide characteristics by nano-HPLC-MS/MS and in silico analysis of complex protein mixtures.

PubMed

Le Bihan, Thierry; Robinson, Mark D; Stewart, Ian I; Figeys, Daniel

2004-01-01

Although HPLC-ESI-MS/MS is rapidly becoming an indispensable tool for the analysis of peptides in complex mixtures, the sequence coverage it affords is often quite poor. Low protein expression resulting in peptide signal intensities that fall below the limit of detection of the MS system in combination with differences in peptide ionization efficiency plays a significant role in this. A second important factor stems from differences in physicochemical properties of each peptide and how these properties relate to chromatographic retention and ultimate detection. To identify and understand those properties, we compared data from experimentally identified peptides with data from peptides predicted by in silico digest of all corresponding proteins in the experimental set. Three different complex protein mixtures extracted were used to define a training set to evaluate the amino acid retention coefficients based on linear regression analysis. The retention coefficients were also compared with other previous hydrophobic and retention scale. From this, we have constructed an empirical model that can be readily used to predict peptides that are likely to be observed on our HPLC-ESI-MS/MS system based on their physicochemical properties. Finally, we demonstrated that in silico prediction of peptides and their retention coefficients can be used to generate an inclusion list for a targeted mass spectrometric identification of low abundance proteins in complex protein samples. This approach is based on experimentally derived data to calibrate the method and therefore may theoretically be applied to any HPLC-MS/MS system on which data are being generated.
Variation of the chemical reactivity of Thermus thermophilus HB8 ribosomal proteins as a function of pH.

PubMed

Running, William E; Reilly, James P

2010-10-01

Ribosomes occupy a central position in cellular metabolism, converting stored genetic information into active cellular machinery. Ribosomal proteins modulate both the intrinsic function of the ribosome and its interaction with other cellular complexes, such as chaperonins or the signal recognition particle. Chemical modification of proteins combined with mass spectrometric detection of the extent and position of covalent modifications is a rapid, sensitive method for the study of protein structure and flexibility. By altering the pH of the solution, we have induced non-denaturing changes in the structure of bacterial ribosomal proteins and detected these conformational changes by covalent labeling. Changes in ribosomal protein modification across a pH range from 6.6 to 8.3 are unique to each protein, and correlate with their structural environment in the ribosome. Lysine residues whose extent of modification increases as a function of increasing pH are on the surface of proteins, but in close proximity either to glutamate and aspartate residues, or to rRNA backbone phosphates. Increasing pH disrupts tertiary and quaternary interactions mediated by hydrogen bonding or ionic interactions, and regions of protein structure whose conformations are sensitive to these changes are of potential importance in modulating the flexibility of the ribosome or its interaction with other cellular complexes.
Ultrastructural localisation of protein interactions using conditionally stable nanobodies.

PubMed

Ariotti, Nicholas; Rae, James; Giles, Nichole; Martel, Nick; Sierecki, Emma; Gambin, Yann; Hall, Thomas E; Parton, Robert G

2018-04-01

We describe the development and application of a suite of modular tools for high-resolution detection of proteins and intracellular protein complexes by electron microscopy (EM). Conditionally stable GFP- and mCherry-binding nanobodies (termed csGBP and csChBP, respectively) are characterized using a cell-free expression and analysis system and subsequently fused to an ascorbate peroxidase (APEX) enzyme. Expression of these cassettes alongside fluorescently labelled proteins results in recruitment and stabilisation of APEX, whereas unbound APEX nanobodies are efficiently degraded by the proteasome. This greatly simplifies correlative analyses, enables detection of less-abundant proteins, and eliminates the need to balance expression levels between fluorescently labelled and APEX nanobody proteins. Furthermore, we demonstrate the application of this system to bimolecular complementation ('EM split-fluorescent protein'), for localisation of protein-protein interactions at the ultrastructural level.
Alignment and integration of complex networks by hypergraph-based spectral clustering

NASA Astrophysics Data System (ADS)

Michoel, Tom; Nachtergaele, Bruno

2012-11-01

Complex networks possess a rich, multiscale structure reflecting the dynamical and functional organization of the systems they model. Often there is a need to analyze multiple networks simultaneously, to model a system by more than one type of interaction, or to go beyond simple pairwise interactions, but currently there is a lack of theoretical and computational methods to address these problems. Here we introduce a framework for clustering and community detection in such systems using hypergraph representations. Our main result is a generalization of the Perron-Frobenius theorem from which we derive spectral clustering algorithms for directed and undirected hypergraphs. We illustrate our approach with applications for local and global alignment of protein-protein interaction networks between multiple species, for tripartite community detection in folksonomies, and for detecting clusters of overlapping regulatory pathways in directed networks.
Alignment and integration of complex networks by hypergraph-based spectral clustering.

PubMed

Michoel, Tom; Nachtergaele, Bruno

2012-11-01

Complex networks possess a rich, multiscale structure reflecting the dynamical and functional organization of the systems they model. Often there is a need to analyze multiple networks simultaneously, to model a system by more than one type of interaction, or to go beyond simple pairwise interactions, but currently there is a lack of theoretical and computational methods to address these problems. Here we introduce a framework for clustering and community detection in such systems using hypergraph representations. Our main result is a generalization of the Perron-Frobenius theorem from which we derive spectral clustering algorithms for directed and undirected hypergraphs. We illustrate our approach with applications for local and global alignment of protein-protein interaction networks between multiple species, for tripartite community detection in folksonomies, and for detecting clusters of overlapping regulatory pathways in directed networks.
Elastin-like polypeptide switches: A design strategy to detect multimeric proteins.

PubMed

Dhandhukia, Jugal P; Brill, Dab A; Kouhi, Aida; Pastuszka, Martha K; MacKay, J Andrew

2017-09-01

Elastin-Like Polypeptides (ELPs) reversibly phase separate in response to changes in temperature, pressure, concentration, pH, and ionic species. While powerful triggers, biological microenvironments present a multitude of more specific biological cues, such as antibodies, cytokines, and cell-surface receptors. To develop better biosensors and bioresponsive drug carriers, rational strategies are required to sense and respond to these target proteins. We recently reported that noncovalent association of two ELP fusion proteins to a "chemical inducer of dimerization" small molecule (1.5 kDa) induces phase separation at physiological temperatures. Having detected a small molecule, here we present the first evidence that ELP multimerization can also detect a much larger (60 kDa) protein target. To demonstrate this strategy, ELPs were biotinylated at their amino terminus and mixed with tetrameric streptavidin. At a stoichiometric ratio of [4:1], two to three biotin-ELPs associate with streptavidin into multimeric complexes with an apparent K d of 5 nM. The increased ELP density around a streptavidin core strongly promotes isothermal phase separation, which was tuned to occur at physiological temperature. This phase separation reverses upon saturation with excess streptavidin, which only favors [1:1] complexes. Together, these findings suggest that ELP association with multimeric biomolecules is a viable strategy to deliberately engineer ELPs that respond to multimeric protein substrates. © 2017 The Protein Society.
Fluorescence correlation spectroscopy as a method for assessment of interactions between phage displaying antibodies and soluble antigen

PubMed Central

Lagerkvist, Ann Catrin; Földes-Papp, Zeno; Persson, Mats A.A.; Rigler, Rudolf

2001-01-01

Phage display is widely used for expression of combinatorial libraries, not least for protein engineering purposes. Precise selection at the single molecule level will provide an improved tool for generating proteins with complex and distinct properties from large molecular libraries. To establish such an improved selection system, we here report the detection of specific interactions between phage with displayed antibody fragments and fluorescently labeled soluble antigen based on Fluorescence Correlation Spectroscopy (FCS). Our novel strategy comprises the use of two separate fluorochromes for detection of the phage–antigen complex, either with labeled antiphage antibody or using a labeled antigen. As a model system, we studied a human monoclonal antibody to the hepatitis-C virus (HCV) envelope protein E2 and its cognate antigen (rE2 or rE1/E2). We could thus assess the specific interactions and determine the fraction of specific versus background phage (26% specific phage). Aggregation of these particular antigens made it difficult to reliably utilize the full potential of cross-correlation studies using the two labels simultaneously. However, with true monomeric proteins, this will certainly be possible, offering a great advantage in a safer and highly specific detection system. PMID:11468349
Triangle network motifs predict complexes by complementing high-error interactomes with structural information.

PubMed

Andreopoulos, Bill; Winter, Christof; Labudde, Dirk; Schroeder, Michael

2009-06-27

A lot of high-throughput studies produce protein-protein interaction networks (PPINs) with many errors and missing information. Even for genome-wide approaches, there is often a low overlap between PPINs produced by different studies. Second-level neighbors separated by two protein-protein interactions (PPIs) were previously used for predicting protein function and finding complexes in high-error PPINs. We retrieve second level neighbors in PPINs, and complement these with structural domain-domain interactions (SDDIs) representing binding evidence on proteins, forming PPI-SDDI-PPI triangles. We find low overlap between PPINs, SDDIs and known complexes, all well below 10%. We evaluate the overlap of PPI-SDDI-PPI triangles with known complexes from Munich Information center for Protein Sequences (MIPS). PPI-SDDI-PPI triangles have ~20 times higher overlap with MIPS complexes than using second-level neighbors in PPINs without SDDIs. The biological interpretation for triangles is that a SDDI causes two proteins to be observed with common interaction partners in high-throughput experiments. The relatively few SDDIs overlapping with PPINs are part of highly connected SDDI components, and are more likely to be detected in experimental studies. We demonstrate the utility of PPI-SDDI-PPI triangles by reconstructing myosin-actin processes in the nucleus, cytoplasm, and cytoskeleton, which were not obvious in the original PPIN. Using other complementary datatypes in place of SDDIs to form triangles, such as PubMed co-occurrences or threading information, results in a similar ability to find protein complexes. Given high-error PPINs with missing information, triangles of mixed datatypes are a promising direction for finding protein complexes. Integrating PPINs with SDDIs improves finding complexes. Structural SDDIs partially explain the high functional similarity of second-level neighbors in PPINs. We estimate that relatively little structural information would be sufficient for finding complexes involving most of the proteins and interactions in a typical PPIN.
Triangle network motifs predict complexes by complementing high-error interactomes with structural information

PubMed Central

Andreopoulos, Bill; Winter, Christof; Labudde, Dirk; Schroeder, Michael

2009-01-01

Background A lot of high-throughput studies produce protein-protein interaction networks (PPINs) with many errors and missing information. Even for genome-wide approaches, there is often a low overlap between PPINs produced by different studies. Second-level neighbors separated by two protein-protein interactions (PPIs) were previously used for predicting protein function and finding complexes in high-error PPINs. We retrieve second level neighbors in PPINs, and complement these with structural domain-domain interactions (SDDIs) representing binding evidence on proteins, forming PPI-SDDI-PPI triangles. Results We find low overlap between PPINs, SDDIs and known complexes, all well below 10%. We evaluate the overlap of PPI-SDDI-PPI triangles with known complexes from Munich Information center for Protein Sequences (MIPS). PPI-SDDI-PPI triangles have ~20 times higher overlap with MIPS complexes than using second-level neighbors in PPINs without SDDIs. The biological interpretation for triangles is that a SDDI causes two proteins to be observed with common interaction partners in high-throughput experiments. The relatively few SDDIs overlapping with PPINs are part of highly connected SDDI components, and are more likely to be detected in experimental studies. We demonstrate the utility of PPI-SDDI-PPI triangles by reconstructing myosin-actin processes in the nucleus, cytoplasm, and cytoskeleton, which were not obvious in the original PPIN. Using other complementary datatypes in place of SDDIs to form triangles, such as PubMed co-occurrences or threading information, results in a similar ability to find protein complexes. Conclusion Given high-error PPINs with missing information, triangles of mixed datatypes are a promising direction for finding protein complexes. Integrating PPINs with SDDIs improves finding complexes. Structural SDDIs partially explain the high functional similarity of second-level neighbors in PPINs. We estimate that relatively little structural information would be sufficient for finding complexes involving most of the proteins and interactions in a typical PPIN. PMID:19558694
The eighth fibronectin type III domain of protein tyrosine phosphatase receptor J influences the formation of protein complexes and cell localization.

PubMed

Iuliano, Rodolfo; Raso, Cinzia; Quintiero, Alfina; Pera, Ilaria Le; Pichiorri, Flavia; Palumbo, Tiziana; Palmieri, Dario; Pattarozzi, Alessandra; Florio, Tullio; Viglietto, Giuseppe; Trapasso, Francesco; Croce, Carlo Maria; Fusco, Alfredo

2009-03-01

Regulation of receptor-type phosphatases can involve the formation of higher-order structures, but the exact role played in this process by protein domains is not well understood. In this study we show the formation of different higher-order structures of the receptor-type phosphatase PTPRJ, detected in HEK293A cells transfected with different PTPRJ expression constructs. In the plasma membrane PTPRJ forms dimers detectable by treatment with the cross-linking reagent BS(3) (bis[sulfosuccinimidyl]suberate). However, other PTPRJ complexes, dependent on the formation of disulfide bonds, are detected by treatment with the oxidant agent H(2)O(2) or by a mutation Asp872Cys, located in the eighth fibronectin type III domain of PTPRJ. A deletion in the eighth fibronectin domain of PTPRJ impairs its dimerization in the plasma membrane and increases the formation of PTPRJ complexes dependent on disulfide bonds that remain trapped in the cytoplasm. The deletion mutant maintains the catalytic activity but is unable to carry out inhibition of proliferation on HeLa cells, achieved by the wild type form, since it does not reach the plasma membrane. Therefore, the intact structure of the eighth fibronectin domain of PTPRJ is critical for its localization in plasma membrane and biological function.
Detection and quantitation of single nucleotide polymorphisms, DNA sequence variations, DNA mutations, DNA damage and DNA mismatches

DOEpatents

McCutchen-Maloney, Sandra L.

2002-01-01

DNA mutation binding proteins alone and as chimeric proteins with nucleases are used with solid supports to detect DNA sequence variations, DNA mutations and single nucleotide polymorphisms. The solid supports may be flow cytometry beads, DNA chips, glass slides or DNA dips sticks. DNA molecules are coupled to solid supports to form DNA-support complexes. Labeled DNA is used with unlabeled DNA mutation binding proteins such at TthMutS to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by binding which gives an increase in signal. Unlabeled DNA is utilized with labeled chimeras to detect DNA sequence variations, DNA mutations and single nucleotide length polymorphisms by nuclease activity of the chimera which gives a decrease in signal.
Sensitive detection of C-reactive protein in serum by immunoprecipitation-microchip capillary gel electrophoresis.

PubMed

Herwig, Ela; Marchetti-Deschmann, Martina; Wenz, Christian; Rüfer, Andreas; Redl, Heinz; Bahrami, Soheyl; Allmaier, Günter

2015-06-01

Sepsis represents a significant cause of mortality in intensive care units. Early diagnosis of sepsis is essential to increase the survival rate of patients. Among others, C-reactive protein (CRP) is commonly used as a sepsis marker. In this work we introduce immune precipitation combined with microchip capillary gel electrophoresis (IP-MCGE) for the detection and quantification of CRP in serum samples. First high-abundance proteins (HSA, IgG) are removed from serum samples using affinity spin cartridges, and then the remaining proteins are labeled with a fluorescence dye and incubated with an anti-CRP antibody, and the antigen/antibody complex is precipitated with protein G-coated magnetic beads. After precipitation the complex is eluted from the beads and loaded onto the MCGE system. CRP could be reliably detected and quantified, with a detection limit of 25 ng/μl in serum samples and 126 pg/μl in matrix-free samples. The overall sensitivity (LOQ = 75 ng/μl, R(2) = 0.9668) of the method is lower than that of some specially developed methods (e.g., immune radiometric assay) but is comparable to those of clinically accepted ELISA methods. The straightforward sample preparation (not prone to mistakes), reduced sample and reagent volumes (including the antibodies), and high throughput (10 samples/3 h) are advantages and therefore IP-MCGE bears potential for point-of-care diagnosis. Copyright © 2015 Elsevier Inc. All rights reserved.
Detecting microRNAs of high influence on protein functional interaction networks: a prostate cancer case study

PubMed Central

2012-01-01

Background The use of biological molecular network information for diagnostic and prognostic purposes and elucidation of molecular disease mechanism is a key objective in systems biomedicine. The network of regulatory miRNA-target and functional protein interactions is a rich source of information to elucidate the function and the prognostic value of miRNAs in cancer. The objective of this study is to identify miRNAs that have high influence on target protein complexes in prostate cancer as a case study. This could provide biomarkers or therapeutic targets relevant for prostate cancer treatment. Results Our findings demonstrate that a miRNA’s functional role can be explained by its target protein connectivity within a physical and functional interaction network. To detect miRNAs with high influence on target protein modules, we integrated miRNA and mRNA expression profiles with a sequence based miRNA-target network and human functional and physical protein interactions (FPI). miRNAs with high influence on target protein complexes play a role in prostate cancer progression and are promising diagnostic or prognostic biomarkers. We uncovered several miRNA-regulated protein modules which were enriched in focal adhesion and prostate cancer genes. Several miRNAs such as miR-96, miR-182, and miR-143 demonstrated high influence on their target protein complexes and could explain most of the gene expression changes in our analyzed prostate cancer data set. Conclusions We describe a novel method to identify active miRNA-target modules relevant to prostate cancer progression and outcome. miRNAs with high influence on protein networks are valuable biomarkers that can be used in clinical investigations for prostate cancer treatment. PMID:22929553
Paramagnetic particles coupled with an automated flow injection analysis as a tool for influenza viral protein detection.

PubMed

Krejcova, Ludmila; Dospivova, Dana; Ryvolova, Marketa; Kopel, Pavel; Hynek, David; Krizkova, Sona; Hubalek, Jaromir; Adam, Vojtech; Kizek, Rene

2012-11-01

Currently, the influenza virus infects millions of individuals every year. Since the influenza virus represents one of the greatest threats, it is necessary to develop a diagnostic technique that can quickly, inexpensively, and accurately detect the virus to effectively treat and control seasonal and pandemic strains. This study presents an alternative to current detection methods. The flow-injection analysis-based biosensor, which can rapidly and economically analyze a wide panel of influenza virus strains by using paramagnetic particles modified with glycan, can selectively bind to specific viral A/H5N1/Vietnam/1203/2004 protein-labeled quantum dots. Optimized detection of cadmium sulfide quantum dots (CdS QDs)-protein complexes connected to paramagnetic microbeads was performed using differential pulse voltammetry on the surface of a hanging mercury drop electrode (HMDE) and/or glassy carbon electrode (GCE). Detection limit (3 S/N) estimations based on cadmium(II) ions quantification were 0.1 μg/mL or 10 μg/mL viral protein at HMDE or GCE, respectively. Viral protein detection was directly determined using differential pulse voltammetry Brdicka reaction. The limit detection (3 S/N) of viral protein was estimated as 0.1 μg/mL. Streptavidin-modified paramagnetic particles were mixed with biotinylated selective glycan to modify their surfaces. Under optimized conditions (250 μg/mL of glycan, 30-min long interaction with viral protein, 25°C and 400 rpm), the viral protein labeled with quantum dots was selectively isolated and its cadmium(II) content was determined. Cadmium was present in detectable amounts of 10 ng per mg of protein. Using this method, submicrogram concentrations of viral proteins can be identified. © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Fitting new technologies into the safety paradigm: use of microarrays in transfusion.

PubMed

Fournier-Wirth, C; Coste, J

2007-01-01

Until the late 1990s, mandatory blood screening for transmissible infectious agents depended entirely on antigen/antibody-based detection assays. The recent emergence of Nucleic acid Amplification Technologies (NAT) has revolutionised viral diagnosis, not only by increasing the level of sensitivity but also by facilitating the detection of several viruses in parallel by multiplexing specific primers. In more complex biological situations, when a broad spectrum of pathogens must be screened, the limitations of these first generation technologies became apparent. High throughput systems, such as DNA Arrays, permit a conceptually new approach. These miniaturised micro systems allow the detection of hundreds of different targets simultaneously, inducing a dramatic decrease in reagent consumption, a reduction in the number of confirmation tests and a simplification of data interpretation. However, the systems currently available require additional instrumentation and reagents for sample preparation and target amplification prior to detection on the DNA array. A major challenge in the area of DNA detection is the development of methods that do not rely on target amplification systems. Likewise, the advances of protein microarrays have lagged because of poor stability of proteins, complex coupling chemistry and weak detection signals. Emerging technologies like Biosensors and nano-particle based DNA or Protein Bio-Barcode Amplification Assays are promising diagnostic tools for a wide range of clinical applications, including blood donation screening.
Western blotting using chemiluminescent substrates.

PubMed

Alegria-Schaffer, Alice

2014-01-01

Western blotting is a powerful and commonly used tool to identify and quantify a specific protein in a complex mixture (Towbin et al., 1979). The technique enables indirect detection of protein samples immobilized on a nitrocellulose or polyvinylidene fluoride (PVDF) membrane. Copyright © 2014 Elsevier Inc. All rights reserved.
Recruitment of Fanconi Anemia and Breast Cancer Proteins to DNA Damage Sites is differentially Governed by Replication

PubMed Central

Shen, Xi; Do, Huong; Li, Yongjian; Chung, Woo-Hyun; Tomasz, Maria; de Winter, Johan P.; Xia, Bing; Elledge, Stephen J.; Wang, Weidong; Li, Lei

2009-01-01

Summary Fanconi anemia (FA) is characterized by cellular hypersensivity to DNA crosslinking agents, but how the Fanconi pathway protects cells from DNA crosslinks and whether FA proteins act directly on crosslinks remains unclear. We developed a chromatin-IP-based strategy termed eChIP and detected association of multiple FA proteins with DNA crosslinks in vivo. Inter-dependence analyses revealed that crosslink-specific enrichment of various FA proteins is controlled by distinct mechanisms. BRCA-related FA proteins (BRCA2, FANCJ/BACH1, and FANCN/PALB2), but not FA core and I/D2 complexes, require replication for their crosslink association. FANCD2, but not FANCJ and FANCN, requires the FA core complex for its recruitment. FA core complex requires nucleotide excision repair proteins XPA and XPC for its association. Consistent with the distinct recruitment mechanism, recombination-independent crosslink repair was inversely affected in cells deficient of FANC-core versus BRCA-related FA proteins. Thus, FA proteins participate in distinct DNA damage response mechanisms governed by DNA replication status. PMID:19748364
Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells.

PubMed

Kerppola, Tom K

2006-01-01

Bimolecular fluorescence complementation (BiFC) analysis enables direct visualization of protein interactions in living cells. The BiFC assay is based on the discoveries that two non-fluorescent fragments of a fluorescent protein can form a fluorescent complex and that the association of the fragments can be facilitated when they are fused to two proteins that interact with each other. BiFC must be confirmed by parallel analysis of proteins in which the interaction interface has been mutated. It is not necessary for the interaction partners to juxtapose the fragments within a specific distance of each other because they can associate when they are tethered to a complex with flexible linkers. It is also not necessary for the interaction partners to form a complex with a long half-life or a high occupancy since the fragments can associate in a transient complex and un-associated fusion proteins do not interfere with detection of the complex. Many interactions can be visualized when the fusion proteins are expressed at levels comparable to their endogenous counterparts. The BiFC assay has been used for the visualization of interactions between many types of proteins in different subcellular locations and in different cell types and organisms. It is technically straightforward and can be performed using a regular fluorescence microscope and standard molecular biology and cell culture reagents.
Experimental Methods for Protein Interaction Identification and Characterization

NASA Astrophysics Data System (ADS)

Uetz, Peter; Titz, Björn; Cagney, Gerard

There are dozens of methods for the detection of protein-protein interactions but they fall into a few broad categories. Fragment complementation assays such as the yeast two-hybrid (Y2H) system are based on split proteins that are functionally reconstituted by fusions of interacting proteins. Biophysical methods include structure determination and mass spectrometric (MS) identification of proteins in complexes. Biochemical methods include methods such as far western blotting and peptide arrays. Only the Y2H and protein complex purification combined with MS have been used on a larger scale. Due to the lack of data it is still difficult to compare these methods with respect to their efficiency and error rates. Current data does not favor any particular method and thus multiple experimental approaches are necessary to maximally cover the interactome of any target cell or organism.

Protein-protein interaction predictions using text mining methods.

PubMed

Papanikolaou, Nikolas; Pavlopoulos, Georgios A; Theodosiou, Theodosios; Iliopoulos, Ioannis

2015-03-01

It is beyond any doubt that proteins and their interactions play an essential role in most complex biological processes. The understanding of their function individually, but also in the form of protein complexes is of a great importance. Nowadays, despite the plethora of various high-throughput experimental approaches for detecting protein-protein interactions, many computational methods aiming to predict new interactions have appeared and gained interest. In this review, we focus on text-mining based computational methodologies, aiming to extract information for proteins and their interactions from public repositories such as literature and various biological databases. We discuss their strengths, their weaknesses and how they complement existing experimental techniques by simultaneously commenting on the biological databases which hold such information and the benchmark datasets that can be used for evaluating new tools. Copyright © 2014 Elsevier Inc. All rights reserved.
A Simple Method for Rapid Depletion of Rubisco from Soybean (Glycine max) Leaf for Proteomic Analysis of Lower Abundance Proteins

USDA-ARS?s Scientific Manuscript database

2-DE analysis of complex plant proteomes has limited dynamic resolution because only abundant proteins can be detected. Proteomic assessment of the low abundance proteins within leaf tissue is difficult when it is comprised of 30 – 50% of the CO2 fixation enzyme Rubisco. Resolution can be improved t...
Detection of Mycobacterium tuberculosis peptides in the exosomes of patients with active and latent M. tuberculosis infection using MRM-MS.

PubMed

Kruh-Garcia, Nicole A; Wolfe, Lisa M; Chaisson, Lelia H; Worodria, William O; Nahid, Payam; Schorey, Jeff S; Davis, J Lucian; Dobos, Karen M

2014-01-01

The identification of easily measured, accurate diagnostic biomarkers for active tuberculosis (TB) will have a significant impact on global TB control efforts. Because of the host and pathogen complexities involved in TB pathogenesis, identifying a single biomarker that is adequately sensitive and specific continues to be a major hurdle. Our previous studies in models of TB demonstrated that exosomes, such as those released from infected macrophages, contain mycobacterial products, including many Mtb proteins. In this report, we describe the development of targeted proteomics assays employing multiplexed multiple reaction monitoring mass spectrometry (MRM-MS) in order to allow us to follow those proteins previously identified by western blot or shotgun mass spectrometry, and enhance biomarker discovery to include detection of Mtb proteins in human serum exosomes. Targeted MRM-MS assays were applied to exosomes isolated from human serum samples obtained from culture-confirmed active TB patients to detect 76 peptides representing 33 unique Mtb proteins. Our studies revealed the first identification of bacteria-derived biomarker candidates of active TB in exosomes from human serum. Twenty of the 33 proteins targeted for detection were found in the exosomes of TB patients, and included multiple peptides from 8 proteins (Antigen 85B, Antigen 85C, Apa, BfrB, GlcB, HspX, KatG, and Mpt64). Interestingly, all of these proteins are known mycobacterial adhesins and/or proteins that contribute to the intracellular survival of Mtb. These proteins will be included as target analytes in future validation studies as they may serve as markers for persistent active and latent Mtb infection. In summary, this work is the first step in identifying a unique and specific panel of Mtb peptide biomarkers encapsulated in exosomes and reveals complex biomarker patterns across a spectrum of TB disease states.
Detection of Mycobacterium tuberculosis Peptides in the Exosomes of Patients with Active and Latent M. tuberculosis Infection Using MRM-MS

PubMed Central

Kruh-Garcia, Nicole A.; Wolfe, Lisa M.; Chaisson, Lelia H.; Worodria, William O.; Nahid, Payam; Schorey, Jeff S.; Davis, J. Lucian; Dobos, Karen M.

2014-01-01

The identification of easily measured, accurate diagnostic biomarkers for active tuberculosis (TB) will have a significant impact on global TB control efforts. Because of the host and pathogen complexities involved in TB pathogenesis, identifying a single biomarker that is adequately sensitive and specific continues to be a major hurdle. Our previous studies in models of TB demonstrated that exosomes, such as those released from infected macrophages, contain mycobacterial products, including many Mtb proteins. In this report, we describe the development of targeted proteomics assays employing multiplexed multiple reaction monitoring mass spectrometry (MRM-MS) in order to allow us to follow those proteins previously identified by western blot or shotgun mass spectrometry, and enhance biomarker discovery to include detection of Mtb proteins in human serum exosomes. Targeted MRM-MS assays were applied to exosomes isolated from human serum samples obtained from culture-confirmed active TB patients to detect 76 peptides representing 33 unique Mtb proteins. Our studies revealed the first identification of bacteria-derived biomarker candidates of active TB in exosomes from human serum. Twenty of the 33 proteins targeted for detection were found in the exosomes of TB patients, and included multiple peptides from 8 proteins (Antigen 85B, Antigen 85C, Apa, BfrB, GlcB, HspX, KatG, and Mpt64). Interestingly, all of these proteins are known mycobacterial adhesins and/or proteins that contribute to the intracellular survival of Mtb. These proteins will be included as target analytes in future validation studies as they may serve as markers for persistent active and latent Mtb infection. In summary, this work is the first step in identifying a unique and specific panel of Mtb peptide biomarkers encapsulated in exosomes and reveals complex biomarker patterns across a spectrum of TB disease states. PMID:25080351
Ultrastructural localisation of protein interactions using conditionally stable nanobodies

PubMed Central

Ariotti, Nicholas; Rae, James; Giles, Nichole; Martel, Nick; Sierecki, Emma; Gambin, Yann; Parton, Robert G.

2018-01-01

We describe the development and application of a suite of modular tools for high-resolution detection of proteins and intracellular protein complexes by electron microscopy (EM). Conditionally stable GFP- and mCherry-binding nanobodies (termed csGBP and csChBP, respectively) are characterized using a cell-free expression and analysis system and subsequently fused to an ascorbate peroxidase (APEX) enzyme. Expression of these cassettes alongside fluorescently labelled proteins results in recruitment and stabilisation of APEX, whereas unbound APEX nanobodies are efficiently degraded by the proteasome. This greatly simplifies correlative analyses, enables detection of less-abundant proteins, and eliminates the need to balance expression levels between fluorescently labelled and APEX nanobody proteins. Furthermore, we demonstrate the application of this system to bimolecular complementation (‘EM split-fluorescent protein’), for localisation of protein–protein interactions at the ultrastructural level. PMID:29621251
The putative Agrobacterium transcriptional activator-like virulence protein VirD5 may target T-complex to prevent the degradation of coat proteins in the plant cell nucleus.

PubMed

Wang, Yafei; Peng, Wei; Zhou, Xu; Huang, Fei; Shao, Lingyun; Luo, Meizhong

2014-09-01

Agrobacterium exports at least five virulence proteins (VirE2, VirE3, VirF, VirD2, VirD5) into host cells and hijacks some host plant factors to facilitate its transformation process. Random DNA binding selection assays (RDSAs), electrophoretic mobility shift assays (EMSAs) and yeast one-hybrid systems were used to identify protein-bound DNA elements. Bimolecular fluorescence complementation, glutathione S-transferase pull-down and yeast two-hybrid assays were used to detect protein interactions. Protoplast transformation, coprecipitation, competitive binding and cell-free degradation assays were used to analyze the relationships among proteins. We found that Agrobacterium VirD5 exhibits transcriptional activation activity in yeast, is located in the plant cell nucleus, and forms homodimers. A specific VirD5-bound DNA element designated D5RE (VirD5 response element) was identified. VirD5 interacted directly with Arabidopsis VirE2 Interacting Protein 1 (AtVIP1). However, the ternary complex of VirD5-AtVIP1-VirE2 could be detected, whereas that of VirD5-AtVIP1-VBF (AtVIP1 Binding F-box protein) could not. We demonstrated that VirD5 competes with VBF for binding to AtVIP1 and stabilizes AtVIP1 and VirE2 in the cell-free degradation system. Our results indicated that VirD5 may act as both a transcriptional activator-like effector to regulate host gene expression and a protector preventing the coat proteins of the T-complex from being quickly degraded by the host's ubiquitin proteasome system (UPS). © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.
BindML/BindML+: Detecting Protein-Protein Interaction Interface Propensity from Amino Acid Substitution Patterns.

PubMed

Wei, Qing; La, David; Kihara, Daisuke

2017-01-01

Prediction of protein-protein interaction sites in a protein structure provides important information for elucidating the mechanism of protein function and can also be useful in guiding a modeling or design procedures of protein complex structures. Since prediction methods essentially assess the propensity of amino acids that are likely to be part of a protein docking interface, they can help in designing protein-protein interactions. Here, we introduce BindML and BindML+ protein-protein interaction sites prediction methods. BindML predicts protein-protein interaction sites by identifying mutation patterns found in known protein-protein complexes using phylogenetic substitution models. BindML+ is an extension of BindML for distinguishing permanent and transient types of protein-protein interaction sites. We developed an interactive web-server that provides a convenient interface to assist in structural visualization of protein-protein interactions site predictions. The input data for the web-server are a tertiary structure of interest. BindML and BindML+ are available at http://kiharalab.org/bindml/ and http://kiharalab.org/bindml/plus/ .
Functioning of the Drosophila Wilms'-Tumor-1-Associated Protein Homolog, Fl(2)d, in Sex-Lethal-Dependent Alternative Splicing

PubMed Central

Penn, Jill K. M.; Graham, Patricia; Deshpande, Girish; Calhoun, Gretchen; Chaouki, Ahmad Sami; Salz, Helen K.; Schedl, Paul

2008-01-01

fl(2)d, the Drosophila homolog of Wilms'-tumor-1-associated protein (WTAP), regulates the alternative splicing of Sex-lethal (Sxl), transformer (tra), and Ultrabithorax (Ubx). Although WTAP has been found in functional human spliceosomes, exactly how it contributes to the splicing process remains unknown. Here we attempt to identify factors that interact genetically and physically with fl(2)d. We begin by analyzing the Sxl-Fl(2)d protein–protein interaction in detail and present evidence suggesting that the female-specific fl(2)d1 allele is antimorphic with respect to the process of sex determination. Next we show that fl(2)d interacts genetically with early acting general splicing regulators and that Fl(2)d is present in immunoprecipitable complexes with Snf, U2AF50, U2AF38, and U1-70K. By contrast, we could not detect Fl(2)d complexes containing the U5 snRNP protein U5-40K or with a protein that associates with the activated B spliceosomal complex SKIP. Significantly, the genetic and molecular interactions observed for Sxl are quite similar to those detected for fl(2)d. Taken together, our findings suggest that Sxl and fl(2)d function to alter splice-site selection at an early step in spliceosome assembly. PMID:18245840
Measuring Protein Concentration on Nitrocellulose and After the Electrophoretic Transfer of Protein to Nitrocellulose.

PubMed

Goldring, J P Dean

2015-01-01

Proteins bind to nitrocellulose membranes when applied directly or after electrophoretic transfer from polyacrylamide electrophoresis gels. Proteins can be stained for visualization with organic dyes Ponceau S, amido black, Coomassie Blue, and colloidal silver/gold and the intensity of the stain is directly proportional to the amount of protein present. Chemicals that interfere with dye/protein interactions in solution can be removed by washing the nitrocellulose after protein application. A method is described whereby protein-dye complexes attached to the nitrocellulose can be solubilized, dissolving the nitrocellulose and releasing dye into solution for detection by a spectrophotometer. The concentration of the dyes Ponceau S, amido black, and colloidal silver is proportional to the concentration of protein. Proteins transferred electrophoretically from SDS-PAGE, isoelectric focusing, or 2D gels to nitrocellulose can be stained with amido black, protein bands excised, and the bound dye detected in a spectrophotometer to quantify proteins in the individual protein bands.
Western Blot of Stained Proteins from Dried Polyacrylamide Gels

NASA Technical Reports Server (NTRS)

Gruber, Claudia; Stan-Lotter, Helga

1996-01-01

Western blotting of proteins is customarily performed following their separation on polyacrylamide gels, either prior to staining (1) or, as recently reported, following staining (2). We describe here Western blotting with stained gels, which had been dried and some of which had been stored for years. This procedure permits immunological analysis of proteins, to which antisera may have become available only later, or where the application of newly developed sensitive detection methods is desired. Once rehydration of the gels is achieved, proteins can be-transferred to blotting membranes by any appropriate protocol. Proteins stained with Coomassie Blue have to be detected with a non-chromogenic method, such as the film-based enhanced chemiluminescence (ECL)2) procedure (3). Silver stained proteins, which transfer in the colorless form, may be visualized by any detection method, although, because of the usually very low amounts of proteins, detection by ECL is preferable. Blotting of stained proteins from rehydrated gels is as rapid and as quantitative as from freshly prepared gels, in contrast to blotting from wet stained gels, which requires extensive washing and results in low transfer efficiency (2). Together with a photographic record of the gel pattern, unambiguous identification of immunoreactive proteins from complex mixtures is possible. Some further applications of this work are discussed.
Protein markers for identification of Yersinia pestis and their variation related to culture

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wunschel, David S.; Engelmann, Heather E.; Victry, Kristin D.

2013-12-11

The detection of high consequence pathogens, such as Yersinia pestis, is well established in biodefense laboratories for bioterror situations. Laboratory protocols are well established using specified culture media and a growth temperature of 37 °C for expression of specific antigens. Direct detection of Y. pestis protein markers, without prior culture, depends on their expression. Unfortunately protein expression can be impacted by the culture medium which cannot be predicted ahead of time. Furthermore, higher biomass yields are obtained at the optimal growth temperature (i.e. 28 °C–30 °C) and therefore are more likely to be used for bulk production. Analysis of Y.more » pestis grown on several types of media at 30 °C showed that several protein markers were found to be differentially detected in different media. Analysis of the identified proteins against a comprehensive database provided an additional level of organism identification. Peptides corresponding to variable regions of some proteins could separate large groups of strains and aid in organism identification. This work illustrates the need to understand variability of protein expression for detection targets. The potential for relating expression changes of known proteins to specific media factors, even in nutrient rich and chemically complex culture medium, may provide the opportunity to draw forensic information from protein profiles.« less
Optical switches for remote and noninvasive control of cell signaling.

PubMed

Gorostiza, Pau; Isacoff, Ehud Y

2008-10-17

Although the identity and interactions of signaling proteins have been studied in great detail, the complexity of signaling networks cannot be fully understood without elucidating the timing and location of activity of individual proteins. To do this, one needs a means for detecting and controlling specific signaling events. An attractive approach is to use light, both to report on and control signaling proteins in cells, because light can probe cells in real time with minimal damage. Although optical detection of signaling events has been successful for some time, the development of the means for optical control has accelerated only recently. Of particular interest is the development of chemically engineered proteins that are directly sensitive to light.
Western Blotting of the Endocannabinoid System.

PubMed

Wager-Miller, Jim; Mackie, Ken

2016-01-01

Measuring expression levels of G protein-coupled receptors (GPCRs) is an important step for understanding the distribution, function, and regulation of these receptors. A common approach for detecting proteins from complex biological systems is Western blotting. In this chapter, we describe a general approach to Western blotting protein components of the endocannabinoid system using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and nitrocellulose membranes, with a focus on detecting type 1 cannabinoid (CB1) receptors. When this technique is carefully used, specifically with validation of the primary antibodies, it can provide quantitative information on protein expression levels. Additional information can also be inferred from Western blotting such as potential posttranslational modifications that can be further evaluated by specific analytical techniques.
Evaluation of a novel ultra-sensitive nanoparticle probe-based assay for ricin detection.

PubMed

Yin, Hui-qiong; Jia, Min-xian; Shi, Li-jun; Liu, Jun; Wang, Rui; Lv, Mao-min; Ma, Yu-yuan; Zhao, Xiong; Zhang, Jin-gang

2014-01-01

A gold nanoparticle (GNP) probe-based assay (GNPA) modified from the bio-barcode assay (BCA) was developed for ultrasensitive detection of ricin, a potential biothreat agent. In the GNPA, a chain of ricin was captured by a GNP probe coated with polyclonal antibodies and single-stranded signal DNA. A magnetic microparticle (MMP) probe coated with ricin A chain monoclonal antibody was then added to form an immuno-complex. After being magnetically separated, the immuno-complex containing the single-stranded signal DNA was characterized by PCR and real-time PCR. A detection limit of 10(-2) fg/ml was determined for the ricin A chain; this is eight orders of magnitude more sensitive than that achieved with an ELISA and two orders more sensitive than that obtained with the BCA. The coefficients of variation (CV) of the intra- and inter-assay values ranged from 3.82-6.46%. The results here show that this novel assay is an ultrasensitive method for detection of ricin proteins and may be suitable for the ultrasensitive detection of other proteins.
Electrochemically mediated polymerization for highly sensitive detection of protein kinase activity.

PubMed

Hu, Qiong; Wang, Qiangwei; Jiang, Cuihua; Zhang, Jian; Kong, Jinming; Zhang, Xueji

2018-07-01

Protein kinases play a pivotal role in cellular regulation and signal transduction, the detection of protein kinase activity and inhibition is therefore of great importance to clinical diagnosis and drug discovery. In this work, a novel electrochemical platform using the electrochemically mediated polymerization as an efficient and cost-effective signal amplification strategy is described for the highly sensitive detection of protein kinase activity. This platform involves 1) the phosphorylation of substrate peptide by protein kinase, 2) the attachment of alkyl halide to the phosphorylated sites via the carboxylate-Zr 4+ -phosphate chemistry, and 3) the in situ grafting of electroactive polymers from the phosphorylated sites through the electrochemically mediated atom transfer radical polymerization (eATRP) at a negative potential, in the presence of the surface-attached alkyl halide as the initiator and the electroactive tag-conjugated acrylate as the monomer, respectively. Due to the electrochemically mediated polymerization, a large number of electroactive tags can be linked to each phosphorylated site, thereby greatly improving the detection sensitivity. This platform has been successfully applied to detect the activity of cAMP-dependent protein kinase (PKA) with a detection limit down to 1.63 mU mL -1 . Results also demonstrate that it is highly selective and can be used for the screening of protein kinase inhibitors. The potential application of our platform for protein kinase activity detection in complex biological samples has been further verified using normal human serum and HepG2 cell lysate. Moreover, our platform is operationally simple, highly efficient and cost-effective, thus holding great potential in protein kinase detection and inhibitor screening. Copyright © 2018 Elsevier B.V. All rights reserved.
Mass amplifying probe for sensitive fluorescence anisotropy detection of small molecules in complex biological samples.

PubMed

Cui, Liang; Zou, Yuan; Lin, Ninghang; Zhu, Zhi; Jenkins, Gareth; Yang, Chaoyong James

2012-07-03

Fluorescence anisotropy (FA) is a reliable and excellent choice for fluorescence sensing. One of the key factors influencing the FA value for any molecule is the molar mass of the molecule being measured. As a result, the FA method with functional nucleic acid aptamers has been limited to macromolecules such as proteins and is generally not applicable for the analysis of small molecules because their molecular masses are relatively too small to produce observable FA value changes. We report here a molecular mass amplifying strategy to construct anisotropy aptamer probes for small molecules. The probe is designed in such a way that only when a target molecule binds to the probe does it activate its binding ability to an anisotropy amplifier (a high molecular mass molecule such as protein), thus significantly increasing the molecular mass and FA value of the probe/target complex. Specifically, a mass amplifying probe (MAP) consists of a targeting aptamer domain against a target molecule and molecular mass amplifying aptamer domain for the amplifier protein. The probe is initially rendered inactive by a small blocking strand partially complementary to both target aptamer and amplifier protein aptamer so that the mass amplifying aptamer domain would not bind to the amplifier protein unless the probe has been activated by the target. In this way, we prepared two probes that constitute a target (ATP and cocaine respectively) aptamer, a thrombin (as the mass amplifier) aptamer, and a fluorophore. Both probes worked well against their corresponding small molecule targets, and the detection limits for ATP and cocaine were 0.5 μM and 0.8 μM, respectively. More importantly, because FA is less affected by environmental interferences, ATP in cell media and cocaine in urine were directly detected without any tedious sample pretreatment. Our results established that our molecular mass amplifying strategy can be used to design aptamer probes for rapid, sensitive, and selective detection of small molecules by means of FA in complex biological samples.
A conservation and biophysics guided stochastic approach to refining docked multimeric proteins.

PubMed

Akbal-Delibas, Bahar; Haspel, Nurit

2013-01-01

We introduce a protein docking refinement method that accepts complexes consisting of any number of monomeric units. The method uses a scoring function based on a tight coupling between evolutionary conservation, geometry and physico-chemical interactions. Understanding the role of protein complexes in the basic biology of organisms heavily relies on the detection of protein complexes and their structures. Different computational docking methods are developed for this purpose, however, these methods are often not accurate and their results need to be further refined to improve the geometry and the energy of the resulting complexes. Also, despite the fact that complexes in nature often have more than two monomers, most docking methods focus on dimers since the computational complexity increases exponentially due to the addition of monomeric units. Our results show that the refinement scheme can efficiently handle complexes with more than two monomers by biasing the results towards complexes with native interactions, filtering out false positive results. Our refined complexes have better IRMSDs with respect to the known complexes and lower energies than those initial docked structures. Evolutionary conservation information allows us to bias our results towards possible functional interfaces, and the probabilistic selection scheme helps us to escape local energy minima. We aim to incorporate our refinement method in a larger framework which also enables docking of multimeric complexes given only monomeric structures.
Optimization of Formaldehyde Cross-Linking for Protein Interaction Analysis of Non-Tagged Integrin β1

PubMed Central

Klockenbusch, Cordula; Kast, Juergen

2010-01-01

Formaldehyde cross-linking of protein complexes combined with immunoprecipitation and mass spectrometry analysis is a promising technique for analysing protein-protein interactions, including those of transient nature. Here we used integrin β1 as a model to describe the application of formaldehyde cross-linking in detail, particularly focusing on the optimal parameters for cross-linking, the detection of formaldehyde cross-linked complexes, the utility of antibodies, and the identification of binding partners. Integrin β1 was found in a high molecular weight complex after formaldehyde cross-linking. Eight different anti-integrin β1 antibodies were used for pull-down experiments and no loss in precipitation efficiency after cross-linking was observed. However, two of the antibodies could not precipitate the complex, probably due to hidden epitopes. Formaldehyde cross-linked complexes, precipitated from Jurkat cells or human platelets and analyzed by mass spectrometry, were found to be composed of integrin β1, α4 and α6 or β1, α6, α2, and α5, respectively. PMID:20634879
In Planta Single-Molecule Pull-Down Reveals Tetrameric Stoichiometry of HD-ZIPIII:LITTLE ZIPPER Complexes.

PubMed

Husbands, Aman Y; Aggarwal, Vasudha; Ha, Taekjip; Timmermans, Marja C P

2016-08-01

Deciphering complex biological processes markedly benefits from approaches that directly assess the underlying biomolecular interactions. Most commonly used approaches to monitor protein-protein interactions typically provide nonquantitative readouts that lack statistical power and do not yield information on the heterogeneity or stoichiometry of protein complexes. Single-molecule pull-down (SiMPull) uses single-molecule fluorescence detection to mitigate these disadvantages and can quantitatively interrogate interactions between proteins and other compounds, such as nucleic acids, small molecule ligands, and lipids. Here, we establish SiMPull in plants using the HOMEODOMAIN LEUCINE ZIPPER III (HD-ZIPIII) and LITTLE ZIPPER (ZPR) interaction as proof-of-principle. Colocalization analysis of fluorophore-tagged HD-ZIPIII and ZPR proteins provides strong statistical evidence of complex formation. In addition, we use SiMPull to directly quantify YFP and mCherry maturation probabilities, showing these differ substantially from values obtained in mammalian systems. Leveraging these probabilities, in conjunction with fluorophore photobleaching assays on over 2000 individual complexes, we determined HD-ZIPIII:ZPR stoichiometry. Intriguingly, these complexes appear as heterotetramers, comprising two HD-ZIPIII and two ZPR molecules, rather than heterodimers as described in the current model. This surprising result raises new questions about the regulation of these key developmental factors and is illustrative of the unique contribution SiMPull is poised to make to in planta protein interaction studies. © 2016 American Society of Plant Biologists. All rights reserved.
Molecular Basis for Structural Heterogeneity of an Intrinsically Disordered Protein Bound to a Partner by Combined ESI-IM-MS and Modeling

NASA Astrophysics Data System (ADS)

D'Urzo, Annalisa; Konijnenberg, Albert; Rossetti, Giulia; Habchi, Johnny; Li, Jinyu; Carloni, Paolo; Sobott, Frank; Longhi, Sonia; Grandori, Rita

2015-03-01

Intrinsically disordered proteins (IDPs) form biologically active complexes that can retain a high degree of conformational disorder, escaping structural characterization by conventional approaches. An example is offered by the complex between the intrinsically disordered NTAIL domain and the phosphoprotein X domain (PXD) from measles virus (MeV). Here, distinct conformers of the complex are detected by electrospray ionization-mass spectrometry (ESI-MS) and ion mobility (IM) techniques yielding estimates for the solvent-accessible surface area (SASA) in solution and the average collision cross-section (CCS) in the gas phase. Computational modeling of the complex in solution, based on experimental constraints, provides atomic-resolution structural models featuring different levels of compactness. The resulting models indicate high structural heterogeneity. The intermolecular interactions are predominantly hydrophobic, not only in the ordered core of the complex, but also in the dynamic, disordered regions. Electrostatic interactions become involved in the more compact states. This system represents an illustrative example of a hydrophobic complex that could be directly detected in the gas phase by native mass spectrometry. This work represents the first attempt to modeling the entire NTAIL domain bound to PXD at atomic resolution.

Cranberry Proanthocyanidins - Protein complexes for macrophage activation.

PubMed

Carballo, Sergio M; Haas, Linda; Krueger, Christian G; Reed, Jess D

2017-09-20

In this work we characterize the interaction of cranberry (Vaccinium macrocarpon) proanthocyanidins (PAC) with bovine serum albumin (BSA) and hen egg-white lysozyme (HEL) and determine the effects of these complexes on macrophage activation and antigen presentation. We isolated PAC from cranberry and complexed the isolated PAC with BSA and HEL. The properties of the PAC-protein complexes were studied by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), gel electrophoresis and zeta-potential. The effects of PAC-BSA complexes on macrophage activation were studied in RAW 264.7 macrophage like cells after treatment with lipopolysaccharide (LPS). Fluorescence microscopy was used to study the endocytosis of PAC-BSA complexes. The effects of the PAC complexes on macrophage antigen presentation were studied in an in vitro model of HEL antigen presentation by mouse peritoneal mononuclear cells to a T-cell hybridoma. The mass spectra of the PAC complexes with BSA and HEL differed from the spectra of the proteins alone by the presence of broad shoulders on the singly and doubly charged protein peaks. Complexation with PAC altered the electrophoretic mobility shift assay in native agarose gel and the electrophoretic mobility (ζ-potential) values. These results indicate that the PAC-protein complexes are stable and alter the protein structure without precipitating the protein. Fluorescence microscopy showed that the RAW 264.7 macrophages endocytosed BSA and PAC-BSA complexes in discrete vesicles that surrounded the nucleus. Macrophages treated with increasing amounts of PAC-BSA complexes had significantly reduced COX-2 and iNOS expression in response to treatment with lipopolysaccharide (LPS) in comparison to the controls. The PAC-HEL complexes modulated antigen uptake, processing and presentation in murine peritoneal macrophages. After 4 h of pre-incubation, only trace amounts of IL-2 were detected in the co-cultures treated with HEL alone, whereas the PAC-HEL complex had already reached the maximum IL-2 expression. Cranberry PAC may increase the rate of endocytosis of HEL and subsequent expression of IL-2 by the T-cell hybridomas. These results suggest that PAC-protein complexes modulate aspects of innate and acquired immune responses in macrophages.
Site-specific quantitative analysis of cardiac mitochondrial protein phosphorylation.

PubMed

Lam, Maggie P Y; Lau, Edward; Scruggs, Sarah B; Wang, Ding; Kim, Tae-Young; Liem, David A; Zhang, Jun; Ryan, Christopher M; Faull, Kym F; Ping, Peipei

2013-04-09

We report the development of a multiple-reaction monitoring (MRM) strategy specifically tailored to the detection and quantification of mitochondrial protein phosphorylation. We recently derived 68 MRM transitions specific to protein modifications in the respiratory chain, voltage-dependent anion channel, and adenine nucleotide translocase. Here, we have now expanded the total number of MRM transitions to 176 to cover proteins from the tricarboxylic acid cycle, pyruvate dehydrogenase complex, and branched-chain alpha-keto acid dehydrogenase complex. We utilized the transition set to analyze endogenous protein phosphorylation in human heart, mouse heart, and mouse liver. The data demonstrate the potential utility of the MRM workflow for studying the functional details of mitochondrial phosphorylation signaling. This article is part of a Special Issue entitled: From protein structures to clinical applications. Copyright © 2012 Elsevier B.V. All rights reserved.
Testing of gastric contents for peanut proteins in a 13-year old anaphylaxis victim.

PubMed

Beavers, Charles; Stauble, M Elaine; Jortani, Saeed A

2014-02-15

We report the case of a 13-y female who went into anaphylactic shock following the ingestion of a meal suspected to be contaminated by peanuts. The teenager had a known sensitivity to peanuts, however, the restaurant claimed that no peanut products were used in the preparation of her meal. The gastric contents of the decedent were retained and tested for peanut proteins due to the possible legal liability of the proprietor. Using antibodies against peanut proteins (roasted and unroasted), we optimized a method to detect total soluble peanut proteins by Western-blot analysis in gastric contents. In addition, we validated two commercially available tests which were originally intended for detection of peanut proteins in food matrices to examine the same gastric sample. One was an enzyme-linked immunosorbent assay (ELISA) that utilized polyclonal antibodies against Ara h 1 (Tepnel Life Sciences). The other was a laminar-flow assay directed against Ara h 1, Ara h 2 and Ara h 3 (R-Biopharm). A positive food-based control was created by reducing bread and peanuts (1:1, w/w) with water (1:1, w/v) using a mortar and pestle. A food-based negative food control was created similar to the positive control, except the peanuts were omitted and the amount of bread was doubled. The Western-blot assay was sensitive down to 2.5ng/ml of total peanut protein. The laminar flow was the most rapid and least complex. The ELISA was the most analytically sensitive with a cut-off of 1ng/ml of Ara h 1 protein compared to the laminar flow which had a cut-off of 4ng/ml Ara h 1 equivalent. Both ELISA and laminar flow assays were able to detect peanut proteins in the food matrices and positive controls, and not in negative controls. No peanut related proteins were detected in the decedent's gastric sample. The gastric sample spiked with peanuts was reliably detectable. The anaphylaxis patient had no peanut allergens detected in her gastric contents by any of the three methods employed. Both commercially available assays are easily adaptable for testing peanut allergens in the gastric contents as judged by the results of the immunoassays as well as the Western blot analysis. Due to the rising need for detecting peanut proteins in various heterogeneous and complex matrices, the use of appropriate controls should be also considered in these unique investigations. Copyright © 2013 Elsevier B.V. All rights reserved.
Carbene footprinting accurately maps binding sites in protein-ligand and protein-protein interactions

NASA Astrophysics Data System (ADS)

Manzi, Lucio; Barrow, Andrew S.; Scott, Daniel; Layfield, Robert; Wright, Timothy G.; Moses, John E.; Oldham, Neil J.

2016-11-01

Specific interactions between proteins and their binding partners are fundamental to life processes. The ability to detect protein complexes, and map their sites of binding, is crucial to understanding basic biology at the molecular level. Methods that employ sensitive analytical techniques such as mass spectrometry have the potential to provide valuable insights with very little material and on short time scales. Here we present a differential protein footprinting technique employing an efficient photo-activated probe for use with mass spectrometry. Using this methodology the location of a carbohydrate substrate was accurately mapped to the binding cleft of lysozyme, and in a more complex example, the interactions between a 100 kDa, multi-domain deubiquitinating enzyme, USP5 and a diubiquitin substrate were located to different functional domains. The much improved properties of this probe make carbene footprinting a viable method for rapid and accurate identification of protein binding sites utilizing benign, near-UV photoactivation.
Large-scale inference of protein tissue origin in gram-positive sepsis plasma using quantitative targeted proteomics

PubMed Central

Malmström, Erik; Kilsgård, Ola; Hauri, Simon; Smeds, Emanuel; Herwald, Heiko; Malmström, Lars; Malmström, Johan

2016-01-01

The plasma proteome is highly dynamic and variable, composed of proteins derived from surrounding tissues and cells. To investigate the complex processes that control the composition of the plasma proteome, we developed a mass spectrometry-based proteomics strategy to infer the origin of proteins detected in murine plasma. The strategy relies on the construction of a comprehensive protein tissue atlas from cells and highly vascularized organs using shotgun mass spectrometry. The protein tissue atlas was transformed to a spectral library for highly reproducible quantification of tissue-specific proteins directly in plasma using SWATH-like data-independent mass spectrometry analysis. We show that the method can determine drastic changes of tissue-specific protein profiles in blood plasma from mouse animal models with sepsis. The strategy can be extended to several other species advancing our understanding of the complex processes that contribute to the plasma proteome dynamics. PMID:26732734
A universal label-free fluorescent aptasensor based on Ru complex and quantum dots for adenosine, dopamine and 17β-estradiol detection.

PubMed

Huang, Hailiang; Shi, Shuo; Gao, Xing; Gao, Ruru; Zhu, Ying; Wu, Xuewen; Zang, Ruimin; Yao, Tianming

2016-05-15

Based on specific aptamer binding properties, a strategy for adenosine, dopamine and 17β-estradiol detection was realised by employing Ru complex and quantum dots (QDs) as fluorescence probes. Ru complex, which could quench the fluorescence of QDs, preferred to bind with aptamer DNA and resulted in the fluorescence rise of QDs. When the aptamer DNA was incubated with the target first, it could not bind with Ru complex and the fluorescence of QDs was quenched. Under the optimal condition, the fluorescence intensity was linearly proportional to the concentration of adenosine, dopamine and 17β-estradiol with a limit of detection (LOD) of 101 nM, 19 nM and 37 nM, respectively. The experiments in fetal bovine serum were also carried out with good results. This universal method was rapid, label-free, low-cost, easy-operating and highly repeatable for the detection of adenosine, dopamine and 17β-estradiol. Qualitative detection by naked eyes was also available without complex instruments. It could also be extended to detect various analytes, such as metal ions, proteins and small molecules by using appropriate aptamers. Copyright © 2015 Elsevier B.V. All rights reserved.
Network analysis reveals the recognition mechanism for complex formation of mannose-binding lectins

NASA Astrophysics Data System (ADS)

Jian, Yiren; Zhao, Yunjie; Zeng, Chen

The specific carbohydrate binding of lectin makes the protein a powerful molecular tool for various applications including cancer cell detection due to its glycoprotein profile on the cell surface. Most biologically active lectins are dimeric. To understand the structure-function relation of lectin complex, it is essential to elucidate the short- and long-range driving forces behind the dimer formation. Here we report our molecular dynamics simulations and associated dynamical network analysis on a particular lectin, i.e., the mannose-binding lectin from garlic. Our results, further supported by sequence coevolution analysis, shed light on how different parts of the complex communicate with each other. We propose a general framework for deciphering the recognition mechanism underlying protein-protein interactions that may have potential applications in signaling pathways.
Structural study of the Fox-1 RRM protein hydration reveals a role for key water molecules in RRM-RNA recognition

PubMed Central

Blatter, Markus; Cléry, Antoine; Damberger, Fred F.

2017-01-01

Abstract The Fox-1 RNA recognition motif (RRM) domain is an important member of the RRM protein family. We report a 1.8 Å X-ray structure of the free Fox-1 containing six distinct monomers. We use this and the nuclear magnetic resonance (NMR) structure of the Fox-1 protein/RNA complex for molecular dynamics (MD) analyses of the structured hydration. The individual monomers of the X-ray structure show diverse hydration patterns, however, MD excellently reproduces the most occupied hydration sites. Simulations of the protein/RNA complex show hydration consistent with the isolated protein complemented by hydration sites specific to the protein/RNA interface. MD predicts intricate hydration sites with water-binding times extending up to hundreds of nanoseconds. We characterize two of them using NMR spectroscopy, RNA binding with switchSENSE and free-energy calculations of mutant proteins. Both hydration sites are experimentally confirmed and their abolishment reduces the binding free-energy. A quantitative agreement between theory and experiment is achieved for the S155A substitution but not for the S122A mutant. The S155 hydration site is evolutionarily conserved within the RRM domains. In conclusion, MD is an effective tool for predicting and interpreting the hydration patterns of protein/RNA complexes. Hydration is not easily detectable in NMR experiments but can affect stability of protein/RNA complexes. PMID:28505313
Analysis of sDMA modifications of PIWI proteins

PubMed Central

Honda, Shozo; Kirino, Yoriko; Kirino, Yohei

2015-01-01

Summary Arginine methylation is an important post-translational protein modification that modulates protein function for a wide range of biological processes. PIWI proteins, a subclade of the Argonaute family proteins, contain evolutionarily conserved symmetrical dimethylarginines (sDMAs). It has become increasingly apparent that the sDMAs of PIWI proteins serve as binding elements for TUDOR-domain containing proteins and that sDMA-dependent protein interactions play crucial roles in the biogenesis and function of PIWI-interacting RNAs (piRNAs). We describe a method for detecting PIWI sDMAs and purifying PIWI/piRNA complexes using anti-sDMA antibodies. PMID:24178562
Protein-protein interactions: an application of Tus-Ter mediated protein microarray system.

PubMed

Sitaraman, Kalavathy; Chatterjee, Deb K

2011-01-01

In this chapter, we present a novel, cost-effective microarray strategy that utilizes expression-ready plasmid DNAs to generate protein arrays on-demand and its use to validate protein-protein interactions. These expression plasmids were constructed in such a way so as to serve a dual purpose of synthesizing the protein of interest as well as capturing the synthesized protein. The microarray system is based on the high affinity binding of Escherichia coli "Tus" protein to "Ter," a 20 bp DNA sequence involved in the regulation of DNA replication. The protein expression is carried out in a cell-free protein synthesis system, with rabbit reticulocyte lysates, and the target proteins are detected either by labeled incorporated tag specific or by gene-specific antibodies. This microarray system has been successfully used for the detection of protein-protein interaction because both the target protein and the query protein can be transcribed and translated simultaneously in the microarray slides. The utility of this system for detecting protein-protein interaction is demonstrated by a few well-known examples: Jun/Fos, FRB/FKBP12, p53/MDM2, and CDK4/p16. In all these cases, the presence of protein complexes resulted in the localization of fluorophores at the specific sites of the immobilized target plasmids. Interestingly, during our interactions studies we also detected a previously unknown interaction between CDK2 and p16. Thus, this Tus-Ter based system of protein microarray can be used for the validation of known protein interactions as well as for identifying new protein-protein interactions. In addition, it can be used to examine and identify targets of nucleic acid-protein, ligand-receptor, enzyme-substrate, and drug-protein interactions.
Recommended Mass Spectrometry-Based Strategies to Identify Ricin-Containing Samples.

PubMed

Kalb, Suzanne R; Schieltz, David M; Becher, François; Astot, Crister; Fredriksson, Sten-Åke; Barr, John R

2015-11-25

Ricin is a protein toxin produced by the castor bean plant (Ricinus communis) together with a related protein known as R. communis agglutinin (RCA120). Mass spectrometric (MS) assays have the capacity to unambiguously identify ricin and to detect ricin's activity in samples with complex matrices. These qualitative and quantitative assays enable detection and differentiation of ricin from the less toxic RCA120 through determination of the amino acid sequence of the protein in question, and active ricin can be monitored by MS as the release of adenine from the depurination of a nucleic acid substrate. In this work, we describe the application of MS-based methods to detect, differentiate and quantify ricin and RCA120 in nine blinded samples supplied as part of the EQuATox proficiency test. Overall, MS-based assays successfully identified all samples containing ricin or RCA120 with the exception of the sample spiked with the lowest concentration (0.414 ng/mL). In fact, mass spectrometry was the most successful method for differentiation of ricin and RCA120 based on amino acid determination. Mass spectrometric methods were also successful at ranking the functional activities of the samples, successfully yielding semi-quantitative results. These results indicate that MS-based assays are excellent techniques to detect, differentiate, and quantify ricin and RCA120 in complex matrices.
A Polypyrimidine Tract Binding Protein, Pumpkin RBP50, Forms the Basis of a Phloem-Mobile Ribonucleoprotein Complex[W

PubMed Central

Ham, Byung-Kook; Brandom, Jeri L.; Xoconostle-Cázares, Beatriz; Ringgold, Vanessa; Lough, Tony J.; Lucas, William J.

2009-01-01

RNA binding proteins (RBPs) are integral components of ribonucleoprotein (RNP) complexes and play a central role in RNA processing. In plants, some RBPs function in a non-cell-autonomous manner. The angiosperm phloem translocation stream contains a unique population of RBPs, but little is known regarding the nature of the proteins and mRNA species that constitute phloem-mobile RNP complexes. Here, we identified and characterized a 50-kD pumpkin (Cucurbita maxima cv Big Max) phloem RNA binding protein (RBP50) that is evolutionarily related to animal polypyrimidine tract binding proteins. In situ hybridization studies indicated a high level of RBP50 transcripts in companion cells, while immunolocalization experiments detected RBP50 in both companion cells and sieve elements. A comparison of the levels of RBP50 present in vascular bundles and phloem sap indicated that this protein is highly enriched in the phloem sap. Heterografting experiments confirmed that RBP50 is translocated from source to sink tissues. Collectively, these findings established that RBP50 functions as a non-cell-autonomous RBP. Protein overlay, coimmunoprecipitation, and cross-linking experiments identified the phloem proteins and mRNA species that constitute RBP50-based RNP complexes. Gel mobility-shift assays demonstrated that specificity, with respect to the bound mRNA, is established by the polypyrimidine tract binding motifs within such transcripts. We present a model for RBP50-based RNP complexes within the pumpkin phloem translocation stream. PMID:19122103
Functional and genomic analyses of alpha-solenoid proteins.

PubMed

Fournier, David; Palidwor, Gareth A; Shcherbinin, Sergey; Szengel, Angelika; Schaefer, Martin H; Perez-Iratxeta, Carol; Andrade-Navarro, Miguel A

2013-01-01

Alpha-solenoids are flexible protein structural domains formed by ensembles of alpha-helical repeats (Armadillo and HEAT repeats among others). While homology can be used to detect many of these repeats, some alpha-solenoids have very little sequence homology to proteins of known structure and we expect that many remain undetected. We previously developed a method for detection of alpha-helical repeats based on a neural network trained on a dataset of protein structures. Here we improved the detection algorithm and updated the training dataset using recently solved structures of alpha-solenoids. Unexpectedly, we identified occurrences of alpha-solenoids in solved protein structures that escaped attention, for example within the core of the catalytic subunit of PI3KC. Our results expand the current set of known alpha-solenoids. Application of our tool to the protein universe allowed us to detect their significant enrichment in proteins interacting with many proteins, confirming that alpha-solenoids are generally involved in protein-protein interactions. We then studied the taxonomic distribution of alpha-solenoids to discuss an evolutionary scenario for the emergence of this type of domain, speculating that alpha-solenoids have emerged in multiple taxa in independent events by convergent evolution. We observe a higher rate of alpha-solenoids in eukaryotic genomes and in some prokaryotic families, such as Cyanobacteria and Planctomycetes, which could be associated to increased cellular complexity. The method is available at http://cbdm.mdc-berlin.de/~ard2/.
Differential antigenic protein recovery from Taenia solium cyst tissues using several detergents.

PubMed

Navarrete-Perea, José; Orozco-Ramírez, Rodrigo; Moguel, Bárbara; Sciutto, Edda; Bobes, Raúl J; Laclette, Juan P

2015-07-01

Human and porcine cysticercosis is caused by the larval stage of the flatworm Taenia solium (Cestoda). The protein extracts of T. solium cysts are complex mixtures including cyst's and host proteins. Little is known about the influence of using different detergents in the efficiency of solubilization-extraction of these proteins, including relevant antigens. Here, we describe the use of CHAPS, ASB-14 and Triton X-100, alone or in combination in the extraction buffers, as a strategy to notably increase the recovery of proteins that are usually left aside in insoluble fractions of cysts. Using buffer with CHAPS alone, 315 protein spots were detected through 2D-PAGE. A total of 255 and 258 spots were detected using buffers with Triton X-100 or ASB-14, respectively. More protein spots were detected when detergents were combined, i.e., 2% CHAPS, 1% Triton X-100 and 1% ASB-14 allowed detection of up to 368 spots. Our results indicated that insoluble fractions of T. solium cysts were rich in antigens, including several glycoproteins that were sensitive to metaperiodate treatment. Host proteins, a common component in protein extracts of cysts, were present in larger amounts in soluble than insoluble fractions of cysts proteins. Finally, antigens present in the insoluble fraction were more appropriate as a source of antigens for diagnostic procedures. Copyright © 2015 Elsevier B.V. All rights reserved.
Size-matched alkyne-conjugated cyanine fluorophores to identify differences in protein glycosylation.

PubMed

Burnham-Marusich, Amanda R; Plechaty, Anna M; Berninsone, Patricia M

2014-09-01

Currently, there are few methods to detect differences in posttranslational modifications (PTMs) in a specific manner from complex mixtures. Thus, we developed an approach that combines the sensitivity and specificity of click chemistry with the resolution capabilities of 2D-DIGE. In "Click-DIGE", posttranslationally modified proteins are metabolically labeled with azido-substrate analogs, then size- and charge-matched alkyne-Cy3 or alkyne-Cy5 dyes are covalently attached to the azide of the PTM by click chemistry. The fluorescently-tagged protein samples are then multiplexed for 2DE analysis. Whereas standard DIGE labels all proteins, Click-DIGE focuses the analysis of protein differences to a targeted subset of posttranslationally modified proteins within a complex sample (i.e. specific labeling and analysis of azido glycoproteins within a cell lysate). Our data indicate that (i) Click-DIGE specifically labels azido proteins, (ii) the resulting Cy-protein conjugates are spectrally distinct, and (iii) the conjugates are size- and charge-matched at the level of 2DE. We demonstrate the utility of this approach by detecting multiple differentially expressed glycoproteins between a mutant cell line defective in UDP-galactose transport and the parental cell line. We anticipate that the diversity of azido substrates already available will enable Click-DIGE to be compatible with analysis of a wide range of PTMs. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Protein Analysis by Dynamic Light Scattering: Methods and Techniques for Students

ERIC Educational Resources Information Center

Lorber, Bernard; Fischer, Frederic; Bailly, Marc; Roy, Herve; Kern, Daniel

2012-01-01

Dynamic light scattering (DLS) analyses are routinely used in biology laboratories to detect aggregates in macromolecular solutions, to determine the size of proteins, nucleic acids, and complexes or to monitor the binding of ligands. This article is written for graduate and undergraduate students with access to DLS and for faculty members who…
Compositions and methods for cancer treatment using targeted carbon nanotubes

DOEpatents

Harrison, Jr., Roger G.; Resasco, Daniel E.; Neves, Luis Filipe Ferreira

2016-11-29

Compositions for detecting and/or destroying cancer tumors and/or cancer cells via photodynamic therapy are disclosed, as well as methods of use thereof. The compositions comprise a linking protein or peptide attached to or otherwise physically associated with a carbon nanotube to form a targeted protein-carbon nanotube complex.
Text Mining for Protein Docking

PubMed Central

Badal, Varsha D.; Kundrotas, Petras J.; Vakser, Ilya A.

2015-01-01

The rapidly growing amount of publicly available information from biomedical research is readily accessible on the Internet, providing a powerful resource for predictive biomolecular modeling. The accumulated data on experimentally determined structures transformed structure prediction of proteins and protein complexes. Instead of exploring the enormous search space, predictive tools can simply proceed to the solution based on similarity to the existing, previously determined structures. A similar major paradigm shift is emerging due to the rapidly expanding amount of information, other than experimentally determined structures, which still can be used as constraints in biomolecular structure prediction. Automated text mining has been widely used in recreating protein interaction networks, as well as in detecting small ligand binding sites on protein structures. Combining and expanding these two well-developed areas of research, we applied the text mining to structural modeling of protein-protein complexes (protein docking). Protein docking can be significantly improved when constraints on the docking mode are available. We developed a procedure that retrieves published abstracts on a specific protein-protein interaction and extracts information relevant to docking. The procedure was assessed on protein complexes from Dockground (http://dockground.compbio.ku.edu). The results show that correct information on binding residues can be extracted for about half of the complexes. The amount of irrelevant information was reduced by conceptual analysis of a subset of the retrieved abstracts, based on the bag-of-words (features) approach. Support Vector Machine models were trained and validated on the subset. The remaining abstracts were filtered by the best-performing models, which decreased the irrelevant information for ~ 25% complexes in the dataset. The extracted constraints were incorporated in the docking protocol and tested on the Dockground unbound benchmark set, significantly increasing the docking success rate. PMID:26650466
Zn(II)-Coordinated Quantum Dot-FRET Nanosensors for the Detection of Protein Kinase Activity

PubMed Central

Lim, Butaek; Park, Ji-In; Lee, Kyung Jin; Lee, Jin-Won; Kim, Tae-Wuk; Kim, Young-Pil

2015-01-01

We report a simple detection of protein kinase activity using Zn(II)-mediated fluorescent resonance energy transfer (FRET) between quantum dots (QDs) and dye-tethered peptides. With neither complex chemical ligands nor surface modification of QDs, Zn(II) was the only metal ion that enabled the phosphorylated peptides to be strongly attached on the carboxyl groups of the QD surface via metal coordination, thus leading to a significant FRET efficiency. As a result, protein kinase activity in intermixed solution was efficiently detected by QD-FRET via Zn(II) coordination, especially when the peptide substrate was combined with affinity-based purification. We also found that mono- and di-phosphorylation in the peptide substrate could be discriminated by the Zn(II)-mediated QD-FRET. Our approach is expected to find applications for studying physiological function and signal transduction with respect to protein kinase activity. PMID:26213934
Zn(II)-Coordinated Quantum Dot-FRET Nanosensors for the Detection of Protein Kinase Activity.

PubMed

Lim, Butaek; Park, Ji-In; Lee, Kyung Jin; Lee, Jin-Won; Kim, Tae-Wuk; Kim, Young-Pil

2015-07-23

We report a simple detection of protein kinase activity using Zn(II)-mediated fluorescent resonance energy transfer (FRET) between quantum dots (QDs) and dye-tethered peptides. With neither complex chemical ligands nor surface modification of QDs, Zn(II) was the only metal ion that enabled the phosphorylated peptides to be strongly attached on the carboxyl groups of the QD surface via metal coordination, thus leading to a significant FRET efficiency. As a result, protein kinase activity in intermixed solution was efficiently detected by QD-FRET via Zn(II) coordination, especially when the peptide substrate was combined with affinity-based purification. We also found that mono- and di-phosphorylation in the peptide substrate could be discriminated by the Zn(II)-mediated QD-FRET. Our approach is expected to find applications for studying physiological function and signal transduction with respect to protein kinase activity.

Protein associations and analytical ultracentrifugation

NASA Astrophysics Data System (ADS)

Laue, Tom

2010-03-01

Analytical ultracentrifugation (AUC) is a first principle method for characterizing the thermodynamics of macromolecules in solution. Since AUC directly assesses mass, it is particularly useful for characterizing both reversible and irreversible binding interactions between macromolecules. The principle measurement in AUC is the concentration as a function of radial position, which may be provided by either absorbance, interference or fluorescence detection. Each of these three different detectors may be used to characterize protein associations using either sedimentation equilibrium or sedimentation velocity analysis. Examples will be shown for characterizing irreversible (aggregate) formation, high-accuracy reversible association analysis, and the detection of protein interactions in complex and concentrated fluids (e.g. serum, cell cytosol).
VGKC complex antibodies in pediatric severe acute encephalitis: a study and literature review.

PubMed

Lin, Jainn-Jim; Lin, Kuang-Lin; Hsia, Shao-Hsuan; Wang, Huei-Shyong; Chiu, Cheng-Hsun; CHEESE Study Group

2013-08-01

Antibodies to surface proteins like voltage-gated potassium channel (VGKC) complexes are increasingly found in different neurologic diseases and encephalitis in adults and recently, in children. Detecting such antibodies can help identify forms of encephalitis that may respond to immuno-therapies. However, there are few reports on VGKC complex antibodies in pediatric severe acute encephalitis. This study retrospectively reviewed antibodies to VGKC, leucine-rich glioma-inactivated 1 (Lgi1), and contactin-associated protein-like 2 (Caspr2) in 46 children with severe acute encephalitis. Published cases of VGKC complex antibodies in pediatric encephalitis in the period of 2000-2012 were also reviewed. Elevated VGKC complex antibodies (>100pM) were detected in one of the 46 children with severe acute encephalitis. The 4-year and 6-month-old girl presented with seizure and disturbed consciousness. Viral PCR/culture and serologic evidence of influenza A infection was noted. She also had complications of epilepsy, impaired cognition, and altered behavior and psychology. Antibodies to Lgi1 and Caspr2 were not detected. Ten previously published reports revealed that VGKC complex antibodies can occur in children with limbic encephalitis and acute or sub-acute encephalitis. The incidence of VGKC complex antibodies in pediatric severe acute encephalitis is not high with only one (2.2%) of 46 children in this study. And, this is the first report on the association of VGKC complex antibodies and patients with influenza A-related severe acute encephalitis. The mechanism of VGKC complex antibodies in pediatric severe acute encephalitis warrants further study. Copyright © 2012 The Japanese Society of Child Neurology. Published by Elsevier B.V. All rights reserved.
[Immunochemical study of the proteins of various tissues in Crustacea (Decapoda): nature, role, origin].

PubMed

Durliat, M

1983-11-01

The main proteins of the haemolymph of Crustacea Decapoda have been identified and analysed: haemocyanin, plasma coagulogen, heteroagglutinins, vitellogenins, and molt-related proteins. All these complex components exhibit a high molecular weight and as oligomeric fractions are able to aggregate or dissociate in subunits according to the composition of medium and experimental procedures. Besides their important rôle in the defense mechanism, some proteins are involved in the edification of diverse tissues. They are detected within different compartments: soft integument, calcified carapace and hepatopancreas. They are either in transit or sequestered or synthetized within these tissues. In the crayfish Astacus leptodactylus, some components have been identified in different compartments: --in aqueous extracts from soft integument: the haemocyanin, coagulogen and both fraction F1 (lipoprotein with an approximate molecular weight of 45 kdal) and fraction F2 related to the molt. Both coagulogen and fraction F2 appear sometimes as melanized. These two latter fractions exhibit some glucose-mannose residues and they occur with a higher relative amount than in the blood. --in soluble extracts from calcified cuticle: among the numerous fractions showing a high molecular weight, the haemocyanin and coagulogen are detected. --in aqueous extracts from hepatopancreas: both haemocyanin and coagulogen appear with a little relative amount. Components termed as Fa and Fb are found with a high concentration. One minor fraction is also detected. --in aqueous extracts from eggs: the haemocyanin and fraction Fb are present. Other proteins showing only some antigenic identities with those of the haemolymph are also detected in all these tissues. The haemolymph proteins are not present within these compartments following a passive diffusion. Indeed, their relative amount varies according to the tissue investigated and is different from that found in the blood. Except the haemocyanin detected in all tissues with different aggregation states, the haemolymph proteins identified vary in the organs studied. A qualitative and quantitative selection occurs when the blood proteins enter the other compartments. Perhaps some other proteins are not detected following alterations underwent either in the epithelial barriers or during the tannage process or the chitino-proteic complex formation or due to experimental procedures. On the other hand, each tissue has its own proteins. The integument contains crustacyanins alpha, beta, gamma; the eggs are mainly constituted of lipovitellins and the hepatopancreas is rich in small molecular weight proteins and digestive enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)
The Tim9p–Tim10p complex binds to the transmembrane domains of the ADP/ATP carrier

PubMed Central

Curran, Sean P.; Leuenberger, Danielle; Oppliger, Wolfgang; Koehler, Carla M.

2002-01-01

The soluble Tim9p–Tim10p (Tim, translocase of inner membrane) complex of the mitochondrial intermembrane space mediates the import of the carrier proteins and is a component of the TIM22 import system. The mechanism by which the Tim9p–Tim10p complex assembles and binds the carriers is not well understood, but previous studies have proposed that the conserved cysteine residues in the ‘twin CX3C’ motif coordinate zinc and potentially generate a zinc-finger-like structure that binds to the matrix loops of the carrier proteins. Here we have purified the native and recombinant Tim9p–Tim10p complex, and show that both complexes resemble each other and consist of three Tim9p and three Tim10p. Results from inductively coupled plasma–mass spectrometry studies failed to detect zinc in the Tim9p–Tim10p complex. Instead, the cysteine residues seemingly formed disulfide linkages. The Tim9p–Tim10p complex bound specifically to the transmembrane domains of the ADP/ATP carrier, but had no affinity for Tim23p, an inner membrane protein that is inserted via the TIM22 complex. The chaperone-like Tim9p–Tim10p complex thus may prevent aggregation of the unfolded carrier proteins in the aqueous intermembrane space. PMID:11867522
A sensor system based on a luminescent protein complex in a biopolymer matrix for detecting small concentrations of hydrogen sulfide in aqueous solutions

NASA Astrophysics Data System (ADS)

Sergeev, A. A.; Leonov, A. A.; Kamenev, D. G.; Voznesenskii, S. S.; Kul'chin, Yu. M.

2017-09-01

We have studied the properties of luminescent protein complexes based on myoglobin with covalently bound CY3 luminophore, which were incorporated into polysaccharide agarose films, as potential elements sensitive to hydrogen sulfide (H2S) in aqueous solutions. The presence of this analyte changes the absorption spectrum of myoglobin, which influences the efficiency of luminophore excitation while having almost no effect on its emission spectrum. This effect shows that a luminescent sensor system with the optical response determined by analyte-induced changes in the efficiency of luminescence excitation in the sensitive element can be created. For the system studied, the limit of detection of H2S dissolved in water amounted to 100 pM.
Identification of Contractile Vacuole Proteins in Trypanosoma cruzi

PubMed Central

Park, Miyoung; Martins, Vicente P.; Atwood, James; Moles, Kristen; Collins, Dalis; Rohloff, Peter; Tarleton, Rick; Moreno, Silvia N. J.; Orlando, Ron; Docampo, Roberto

2011-01-01

Contractile vacuole complexes are critical components of cell volume regulation and have been shown to have other functional roles in several free-living protists. However, very little is known about the functions of the contractile vacuole complex of the parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, other than a role in osmoregulation. Identification of the protein composition of these organelles is important for understanding their physiological roles. We applied a combined proteomic and bioinfomatic approach to identify proteins localized to the contractile vacuole. Proteomic analysis of a T. cruzi fraction enriched for contractile vacuoles and analyzed by one-dimensional gel electrophoresis and LC-MS/MS resulted in the addition of 109 newly detected proteins to the group of expressed proteins of epimastigotes. We also identified different peptides that map to at least 39 members of the dispersed gene family 1 (DGF-1) providing evidence that many members of this family are simultaneously expressed in epimastigotes. Of the proteins present in the fraction we selected several homologues with known localizations in contractile vacuoles of other organisms and others that we expected to be present in these vacuoles on the basis of their potential roles. We determined the localization of each by expression as GFP-fusion proteins or with specific antibodies. Six of these putative proteins (Rab11, Rab32, AP180, ATPase subunit B, VAMP1, and phosphate transporter) predominantly localized to the vacuole bladder. TcSNARE2.1, TcSNARE2.2, and calmodulin localized to the spongiome. Calmodulin was also cytosolic. Our results demonstrate the utility of combining subcellular fractionation, proteomic analysis, and bioinformatic approaches for localization of organellar proteins that are difficult to detect with whole cell methodologies. The CV localization of the proteins investigated revealed potential novel roles of these organelles in phosphate metabolism and provided information on the potential participation of adaptor protein complexes in their biogenesis. PMID:21437209
Evidence that the respiratory syncytial virus polymerase complex associates with lipid rafts in virus-infected cells: a proteomic analysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

McDonald, Terence P.; Pitt, Andrew R.; Brown, Gaie

2004-12-05

The interaction between the respiratory syncytial virus (RSV) polymerase complex and lipid rafts was examined in HEp2 cells. Lipid-raft membranes were prepared from virus-infected cells and their protein content was analysed by Western blotting and mass spectrometry. This analysis revealed the presence of the N, P, L, M2-1 and M proteins. However, these proteins appeared to differ from one another in their association with these structures, with the M2-1 protein showing a greater partitioning into raft membranes compared to that of the N, P or M proteins. Determination of the polymerase activity profile of the gradient fractions revealed that 95%more » of the detectable viral enzyme activity was associated with lipid-raft membranes. Furthermore, analysis of virus-infected cells by confocal microscopy suggested an association between these proteins and the raft-lipid, GM1. Together, these results provide evidence that the RSV polymerase complex is able to associate with lipid rafts in virus-infected cells.« less
Inhibition of the Membrane Attack Complex by Dengue Virus NS1 through Interaction with Vitronectin and Terminal Complement Proteins

PubMed Central

Conde, Jonas Nascimento; da Silva, Emiliana Mandarano; Allonso, Diego; Coelho, Diego Rodrigues; Andrade, Iamara da Silva; de Medeiros, Luciano Neves; Menezes, Joice Lima; Barbosa, Angela Silva

2016-01-01

ABSTRACT Dengue virus (DENV) infects millions of people worldwide and is a major public health problem. DENV nonstructural protein 1 (NS1) is a conserved glycoprotein that associates with membranes and is also secreted into the plasma in DENV-infected patients. The present study describes a novel mechanism by which NS1 inhibits the terminal complement pathway. We first identified the terminal complement regulator vitronectin (VN) as a novel DENV2 NS1 binding partner by using a yeast two-hybrid system. This interaction was further assessed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) assay. The NS1-VN complex was also detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the DENV2 NS1 protein, either by itself or by interacting with VN, hinders the formation of the membrane attack complex (MAC) and C9 polymerization. Finally, we showed that DENV2, West Nile virus (WNV), and Zika virus (ZIKV) NS1 proteins produced in mammalian cells inhibited C9 polymerization. Taken together, our results points to a role for NS1 as a terminal pathway inhibitor of the complement system. IMPORTANCE Dengue is the most important arthropod-borne viral disease nowadays and is caused by dengue virus (DENV). The flavivirus NS1 glycoprotein has been characterized functionally as a complement evasion protein that can attenuate the activation of the classical, lectin, and alternative pathways. The present study describes a novel mechanism by which DENV NS1 inhibits the terminal complement pathway. We identified the terminal complement regulator vitronectin (VN) as a novel DENV NS1 binding partner, and the NS1-VN complex was detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the NS1-VN complex inhibited membrane attack complex (MAC) formation, thus interfering with the complement terminal pathway. Interestingly, NS1 itself also inhibited MAC activity, suggesting a direct role of this protein in the inhibition process. Our findings imply a role for NS1 as a terminal pathway inhibitor of the complement system. PMID:27512066
The Bordetella pertussis Type III Secretion System Tip Complex Protein Bsp22 Is Not a Protective Antigen and Fails To Elicit Serum Antibody Responses during Infection of Humans and Mice

PubMed Central

Villarino Romero, Rodrigo; Bibova, Ilona; Cerny, Ondrej; Vecerek, Branislav; Wald, Tomas; Benada, Oldrich; Zavadilova, Jana; Sebo, Peter

2013-01-01

The type III secretion system (T3SS) of pathogenic bordetellae employs a self-associating tip complex protein Bsp22. This protein is immunogenic during infections by Bordetella bronchiseptica and could be used as a protective antigen to immunize mice against B. bronchiseptica challenge. Since low-passage clinical isolates of the human pathogen Bordetella pertussis produce a highly homologous Bsp22 protein (97% homology), we examined its vaccine and diagnostic potential. No Bsp22-specific antibodies were, however, detected in serum samples from 36 patients with clinically and serologically confirmed whooping cough disease (pertussis syndrome). Moreover, although the induction of Bsp22 secretion by the laboratory-adapted 18323 strain in the course of mice lung infection was observed, the B. pertussis 18323-infected mice did not mount any detectable serum antibody response against Bsp22. Furthermore, immunization with recombinant Bsp22 protein yielded induction of high Bsp22-specific serum antibody titers but did not protect mice against an intranasal challenge with B. pertussis 18323. Unlike for B. bronchiseptica, hence, the Bsp22 protein is nonimmunogenic, and/or the serum antibody response to it is suppressed, during B. pertussis infections of humans and mice. PMID:23690400
Synchrotron Radiation Circular Dichroism (SRCD) Spectroscopy - An Enhanced Method for Examining Protein Conformations and Protein Interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

B Wallace; R Janes

CD (circular dichroism) spectroscopy is a well-established technique in structural biology. SRCD (synchrotron radiation circular dichroism) spectroscopy extends the utility and applications of conventional CD spectroscopy (using laboratory-based instruments) because the high flux of a synchrotron enables collection of data at lower wavelengths (resulting in higher information content), detection of spectra with higher signal-to-noise levels and measurements in the presence of absorbing components (buffers, salts, lipids and detergents). SRCD spectroscopy can provide important static and dynamic structural information on proteins in solution, including secondary structures of intact proteins and their domains, protein stability, the differences between wild-type and mutant proteins,more » the identification of natively disordered regions in proteins, and the dynamic processes of protein folding and membrane insertion and the kinetics of enzyme reactions. It has also been used to effectively study protein interactions, including protein-protein complex formation involving either induced-fit or rigid-body mechanisms, and protein-lipid complexes. A new web-based bioinformatics resource, the Protein Circular Dichroism Data Bank (PCDDB), has been created which enables archiving, access and analyses of CD and SRCD spectra and supporting metadata, now making this information publicly available. To summarize, the developing method of SRCD spectroscopy has the potential for playing an important role in new types of studies of protein conformations and their complexes.« less
Association of Membrane Rafts and Postsynaptic Density: Proteomics, Biochemical, and Ultrastructural Analyses

PubMed Central

Suzuki, Tatsuo; Zhang, Jingping; Miyazawa, Shoko; Liu, Qian; Farzan, Michael R.; Yao, Wei-Dong

2011-01-01

Postsynaptic membrane rafts are believed to play important roles in synaptic signaling, plasticity, and maintenance. However, their molecular identities remain elusive. Further, how they interact with the well-established signaling specialization, the postsynaptic density (PSD), is poorly understood. We previously detected a number of conventional PSD proteins in detergent-resistant membranes (DRMs). Here, we have performed LC-MS/MS (liquid chromatography coupled with tandem mass spectrometry) analyses on postsynaptic membrane rafts and PSDs. Our comparative analysis identified an extensive overlap of protein components in the two structures. This overlapping could be explained, at least partly, by a physical association of the two structures. Meanwhile, a significant number of proteins displayed biased distributions to either rafts or PSDs, suggesting distinct roles for the two postsynaptic specializations. Using biochemical and electron microscopic methods, we directly detected membrane raft-PSD complexes. In vitro reconstitution experiments indicated that the formation of raft-PSD complexes was not due to the artificial reconstruction of once-solubilized membrane components and PSD structures, supporting that these complexes occurred in vivo. Taking together, our results provide evidence that postsynaptic membrane rafts and PSDs may be physically associated. Such association could be important in postsynaptic signal integration, synaptic function, and maintenance. PMID:21797867
A High-Throughput Screening Method for Small-Molecule Inhibitors of the Aberrant Mutant SOD1 and Dynein Complex Interaction

PubMed Central

Tang, Xiaohu; Seyb, Kathleen I.; Huang, Mickey; Schuman, Eli R.; Shi, Ping; Zhu, Haining; Glicksman, Marcie A.

2013-01-01

Aberrant protein-protein interactions are attractive drug targets in a variety of neurodegenerative diseases due to the common pathology of accumulation of protein aggregates. In amyotrophic lateral sclerosis, mutations in SOD1 cause the formation of aggregates and inclusions that may sequester other proteins and disrupt cellular processes. It has been demonstrated that mutant SOD1, but not wild-type SOD1, interacts with the axonal transport motor dynein and that this interaction contributes to motor neuron cell death, suggesting that disrupting this interaction may be a potential therapeutic target. However, it can be challenging to configure a high-throughput screening (HTS)–compatible assay to detect inhibitors of a protein-protein interaction. Here we describe the development and challenges of an HTS for small-molecule inhibitors of the mutant SOD1-dynein interaction. We demonstrate that the interaction can be formed by coexpressing the A4V mutant SOD1 and dynein intermediate complex in cells and that this interaction can be disrupted by compounds added to the cell lysates. Finally, we show that some of the compounds identified from a pilot screen to inhibit the protein-protein interaction with this method specifically disrupt the interaction between the dynein complex and mtSOD1 but not the dynein complex itself when applied to live cells. PMID:22140121
Identification of protein–protein interfaces by decreased amide proton solvent accessibility

PubMed Central

Mandell, Jeffrey G.; Falick, Arnold M.; Komives, Elizabeth A.

1998-01-01

Matrix-assisted laser desorption ionization–time-of-flight mass spectrometry was used to identify peptic fragments from protein complexes that retained deuterium under hydrogen exchange conditions due to decreased solvent accessibility at the interface of the complex. Short deuteration times allowed preferential labeling of rapidly exchanging surface amides so that primarily solvent accessibility changes and not conformational changes were detected. A single mass spectrum of the peptic digest mixture was analyzed to determine the deuterium content of all proteolytic fragments of the protein. The protein–protein interface was reliably indicated by those peptides that retained more deuterons in the complex compared with control experiments in which only one protein was present. The method was used to identify the kinase inhibitor [PKI(5–24)] and ATP-binding sites in the cyclic-AMP-dependent protein kinase. Three overlapping peptides identified the ATP-binding site, three overlapping peptides identified the glycine-rich loop, and two peptides identified the PKI(5–24)-binding site. A complex of unknown structure also was analyzed, human α-thrombin bound to an 83-aa fragment of human thrombomodulin [TMEGF(4–5)]. Five peptides from thrombin showed significantly decreased solvent accessibility in the complex. Three peptides identified the anion-binding exosite I, confirming ligand competition experiments. Two peptides identified a new region of thrombin near the active site providing a potential mechanism of how thrombomodulin alters thrombin substrate specificity. PMID:9843953
Mobilization of Cd from human serum albumin by small molecular weight thiols.

PubMed

Morris, Thomas T; Keir, Jennifer L A; Boshart, Steven J; Lobanov, Victor P; Ruhland, Anthony M A; Bahl, Nishita; Gailer, Jürgen

2014-05-01

Although the toxic metal Cd is an established human nephrotoxin, little is known about the role that interactions with plasma constitutents play in determining its mammalian target organs. To gain insight, a Cd-human serum albumin (HSA) complex was analyzed on a system consisting of size exclusion chromatography (SEC) coupled on-line to a flame atomic absorption spectrometer (FAAS). Using phosphate buffered saline (pH 7.4) as the mobile phase, we investigated the effect of 1-10mM oxidized glutathione (GSSG), l-cysteine (Cys), l-glutathione (GSH), or N-acetyl-l-cysteine (NAC) on the elution of Cd. As expected, GSSG did not mobilize Cd from the Cd-HSA complex up to a concentration of 4mM. With 1.0mM NAC, ∼30% of the injected Cd-HSA complex eluted as such, while the mobilized Cd was lost on the column. With 1.0mM of Cys or GSH, no parent Cd-HSA complex was detected and 88% and 82% of the protein bound Cd eluted close to the elution volume, likely in form of Cd(Cys)2 and a Cd-GSH 1:1 complex. Interestingly, with GSH and NAC concentrations >4.0mM, a Cd double peak was detected, which was rationalized in terms of the elution of a polynuclear Cd complex baseline-separated from a mononuclear Cd complex. In contrast, mobile phases which contained Cys concentrations ≥2mM resulted in the detection of only a single Cd peak, probably Cd(Cys)4. Our results establish SEC-FAAS as a viable tool to probe the mobilization of Cd from binding sites on plasma proteins at near physiological conditions. The detected complexes between Cd and Cys or GSH may be involved in the translocation of Cd to mammalian target organs. Copyright © 2014 Elsevier B.V. All rights reserved.
Molecular assembly and subcellular distribution of ATP-sensitive potassium channel proteins in rat hearts.

PubMed

Kuniyasu, Akihiko; Kaneko, Kazuyoshi; Kawahara, Kohichi; Nakayama, Hitoshi

2003-09-25

Cardiac ATP-sensitive K(+) (K(ATP)) channels are proposed to contribute to cardio-protection and ischemic preconditioning. Although mRNAs for all subunits of K(ATP) channels (Kir6.0 and sulfonylurea receptors SURs) were detected in hearts, subcellular localization of their proteins and the subunit combination are not well elucidated. We address these questions in rat hearts, using anti-peptide antibodies raised against each subunit. By immunoblot analysis, all of the subunits were detected in microsomal fractions including sarcolemmal membranes, while they were not detected in mitochondrial fractions at all. Immunoprecipitation and sucrose gradient sedimentation of the digitonin-solubilized microsomes indicated that Kir6.2 exclusively assembled with SUR2A. The molecular mass of the Kir6.2-SUR2A complex estimated by sucrose sedimentation was 1150 kDa, significantly larger than the calculated value for (Kir6.2)(4)-(SUR2A)(4), suggesting a potential formation of micellar complex with digitonin but no indication of hybrid channel formation under the conditions. These findings provide additional information on the structural and functional relationships of cardiac K(ATP) channel proteins involving subcellular localization and roles for cardioprotection and ischemic preconditioning.
New insights into circulating FABP4: Interaction with cytokeratin 1 on endothelial cell membranes.

PubMed

Saavedra, Paula; Girona, Josefa; Bosquet, Alba; Guaita, Sandra; Canela, Núria; Aragonès, Gemma; Heras, Mercedes; Masana, Lluís

2015-11-01

Fatty acid-binding protein 4 (FABP4) is an adipose tissue-secreted adipokine that is involved in the regulation of energetic metabolism and inflammation. Increased levels of circulating FABP4 have been detected in individuals with cardiovascular risk factors. Recent studies have demonstrated that FABP4 has a direct effect on peripheral tissues, specifically promoting vascular dysfunction; however, its mechanism of action is unknown. The objective of this work was to assess the specific interactions between exogenous FABP4 and the plasma membranes of endothelial cells. Immunofluorescence assays showed that exogenous FABP4 localized along the plasma membranes of human umbilical vein endothelial cells (HUVECs), interacting specifically with plasma membrane proteins. Anti-FABP4 immunoblotting revealed two covalent protein complexes containing FABP4 and its putative receptor; these complexes were approximately 108 kDa and 77 kDa in size. Proteomics and mass spectrometry experiments revealed that cytokeratin 1 (CK1) was the FABP4-binding protein. An anti-CK1 immunoblot confirmed the presence of CK1. FABP4-CK1 complexes were also detected in HAECs, HCASMCs, HepG2 cells and THP-1 cells. Pharmacological FABP4 inhibition by BMS309403 results in a slight decrease in the formation of these complexes, indicating that fatty acids may play a role in FABP4 functionality. In addition, we demonstrated that exogenous FABP4 crosses the plasma membrane to enter the cytoplasm and nucleus in HUVECs. These findings indicate that exogenous FABP4 interacts with plasma membrane proteins, specifically CK1. These data contribute to our current knowledge regarding the mechanism of action of circulating FABP4.
Semiquantitative Multiplexed Tandem PCR for Detection and Differentiation of Four Theileria orientalis Genotypes in Cattle

PubMed Central

Perera, Piyumali K.; Gasser, Robin B.; Firestone, Simon M.; Smith, Lee; Roeber, Florian

2014-01-01

Oriental theileriosis is an emerging, tick-borne disease of bovines in the Asia-Pacific region and is caused by one or more genotypes of the Theileria orientalis complex. This study aimed to establish and validate a multiplexed tandem PCR (MT-PCR) assay using three distinct markers (major piroplasm surface protein, 23-kDa piroplasm membrane protein, and the first internal transcribed spacer of nuclear DNA), for the simultaneous detection and semiquantification of four genotypes (Buffeli, Chitose, Ikeda, and type 5) of the T. orientalis complex. Analytical specificity, analytical sensitivity, and repeatability of the established MT-PCR assay were assessed in a series of experiments. Subsequently, the assay was evaluated using 200 genomic DNA samples collected from cattle from farms on which oriental theileriosis outbreaks had occurred, and 110 samples from a region where no outbreaks had been reported. The results showed the MT-PCR assay specifically and reproducibly detected the expected genotypes (i.e., genotypes Buffeli, Chitose, Ikeda, and type 5) of the T. orientalis complex, reliably differentiated them, and was able to detect as little as 1 fg of genomic DNA from each genotype. The diagnostic specificity and sensitivity of the MT-PCR were estimated at 94.0% and 98.8%, respectively. The MT-PCR assay established here is a practical and effective diagnostic tool for the four main genotypes of T. orientalis complex in Australia and should assist studies of the epidemiology and pathophysiology of oriental theileriosis in the Asia-Pacific region. PMID:25339402
Paper based colorimetric biosensing platform utilizing cross-linked siloxane as probe.

PubMed

Zhou, Miao; Yang, Minghui; Zhou, Feimeng

2014-05-15

Paper based colorimetric biosensing platform utilizing cross-linked siloxane 3-aminopropyltriethoxysilane (APTMS) as probe was developed for the detection of a broad range of targets including H2O2, glucose and protein biomarker. APTMS was extensively used for the modification of filter papers to develop paper based analytical devices. We discovered when APTMS was cross-linked with glutaraldehyde (GA), the resulting complex (APTMS-GA) displays brick-red color, and a visual color change was observed when the complex reacted with H2O2. By integrating the APTMS-GA complex with filter paper, the modified paper enables quantitative detection of H2O2 through the monitoring of the color intensity change of the paper via software Image J. Then, with the immobilization of glucose oxidase (GOx) onto the modified paper, glucose can be detected through the detection of enzymatically generated H2O2. For protein biomarker prostate specific antigen (PSA) assay, we immobilized capture, not captured anti-PSA antibody (Ab1) onto the paper surface and using GOx modified gold nanorod (GNR) as detection anti-PSA antibody (Ab2) label. The detection of PSA was also achieved via the liberated H2O2 when the GOx label reacted with glucose. The results demonstrated the possibility of this paper based sensor for the detection of different analytes with wide linear range. The low cost and simplicity of this paper based sensor could be developed for "point-of-care" analysis and find wide application in different areas. © 2013 Published by Elsevier B.V.
Improving off-line accelerated tryptic digestion. Towards fast-lane proteolysis of complex biological samples.

PubMed

Vukovic, Jadranka; Loftheim, Håvard; Winther, Bjørn; Reubsaet, J Léon E

2008-06-27

Off-line digestion of proteins using immobilized trypsin beads is studied with respect to the format of the digestion reactor, the digestion conditions, the comparison with in-solution digestion and its use in complex biological samples. The use of the filter vial as the most appropriate digestion reactor enables simple, efficient and easy-to-handle off-line digestion of the proteins on trypsin beads. It was shown that complex proteins like bovine serum albumin (BSA) need much longer time (89 min) and elevated temperature (37 degrees C) to be digested to an acceptable level compared to smaller proteins like cytochrome c (5 min, room temperature). Comparing the BSA digestion using immobilized trypsin beads with conventional in-solution digestion (overnight at 37 degrees C), it was shown that comparable results were obtained with respect to sequence coverage (>90%) and amount of missed cleavages (in both cases around 20 peptides with 1 or 2 missed cleavages were detected). However, the digestion using immobilized trypsin beads was considerable less time consuming. Good reproducibility and signal intensities were obtained for the digestion products of BSA in a complex urine sample. In addition to this, peptide products of proteins typically present in urine were identified.
Levels of control exerted by the Isc iron-sulfur cluster system on biosynthesis of the formate hydrogenlyase complex.

PubMed

Pinske, Constanze; Jaroschinsky, Monique; Sawers, R Gary

2013-06-01

The membrane-associated formate hydrogenlyase (FHL) complex of bacteria like Escherichia coli is responsible for the disproportionation of formic acid into the gaseous products carbon dioxide and dihydrogen. It comprises minimally seven proteins including FdhF and HycE, the catalytic subunits of formate dehydrogenase H and hydrogenase 3, respectively. Four proteins of the FHL complex have iron-sulphur cluster ([Fe-S]) cofactors. Biosynthesis of [Fe-S] is principally catalysed by the Isc or Suf systems and each comprises proteins for assembly and for delivery of [Fe-S]. This study demonstrates that the Isc system is essential for biosynthesis of an active FHL complex. In the absence of the IscU assembly protein no hydrogen production or activity of FHL subcomponents was detected. A deletion of the iscU gene also resulted in reduced intracellular formate levels partially due to impaired synthesis of pyruvate formate-lyase, which is dependent on the [Fe-S]-containing regulator FNR. This caused reduced expression of the formate-inducible fdhF gene. The A-type carrier (ATC) proteins IscA and ErpA probably deliver [Fe-S] to specific apoprotein components of the FHL complex because mutants lacking either protein exhibited strongly reduced hydrogen production. Neither ATC protein could compensate for the lack of the other, suggesting that they had independent roles in [Fe-S] delivery to complex components. Together, the data indicate that the Isc system modulates FHL complex biosynthesis directly by provision of [Fe-S] as well as indirectly by influencing gene expression through the delivery of [Fe-S] to key regulators and enzymes that ultimately control the generation and oxidation of formate.

The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate

DOE PAGES

Poudel, Suresh; Giannone, Richard J.; Basen, Mirko; ...

2018-03-23

Background: Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates.Results:Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs),more » ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. In conclusion, this study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii’s utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.« less
The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate

DOE Office of Scientific and Technical Information (OSTI.GOV)

Poudel, Suresh; Giannone, Richard J.; Basen, Mirko

Background: Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates.Results:Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs),more » ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. In conclusion, this study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii’s utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.« less
The diversity and specificity of the extracellular proteome in the cellulolytic bacterium Caldicellulosiruptor bescii is driven by the nature of the cellulosic growth substrate.

PubMed

Poudel, Suresh; Giannone, Richard J; Basen, Mirko; Nookaew, Intawat; Poole, Farris L; Kelly, Robert M; Adams, Michael W W; Hettich, Robert L

2018-01-01

Caldicellulosiruptor bescii is a thermophilic cellulolytic bacterium that efficiently deconstructs lignocellulosic biomass into sugars, which subsequently can be fermented into alcohols, such as ethanol, and other products. Deconstruction of complex substrates by C. bescii involves a myriad of highly abundant, substrate-specific extracellular solute binding proteins (ESBPs) and carbohydrate-active enzymes (CAZymes) containing carbohydrate-binding modules (CBMs). Mass spectrometry-based proteomics was employed to investigate how these substrate recognition proteins and enzymes vary as a function of lignocellulosic substrates. Proteomic analysis revealed several key extracellular proteins that respond specifically to either C5 or C6 mono- and polysaccharides. These include proteins of unknown functions (PUFs), ESBPs, and CAZymes. ESBPs that were previously shown to interact more efficiently with hemicellulose and pectin were detected in high abundance during growth on complex C5 substrates, such as switchgrass and xylan. Some proteins, such as Athe_0614 and Athe_2368, whose functions are not well defined were predicted to be involved in xylan utilization and ABC transport and were significantly more abundant in complex and C5 substrates, respectively. The proteins encoded by the entire glucan degradation locus (GDL; Athe_1857, 1859, 1860, 1865, 1867, and 1866) were highly abundant under all growth conditions, particularly when C. bescii was grown on cellobiose, switchgrass, or xylan. In contrast, the glycoside hydrolases Athe_0609 (Pullulanase) and 0610, which both possess CBM20 and a starch binding domain, appear preferential to C5/complex substrate deconstruction. Some PUFs, such as Athe_2463 and 2464, were detected as highly abundant when grown on C5 substrates (xylan and xylose), also suggesting C5-substrate specificity. This study reveals the protein membership of the C. bescii secretome and demonstrates its plasticity based on the complexity (mono-/disaccharides vs. polysaccharides) and type of carbon (C5 vs. C6) available to the microorganism. The presence or increased abundance of extracellular proteins as a response to specific substrates helps to further elucidate C. bescii 's utilization and conversion of lignocellulosic biomass to biofuel and other valuable products. This includes improved characterization of extracellular proteins that lack discrete functional roles and are poorly/not annotated.
Efficient and Accurate Algorithm for Cleaved Fragments Prediction (CFPA) in Protein Sequences Dataset Based on Consensus and Its Variants: A Novel Degradomics Prediction Application.

PubMed

El-Assaad, Atlal; Dawy, Zaher; Nemer, Georges; Hajj, Hazem; Kobeissy, Firas H

2017-01-01

Degradomics is a novel discipline that involves determination of the proteases/substrate fragmentation profile, called the substrate degradome, and has been recently applied in different disciplines. A major application of degradomics is its utility in the field of biomarkers where the breakdown products (BDPs) of different protease have been investigated. Among the major proteases assessed, calpain and caspase proteases have been associated with the execution phases of the pro-apoptotic and pro-necrotic cell death, generating caspase/calpain-specific cleaved fragments. The distinction between calpain and caspase protein fragments has been applied to distinguish injury mechanisms. Advanced proteomics technology has been used to identify these BDPs experimentally. However, it has been a challenge to identify these BDPs with high precision and efficiency, especially if we are targeting a number of proteins at one time. In this chapter, we present a novel bioinfromatic detection method that identifies BDPs accurately and efficiently with validation against experimental data. This method aims at predicting the consensus sequence occurrences and their variants in a large set of experimentally detected protein sequences based on state-of-the-art sequence matching and alignment algorithms. After detection, the method generates all the potential cleaved fragments by a specific protease. This space and time-efficient algorithm is flexible to handle the different orientations that the consensus sequence and the protein sequence can take before cleaving. It is O(mn) in space complexity and O(Nmn) in time complexity, with N number of protein sequences, m length of the consensus sequence, and n length of each protein sequence. Ultimately, this knowledge will subsequently feed into the development of a novel tool for researchers to detect diverse types of selected BDPs as putative disease markers, contributing to the diagnosis and treatment of related disorders.
On Chip Protein Pre-Concentration for Enhancing the Sensitivity of Porous Silicon Biosensors.

PubMed

Arshavsky-Graham, Sofia; Massad-Ivanir, Naama; Paratore, Federico; Scheper, Thomas; Bercovici, Moran; Segal, Ester

2017-12-22

Porous silicon (PSi) nanomaterials have been widely studied as label-free optical biosensors for protein detection. However, these biosensors' performance, specifically in terms of their sensitivity (which is typically in the micromolar range), is insufficient for many applications. Herein, we present a proof-of-concept application of the electrokinetic isotachophoresis (ITP) technique for real-time preconcentration of a target protein on a PSi biosensor. With ITP, a highly concentrated target zone is delivered to the sensing area, where the protein target is captured by immobilized aptamers. The detection of the binding events is conducted in a label-free manner by reflective interferometric Fourier transformation spectroscopy (RIFTS). Up to 1000-fold enhancement in local concentration of the protein target and the biosensor's sensitivity are achieved, with a measured limit of detection of 7.5 nM. Furthermore, the assay is successfully performed in complex media, such as bacteria lysate samples, while the selectivity of the biosensor is retained. The presented assay could be further utilized for other protein targets, and to promote the development of clinically useful PSi biosensors.
Protein recognition by a pattern-generating fluorescent molecular probe.

PubMed

Pode, Zohar; Peri-Naor, Ronny; Georgeson, Joseph M; Ilani, Tal; Kiss, Vladimir; Unger, Tamar; Markus, Barak; Barr, Haim M; Motiei, Leila; Margulies, David

2017-12-01

Fluorescent molecular probes have become valuable tools in protein research; however, the current methods for using these probes are less suitable for analysing specific populations of proteins in their native environment. In this study, we address this gap by developing a unimolecular fluorescent probe that combines the properties of small-molecule-based probes and cross-reactive sensor arrays (the so-called chemical 'noses/tongues'). On the one hand, the probe can detect different proteins by generating unique identification (ID) patterns, akin to cross-reactive arrays. On the other hand, its unimolecular scaffold and selective binding enable this ID-generating probe to identify combinations of specific protein families within complex mixtures and to discriminate among isoforms in living cells, where macroscopic arrays cannot access. The ability to recycle the molecular device and use it to track several binding interactions simultaneously further demonstrates how this approach could expand the fluorescent toolbox currently used to detect and image proteins.
Identifying DNA-binding proteins using structural motifs and the electrostatic potential

PubMed Central

Shanahan, Hugh P.; Garcia, Mario A.; Jones, Susan; Thornton, Janet M.

2004-01-01

Robust methods to detect DNA-binding proteins from structures of unknown function are important for structural biology. This paper describes a method for identifying such proteins that (i) have a solvent accessible structural motif necessary for DNA-binding and (ii) a positive electrostatic potential in the region of the binding region. We focus on three structural motifs: helix–turn-helix (HTH), helix–hairpin–helix (HhH) and helix–loop–helix (HLH). We find that the combination of these variables detect 78% of proteins with an HTH motif, which is a substantial improvement over previous work based purely on structural templates and is comparable to more complex methods of identifying DNA-binding proteins. Similar true positive fractions are achieved for the HhH and HLH motifs. We see evidence of wide evolutionary diversity for DNA-binding proteins with an HTH motif, and much smaller diversity for those with an HhH or HLH motif. PMID:15356290
Protein recognition by a pattern-generating fluorescent molecular probe

NASA Astrophysics Data System (ADS)

Pode, Zohar; Peri-Naor, Ronny; Georgeson, Joseph M.; Ilani, Tal; Kiss, Vladimir; Unger, Tamar; Markus, Barak; Barr, Haim M.; Motiei, Leila; Margulies, David

2017-12-01

Fluorescent molecular probes have become valuable tools in protein research; however, the current methods for using these probes are less suitable for analysing specific populations of proteins in their native environment. In this study, we address this gap by developing a unimolecular fluorescent probe that combines the properties of small-molecule-based probes and cross-reactive sensor arrays (the so-called chemical 'noses/tongues'). On the one hand, the probe can detect different proteins by generating unique identification (ID) patterns, akin to cross-reactive arrays. On the other hand, its unimolecular scaffold and selective binding enable this ID-generating probe to identify combinations of specific protein families within complex mixtures and to discriminate among isoforms in living cells, where macroscopic arrays cannot access. The ability to recycle the molecular device and use it to track several binding interactions simultaneously further demonstrates how this approach could expand the fluorescent toolbox currently used to detect and image proteins.
[Current Perspective on Voltage-gated Potassium Channel Complex Antibody Associated Diseases].

PubMed

Watanabe, Osamu

2018-04-01

Voltage-gated potassium channel (VGKC) complex auto-antibodies were initially identified in Isaacs' syndrome (IS), which is characterized by muscle cramps and neuromyotonia. These antibodies were subsequently identified in patients with Morvan's syndrome (MoS), which includes IS in conjunction with psychosis, insomnia, and dysautonomia. The antibodies have also been detected in a patient with limbic encephalopathy (LE) presenting with prominent amnesia and frequent seizures. Typical cases of LE have adult-onset, with frequent, brief dystonic seizures that predominantly affect the arms and ipsilateral face, and has recently been termed faciobrachial dystonic seizures. Autoantibodies against the extracellular domains of VGKC complex proteins, leucine-rich glioma-inactivated 1 (LGI1), and contactin-associated protein-2 (Caspr2), occur in patients with IS, MoS, and LE. However, routine testing has detected VGKC complex antibodies without LGI1 or Caspr2 reactivities (double-negative) in patients with other diseases, such as Creutzfeldt-Jakob disease and amyotrophic lateral sclerosis. Furthermore, double-negative VGKC complex antibodies are often directed against cytosolic epitopes of Kv1 subunits. Therefore, these antibodies should no longer be classified as neuronal-surface antibodies and lacking pathogenic potential. Novel information has been generated regarding autoantibody disruption of the physiological functions of target proteins. LGI1 antibodies neutralize the interaction between LGI1 and ADAM22, thereby reducing the synaptic AMPA receptors. It may be that the main action is on inhibitory neurons, explaining why the loss of AMPA receptors causes amnesia, neuronal excitability and seizures.
A survey of single nucleotide polymorphisms identified from whole-genome sequencing and their functional effect in the porcine genome

USDA-ARS?s Scientific Manuscript database

Genetic variants detected from sequence have been used to successfully identify causal variants and map complex traits in several organisms. High and moderate impact variants, those expected to alter or disrupt the protein coded by a gene and those that regulate protein production, likely have a mor...
Background-free, high sensitivity staining of proteins in one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gels using a luminescent ruthenium complex.

PubMed

Berggren, K; Chernokalskaya, E; Steinberg, T H; Kemper, C; Lopez, M F; Diwu, Z; Haugland, R P; Patton, W F

2000-07-01

SYPRO Ruby dye is a permanent stain comprised of ruthenium as part of an organic complex that interacts noncovalently with proteins. SYPRO Ruby Protein Gel Stain provides a sensitive, gentle, fluorescence-based method for detecting proteins in one-dimensional and two-dimensional sodium dodecyl sulfate-polyacrylamide gels. Proteins are fixed, stained from 3h to overnight and then rinsed in deionized water or dilute methanol/acetic acid solution for 30 min. The stain can be visualized using a wide range of excitation sources commonly used in image analysis systems including a 302 nm UV-B transilluminator, 473 nm second harmonic generation (SHG) laser, 488 nm argon-ion laser, 532 nm yttrium-aluminum-garnet (YAG) laser, xenon arc lamp, blue fluorescent light bulb or blue light-emitting diode (LED). The sensitivity of SYPRO Ruby Protein Gel Stain is superior to colloidal Coomassie Brilliant Blue (CBB) stain or monobromobimane labeling and comparable with the highest sensitivity silver or zinc-imidazole staining procedures available. The linear dynamic range of SYPRO Ruby Protein Gel stain extends over three orders of magnitude, which is vastly superior to silver, zinc-imidazole, monobromobimane and CBB stain. The fluorescent stain does not contain superfluous chemicals (formaldehyde, glutaraldehyde, Tween-20) that frequently interfere with peptide identification in mass spectrometry. While peptide mass profiles are severely altered in protein samples prelabeled with monobromobimane, successful identification of proteins by peptide mass profiling using matrix-assisted laser desorption/ionization mass spectrometry was easily performed after protein detection with SYPRO Ruby Protein Gel stain.
Interactions of the Human MCM-BP Protein with MCM Complex Components and Dbf4

PubMed Central

Nguyen, Tin; Jagannathan, Madhav; Shire, Kathy; Frappier, Lori

2012-01-01

MCM-BP was discovered as a protein that co-purified from human cells with MCM proteins 3 through 7; results which were recapitulated in frogs, yeast and plants. Evidence in all of these organisms supports an important role for MCM-BP in DNA replication, including contributions to MCM complex unloading. However the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood. Here we show that human MCM-BP is capable of interacting with individual MCM proteins 2 through 7 when co-expressed in insect cells and can greatly increase the recovery of some recombinant MCM proteins. Glycerol gradient sedimentation analysis indicated that MCM-BP interacts most strongly with MCM4 and MCM7. Similar gradient analyses of human cell lysates showed that only a small amount of MCM-BP overlapped with the migration of MCM complexes and that MCM complexes were disrupted by exogenous MCM-BP. In addition, large complexes containing MCM-BP and MCM proteins were detected at mid to late S phase, suggesting that the formation of specific MCM-BP complexes is cell cycle regulated. We also identified an interaction between MCM-BP and the Dbf4 regulatory component of the DDK kinase in both yeast 2-hybrid and insect cell co-expression assays, and this interaction was verified by co-immunoprecipitation of endogenous proteins from human cells. In vitro kinase assays showed that MCM-BP was not a substrate for DDK but could inhibit DDK phosphorylation of MCM4,6,7 within MCM4,6,7 or MCM2-7 complexes, with little effect on DDK phosphorylation of MCM2. Since DDK is known to activate DNA replication through phosphorylation of these MCM proteins, our results suggest that MCM-BP may affect DNA replication in part by regulating MCM phosphorylation by DDK. PMID:22540012
Interactions of the human MCM-BP protein with MCM complex components and Dbf4.

PubMed

Nguyen, Tin; Jagannathan, Madhav; Shire, Kathy; Frappier, Lori

2012-01-01

MCM-BP was discovered as a protein that co-purified from human cells with MCM proteins 3 through 7; results which were recapitulated in frogs, yeast and plants. Evidence in all of these organisms supports an important role for MCM-BP in DNA replication, including contributions to MCM complex unloading. However the mechanisms by which MCM-BP functions and associates with MCM complexes are not well understood. Here we show that human MCM-BP is capable of interacting with individual MCM proteins 2 through 7 when co-expressed in insect cells and can greatly increase the recovery of some recombinant MCM proteins. Glycerol gradient sedimentation analysis indicated that MCM-BP interacts most strongly with MCM4 and MCM7. Similar gradient analyses of human cell lysates showed that only a small amount of MCM-BP overlapped with the migration of MCM complexes and that MCM complexes were disrupted by exogenous MCM-BP. In addition, large complexes containing MCM-BP and MCM proteins were detected at mid to late S phase, suggesting that the formation of specific MCM-BP complexes is cell cycle regulated. We also identified an interaction between MCM-BP and the Dbf4 regulatory component of the DDK kinase in both yeast 2-hybrid and insect cell co-expression assays, and this interaction was verified by co-immunoprecipitation of endogenous proteins from human cells. In vitro kinase assays showed that MCM-BP was not a substrate for DDK but could inhibit DDK phosphorylation of MCM4,6,7 within MCM4,6,7 or MCM2-7 complexes, with little effect on DDK phosphorylation of MCM2. Since DDK is known to activate DNA replication through phosphorylation of these MCM proteins, our results suggest that MCM-BP may affect DNA replication in part by regulating MCM phosphorylation by DDK.
Quantitative Analysis of Endocytic Recycling of Membrane Proteins by Monoclonal Antibody-Based Recycling Assays.

PubMed

Blagojević Zagorac, Gordana; Mahmutefendić, Hana; Maćešić, Senka; Karleuša, Ljerka; Lučin, Pero

2017-03-01

In this report, we present an analysis of several recycling protocols based on labeling of membrane proteins with specific monoclonal antibodies (mAbs). We analyzed recycling of membrane proteins that are internalized by clathrin-dependent endocytosis, represented by the transferrin receptor, and by clathrin-independent endocytosis, represented by the Major Histocompatibility Class I molecules. Cell surface membrane proteins were labeled with mAbs and recycling of mAb:protein complexes was determined by several approaches. Our study demonstrates that direct and indirect detection of recycled mAb:protein complexes at the cell surface underestimate the recycling pool, especially for clathrin-dependent membrane proteins that are rapidly reinternalized after recycling. Recycling protocols based on the capture of recycled mAb:protein complexes require the use of the Alexa Fluor 488 conjugated secondary antibodies or FITC-conjugated secondary antibodies in combination with inhibitors of endosomal acidification and degradation. Finally, protocols based on the capture of recycled proteins that are labeled with Alexa Fluor 488 conjugated primary antibodies and quenching of fluorescence by the anti-Alexa Fluor 488 displayed the same quantitative assessment of recycling as the antibody-capture protocols. J. Cell. Physiol. 232: 463-476, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
Homogenous assay for protein detection based on proximity DNA hybridization and isothermal circular strand displacement amplification reaction.

PubMed

Zhang, Manjun; Li, Ruimin; Ling, Liansheng

2017-06-01

This work proposed a homogenous fluorescence assay for proteins, based on the target-triggered proximity DNA hybridization in combination with strand displacement amplification (SDA). It benefited from target-triggered proximity DNA hybridization to specifically recognize the target and SDA making recycling signal amplification. The system included a molecular beacon (MB), an extended probe (EP), and an assistant probe (AP), which were not self-assembly in the absence of target proteins, due to the short length of the designed complementary sequence among MB, EP, and AP. Upon addition of the target proteins, EP and AP are bound to the target proteins, which induced the occurrence of proximity hybridization between MB, EP, and AP and followed by strand displacement amplification. Through the primer extension, a tripartite complex of probes and target was displaced and recycled to hybridize with another MB, and the more opened MB enabled the detection signal to amplify. Under optimum conditions, it was used for the detection of streptavidin and thrombin. Fluorescence intensity was proportional to the concentration of streptavidin and thrombin in the range of 0.2-30 and 0.2-35 nmol/L, respectively. Furthermore, this fluorescent method has a good selectivity, in which the fluorescence intensity of thrombin was ~37-fold or even larger than that of the other proteins at the same concentration. It is a new and simple method for SDA-involved target protein detection and possesses a great potential for other protein detection in the future. Graphical abstract A homogenous assay for protein detection is based on proximity DNA hybridization and strand displacement amplification reaction.
Symmetric and asymmetric squarylium dyes as noncovalent protein labels: a study by fluorimetry and capillary electrophoresis.

PubMed

Welder, Frank; Paul, Beverly; Nakazumi, Hiroyuki; Yagi, Shigeyuki; Colyer, Christa L

2003-08-05

Noncovalent interactions between two squarylium dyes and various model proteins have been explored. NN127 and SQ-3 are symmetric and asymmetric squarylium dyes, respectively, the fluorescence emissions of which have been shown to be enhanced upon complexation with proteins such as bovine serum albumin (BSA), human serum albumin (HSA), beta-lactoglobulin A, and trypsinogen. Although these dyes are poorly soluble in aqueous solution, they can be dissolved first in methanol followed by dilution with aqueous buffer without precipitation, and are then suitable for use as fluorescent labels in protein determination studies. The nature of interactions between these dyes and proteins was studied using a variety of buffer systems, and it was found that electrostatic interactions are involved but not dominant. Dye/protein stoichiometries in the noncovalent complexes were found to be 1:1 for SQ-3, although various possible stoichiometries were found for NN127 depending upon pH and protein. Association constants on the order of 10(5) and 10(7) were found for noncovalent complexes of SQ-3 and NN127, respectively, with HSA, indicating stronger interactions of the symmetric dye with proteins. Finally, HSA complexes with NN127 were determined by capillary electrophoresis with laser-induced fluorescence detection (CE-LIF). In particular, NN127 shows promise as a reagent capable of fluorescently labeling analyte proteins for analysis by CE-LIF without itself being significantly fluorescent under the aqueous solution conditions studied herein.
Identification of increased amounts of eppin protein complex components in sperm cells of diabetic and obese individuals by difference gel electrophoresis.

PubMed

Paasch, Uwe; Heidenreich, Falk; Pursche, Theresia; Kuhlisch, Eberhard; Kettner, Karina; Grunewald, Sonja; Kratzsch, Jürgen; Dittmar, Gunnar; Glander, Hans-Jürgen; Hoflack, Bernard; Kriegel, Thomas M

2011-08-01

Metabolic disorders like diabetes mellitus and obesity may compromise the fertility of men and women. To unveil disease-associated proteomic changes potentially affecting male fertility, the proteomes of sperm cells from type-1 diabetic, type-2 diabetic, non-diabetic obese and clinically healthy individuals were comparatively analyzed by difference gel electrophoresis. The adaptation of a general protein extraction procedure to the solubilization of proteins from sperm cells allowed for the resolution of 3187 fluorescent spots in the difference gel electrophoresis image of the master gel, which contained the entirety of solubilized sperm proteins. Comparison of the pathological and reference proteomes by applying an average abundance ratio setting of 1.6 and a p ≤ 0.05 criterion resulted in the identification of 79 fluorescent spots containing proteins that were present at significantly changed levels in the sperm cells. Biometric evaluation of the fluorescence data followed by mass spectrometric protein identification revealed altered levels of 12, 71, and 13 protein species in the proteomes of the type-1 diabetic, type-2 diabetic, and non-diabetic obese patients, respectively, with considerably enhanced amounts of the same set of one molecular form of semenogelin-1, one form of clusterin, and two forms of lactotransferrin in each group of pathologic samples. Remarkably, β-galactosidase-1-like protein was the only protein that was detected at decreased levels in all three pathologic situations. The former three proteins are part of the eppin (epididymal proteinase inhibitor) protein complex, which is thought to fulfill fertilization-related functions, such as ejaculate sperm protection, motility regulation and gain of competence for acrosome reaction, whereas the putative role of the latter protein to function as a glycosyl hydrolase during sperm maturation remains to be explored at the protein/enzyme level. The strikingly similar differences detected in the three groups of pathological sperm proteomes reflect a disease-associated enhanced formation of predominantly proteolytically modified forms of three eppin protein complex components, possibly as a response to enduring hyperglycemia and enhanced oxidative stress.
RNase-assisted RNA chromatography

PubMed Central

Michlewski, Gracjan; Cáceres, Javier F.

2010-01-01

RNA chromatography combined with mass spectrometry represents a widely used experimental approach to identify RNA-binding proteins that recognize specific RNA targets. An important drawback of most of these protocols is the high background due to direct or indirect nonspecific binding of cellular proteins to the beads. In many cases this can hamper the detection of individual proteins due to their low levels and/or comigration with contaminating proteins. Increasing the salt concentration during washing steps can reduce background, but at the cost of using less physiological salt concentrations and the likely loss of important RNA-binding proteins that are less stringently bound to a given RNA, as well as the disassembly of protein or ribonucleoprotein complexes. Here, we describe an improved RNA chromatography method that relies on the use of a cocktail of RNases in the elution step. This results in the release of proteins specifically associated with the RNA ligand and almost complete elimination of background noise, allowing a more sensitive and thorough detection of RNA-binding proteins recognizing a specific RNA transcript. PMID:20571124
Protein-Fragment Complementation Assays for Large-Scale Analysis, Functional Dissection, and Spatiotemporal Dynamic Studies of Protein-Protein Interactions in Living Cells.

PubMed

Michnick, Stephen W; Landry, Christian R; Levy, Emmanuel D; Diss, Guillaume; Ear, Po Hien; Kowarzyk, Jacqueline; Malleshaiah, Mohan K; Messier, Vincent; Tchekanda, Emmanuelle

2016-11-01

Protein-fragment complementation assays (PCAs) comprise a family of assays that can be used to study protein-protein interactions (PPIs), conformation changes, and protein complex dimensions. We developed PCAs to provide simple and direct methods for the study of PPIs in any living cell, subcellular compartments or membranes, multicellular organisms, or in vitro. Because they are complete assays, requiring no cell-specific components other than reporter fragments, they can be applied in any context. PCAs provide a general strategy for the detection of proteins expressed at endogenous levels within appropriate subcellular compartments and with normal posttranslational modifications, in virtually any cell type or organism under any conditions. Here we introduce a number of applications of PCAs in budding yeast, Saccharomyces cerevisiae These applications represent the full range of PPI characteristics that might be studied, from simple detection on a large scale to visualization of spatiotemporal dynamics. © 2016 Cold Spring Harbor Laboratory Press.
Global mapping of rat plasma proteins with a native proteomic approach using nondenaturing micro 2DE and quantitative LC-MS/MS.

PubMed

Chen, Shumin; Wen, Meiling; Bu, Shujie; Wang, Ahui; Jin, Ya; Tan, Wen

2016-12-01

Plasma samples from adult male rats were separated by nondenaturing micro 2DE and a reference gel was selected, on which 136 CBB-stained spots were numbered and subjected to in-gel digestion and quantitative LC-MS/MS. The analysis provided the assignment of 1-25 (average eight) non-redundant proteins in each spot and totally 199 proteins were assigned in the 136 spots. About 40% of the proteins were detected in more than one spot and 15% in more than ten spots. We speculate this complexity arose from multiple causes, including protein heterogeneity, overlapping of protein locations and formation of protein complexes. Consequently, such results could not be appropriately presented as a conventional 2DE map, i.e. a list or a gel pattern with one or a few proteins annotated to each spot. Therefore, the LC-MS/MS quantity data was used to reconstruct the gel distribution of each protein and a library containing 199 native protein maps was established for rat plasma. Since proteins that formed a complex would migrate together during the nondenaturing 2DE and thus show similar gel distributions, correlation analysis was attempted for similarity comparison between the maps. The protein pairs showing high correlation coefficients included some well-known complexes, suggesting the promising application of native protein mapping for interaction analysis. With the importance of rat as the most commonly used laboratory animal in biomedical research, we expect this work would facilitate relevant studies by providing not only a reference library of rat plasma protein maps but a means for functional and interaction analysis. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The use of fluorescent intrabodies to detect endogenous gankyrin in living cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rinaldi, Anne-Sophie; Freund, Guillaume; Desplancq, Dominique

2013-04-01

Expression of antibody fragments in mammalian cells (intrabodies) is used to probe the target protein or interfere with its biological function. We previously described the in vitro characterisation of a single-chain Fv (scFv) antibody fragment (F5) isolated from an intrabody library that binds to the oncoprotein gankyrin (GK) in solution. Here, we have isolated several other scFvs that interact with GK in the presence of F5 and tested whether they allow, when fused to fluorescent proteins, to detect by FRET endogenous GK in living cells. The binding of pairs of scFvs to GK was analysed by gel filtration and themore » ability of each scFv to mediate nuclear import/export of GK was determined. Binding between scFv-EGFP and RFP-labelled GK in living cells was detected by fluorescence lifetime imaging microscopy (FLIM). After co-transfection of two scFvs fused to EGFP and RFP, respectively, which form a tri-molecular complex with GK in vitro, FRET signal was measured. This system allowed us to observe that GK is monomeric and distributed throughout the cytoplasm and nucleus of several cancer cell lines. Our results show that pairs of fluorescently labelled intrabodies can be monitored by FLIM–FRET microscopy and that this technique allows the detection of lowly expressed endogenous proteins in single living cells. Highlights: ► Endogenous GK in living cells was targeted with pairs of fluorescently-tagged scFvs. ► Tri-molecular complexes containing two scFvs and one molecule GK were formed. ► GK was detected using fluorescence lifetime-based FRET imaging. ► GK is monomeric and homogeneously distributed in several cancer cell lines. ► This technique may have many applications in live-cell imaging of endogenous proteins.« less
Functional and Genomic Analyses of Alpha-Solenoid Proteins

PubMed Central

Fournier, David; Palidwor, Gareth A.; Shcherbinin, Sergey; Szengel, Angelika; Schaefer, Martin H.; Perez-Iratxeta, Carol; Andrade-Navarro, Miguel A.

2013-01-01

Alpha-solenoids are flexible protein structural domains formed by ensembles of alpha-helical repeats (Armadillo and HEAT repeats among others). While homology can be used to detect many of these repeats, some alpha-solenoids have very little sequence homology to proteins of known structure and we expect that many remain undetected. We previously developed a method for detection of alpha-helical repeats based on a neural network trained on a dataset of protein structures. Here we improved the detection algorithm and updated the training dataset using recently solved structures of alpha-solenoids. Unexpectedly, we identified occurrences of alpha-solenoids in solved protein structures that escaped attention, for example within the core of the catalytic subunit of PI3KC. Our results expand the current set of known alpha-solenoids. Application of our tool to the protein universe allowed us to detect their significant enrichment in proteins interacting with many proteins, confirming that alpha-solenoids are generally involved in protein-protein interactions. We then studied the taxonomic distribution of alpha-solenoids to discuss an evolutionary scenario for the emergence of this type of domain, speculating that alpha-solenoids have emerged in multiple taxa in independent events by convergent evolution. We observe a higher rate of alpha-solenoids in eukaryotic genomes and in some prokaryotic families, such as Cyanobacteria and Planctomycetes, which could be associated to increased cellular complexity. The method is available at http://cbdm.mdc-berlin.de/~ard2/. PMID:24278209
Combining Ultracentrifugation and Peptide Termini Group-specific Immunoprecipitation for Multiplex Plasma Protein Analysis

PubMed Central

Volk, Sonja; Schreiber, Thomas D.; Eisen, David; Wiese, Calvin; Planatscher, Hannes; Pynn, Christopher J.; Stoll, Dieter; Templin, Markus F.; Joos, Thomas O.; Pötz, Oliver

2012-01-01

Blood plasma is a valuable source of potential biomarkers. However, its complexity and the huge dynamic concentration range of its constituents complicate its analysis. To tackle this problem, an immunoprecipitation strategy was employed using antibodies directed against short terminal epitope tags (triple X proteomics antibodies), which allow the enrichment of groups of signature peptides derived from trypsin-digested plasma. Isolated signature peptides are subsequently detected using MALDI-TOF/TOF mass spectrometry. Sensitivity of the immunoaffinity approach was, however, compromised by the presence of contaminant peaks derived from the peptides of nontargeted high abundant proteins. A closer analysis of the enrichment strategy revealed nonspecific peptide binding to the solid phase affinity matrix as the major source of the contaminating peptides. We therefore implemented a sucrose density gradient ultracentrifugation separation step into the procedure. This yielded a 99% depletion of contaminating peptides from a sucrose fraction containing 70% of the peptide-antibody complexes and enabled the detection of the previously undetected low abundance protein filamin-A. Assessment of this novel approach using 15 different triple X proteomics antibodies demonstrated a more consistent detection of a greater number of targeted peptides and a significant reduction in the intensity of nonspecific peptides. Ultracentrifugation coupled with immunoaffinity MS approaches presents a powerful tool for multiplexed plasma protein analysis without the requirement for demanding liquid chromatography separation techniques. PMID:22527512
A chronic fatigue syndrome – related proteome in human cerebrospinal fluid

PubMed Central

Baraniuk, James N; Casado, Begona; Maibach, Hilda; Clauw, Daniel J; Pannell, Lewis K; Hess S, Sonja

2005-01-01

Background Chronic Fatigue Syndrome (CFS), Persian Gulf War Illness (PGI), and fibromyalgia are overlapping symptom complexes without objective markers or known pathophysiology. Neurological dysfunction is common. We assessed cerebrospinal fluid to find proteins that were differentially expressed in this CFS-spectrum of illnesses compared to control subjects. Methods Cerebrospinal fluid specimens from 10 CFS, 10 PGI, and 10 control subjects (50 μl/subject) were pooled into one sample per group (cohort 1). Cohort 2 of 12 control and 9 CFS subjects had their fluids (200 μl/subject) assessed individually. After trypsin digestion, peptides were analyzed by capillary chromatography, quadrupole-time-of-flight mass spectrometry, peptide sequencing, bioinformatic protein identification, and statistical analysis. Results Pooled CFS and PGI samples shared 20 proteins that were not detectable in the pooled control sample (cohort 1 CFS-related proteome). Multilogistic regression analysis (GLM) of cohort 2 detected 10 proteins that were shared by CFS individuals and the cohort 1 CFS-related proteome, but were not detected in control samples. Detection of ≥1 of a select set of 5 CFS-related proteins predicted CFS status with 80% concordance (logistic model). The proteins were α-1-macroglobulin, amyloid precursor-like protein 1, keratin 16, orosomucoid 2 and pigment epithelium-derived factor. Overall, 62 of 115 proteins were newly described. Conclusion This pilot study detected an identical set of central nervous system, innate immune and amyloidogenic proteins in cerebrospinal fluids from two independent cohorts of subjects with overlapping CFS, PGI and fibromyalgia. Although syndrome names and definitions were different, the proteome and presumed pathological mechanism(s) may be shared. PMID:16321154
AnchorDock for Blind Flexible Docking of Peptides to Proteins.

PubMed

Slutzki, Michal; Ben-Shimon, Avraham; Niv, Masha Y

2017-01-01

Due to increasing interest in peptides as signaling modulators and drug candidates, several methods for peptide docking to their target proteins are under active development. The "blind" docking problem, where the peptide-binding site on the protein surface is unknown, presents one of the current challenges in the field. AnchorDock protocol was developed by Ben-Shimon and Niv to address this challenge.This protocol narrows the docking search to the most relevant parts of the conformational space. This is achieved by pre-folding the free peptide and by computationally detecting anchoring spots on the surface of the unbound protein. Multiple flexible simulated annealing molecular dynamics (SAMD) simulations are subsequently carried out, starting from pre-folded peptide conformations, constrained to the various precomputed anchoring spots.Here, AnchorDock is demonstrated using two known protein-peptide complexes. A PDZ-peptide complex provides a relatively easy case due to the relatively small size of the protein, and a typical peptide conformation and binding region; a more challenging example is a complex between USP7 N-term and a p53-derived peptide, where the protein is larger, and the peptide conformation and a binding site are generally assumed to be unknown. AnchorDock returned native-like solutions ranked first and third for the PDZ and USP7 complexes, respectively. We describe the procedure step by step and discuss possible modifications where applicable.
Rapid Activation of Bone Morphogenic Protein 9 by Receptor-mediated Displacement of Pro-domains*

PubMed Central

Kienast, Yvonne; Jucknischke, Ute; Scheiblich, Stefan; Thier, Martina; de Wouters, Mariana; Haas, Alexander; Lehmann, Christian; Brand, Verena; Bernicke, Dirk; Honold, Konrad; Lorenz, Stefan

2016-01-01

By non-covalent association after proteolytic cleavage, the pro-domains modulate the activities of the mature growth factor domains across the transforming growth factor-β family. In the case of bone morphogenic protein 9 (BMP9), however, the pro-domains do not inhibit the bioactivity of the growth factor, and the BMP9·pro-domain complexes have equivalent biological activities as the BMP9 mature ligand dimers. By using real-time surface plasmon resonance, we could demonstrate that either binding of pro-domain-complexed BMP9 to type I receptor activin receptor-like kinase 1 (ALK1), type II receptors, co-receptor endoglin, or to mature BMP9 domain targeting antibodies leads to immediate and complete displacement of the pro-domains from the complex. Vice versa, pro-domain binding by an anti-pro-domain antibody results in release of the mature BMP9 growth factor. Based on these findings, we adjusted ELISA assays to measure the protein levels of different BMP9 variants. Although mature BMP9 and inactive precursor BMP9 protein were directly detectable by ELISA, BMP9·pro-domain complex could only be measured indirectly as dissociated fragments due to displacement of mature growth factor and pro-domains after antibody binding. Our studies provide a model in which BMP9 can be readily activated upon getting into contact with its receptors. This increases the understanding of the underlying biology of BMP9 activation and also provides guidance for ELISA development for the detection of circulating BMP9 variants. PMID:26677222
Separation and dual detection of prostate cancer cells and protein biomarkers using a microchip device.

PubMed

Huang, Wanfeng; Chang, Chun-Li; Brault, Norman D; Gur, Onur; Wang, Zhe; Jalal, Shadia I; Low, Philip S; Ratliff, Timothy L; Pili, Roberto; Savran, Cagri A

2017-01-31

Current efforts for the detection of prostate cancer using only prostate specific antigen are not ideal and indicate a need to develop new assays - using multiple targets - that can more accurately stratify disease states. We previously introduced a device capable of the concurrent detection of cellular and molecular markers from a single sample fluid. Here, an improved design, which achieves affinity as well as size-based separation of captured targets using antibody-conjugated magnetic beads and a silicon chip containing micro-apertures, is presented. Upon injection of the sample, the integration of magnetic attraction with the micro-aperture chip permits larger cell-bead complexes to be isolated in an upper chamber with the smaller protein-bead complexes and remaining beads passing through the micro-apertures into the lower chamber. This enhances captured cell purity for on chip quantification, allows the separate retrieval of captured cells and proteins for downstream analysis, and enables higher bead concentrations for improved multiplexed ligand targeting. Using LNCaP cells and prostate specific membrane antigen (PSMA) to model prostate cancer, the device was able to detect 34 pM of spiked PSMA and achieve a cell capture efficiency of 93% from culture media. LNCaP cells and PSMA were then spiked into diluted healthy human blood to mimic a cancer patient. The device enabled the detection of spiked PSMA (relative to endogenous PSMA) while recovering 85-90% of LNCaP cells which illustrated the potential of new assays for the diagnosis of prostate cancer.
Proteomic Approaches in Biomarker Discovery: New Perspectives in Cancer Diagnostics

PubMed Central

Kocevar, Nina; Komel, Radovan

2014-01-01

Despite remarkable progress in proteomic methods, including improved detection limits and sensitivity, these methods have not yet been established in routine clinical practice. The main limitations, which prevent their integration into clinics, are high cost of equipment, the need for highly trained personnel, and last, but not least, the establishment of reliable and accurate protein biomarkers or panels of protein biomarkers for detection of neoplasms. Furthermore, the complexity and heterogeneity of most solid tumours present obstacles in the discovery of specific protein signatures, which could be used for early detection of cancers, for prediction of disease outcome, and for determining the response to specific therapies. However, cancer proteome, as the end-point of pathological processes that underlie cancer development and progression, could represent an important source for the discovery of new biomarkers and molecular targets for tailored therapies. PMID:24550697
Tracking serum antibody response to viral antigens with arrayed imaging reflectometry

NASA Astrophysics Data System (ADS)

Mace, Charles R.; Rose, Robert C.; Miller, Benjamin L.

2009-02-01

Arrayed Imaging Reflectometry, or "AIR", is a new label-free technique for detecting proteins that relies on bindinginduced changes in the response of an antireflective coating on the surface of a silicon ship. Because the technique provides high sensitivity, excellent dynamic range, and readily integrates with standard silicon wafer processing technology, it is an exceptionally attractive platform on which to build systems for detecting proteins in complex solutions. In our early research, we used AIR chips bearing secreted receptor proteins from enteropathogenic E. coli to develop sensors for this pathogen. Recently, we have been exploring an alternative strategy: Rather than detecting the pathogen directly, can one immobilize antigens from a pathogen, and employ AIR to detect antibody responses to those antigens? Such a strategy would provide enhanced sensitivity for pathogen detection (as the immune system essentially amplifies the "signal" caused by the presence of an organism to which it responds), and would also potentially prove useful in the process of vaccine development. We describe herein preliminary results in the application of such a strategy to the detection of antibodies to human papillomavirus (HPV).
Combining Whispering-Gallery Mode Optical Biosensors with Microfluidics for Real-Time Detection of Protein Secretion from Living Cells in Complex Media.

PubMed

Chen, Ying-Jen; Schoeler, Ulrike; Huang, Chung-Hsuan Benjamin; Vollmer, Frank

2018-05-01

The noninvasive monitoring of protein secretion of cells responding to drug treatment is an effective and essential tool in latest drug development and for cytotoxicity assays. In this work, a surface functionalization method is demonstrated for specific detection of protein released from cells and a platform that integrates highly sensitive optical devices, called whispering-gallery mode biosensors, with precise microfluidics control to achieve label-free and real-time detection. Cell biomarker release is measured in real time and with nanomolar sensitivity. The surface functionalization method allows for antibodies to be immobilized on the surface for specific detection, while the microfluidics system enables detection in a continuous flow with a negligible compromise between sensitivity and flow control over stabilization and mixing. Cytochrome c detection is used to illustrate the merits of the system. Jurkat cells are treated with the toxin staurosporine to trigger cell apoptosis and cytochrome c released into the cell culture medium is monitored via the newly invented optical microfluidic platform. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Application of mass spectrometry technologies for the discovery of low-molecular weight modulators of enzymes and protein-protein interactions.

PubMed

Zehender, Hartmut; Mayr, Lorenz M

2007-10-01

In recent years, mass spectrometry has gained widespread use as an assay and screening technology in drug discovery because it enables sensitive, label-free detection of low-molecular weight modulators of biomolecules as well as sensitive and accurate detection of high-molecular weight modifications of biomolecules. Electrospray and matrix-assisted laser desorption ionization are the most widely used ionization techniques to identify chemical compounds interfering with enzymatic function, receptor-ligand binding or molecules modulating a protein-protein interaction of interest. Mass spectrometry based techniques are no longer restricted to screening in biochemical assay systems but have now become also applicable to imaging of biomolecules and chemical compounds in cell-based assay systems and even in highly complex tissue sections.
Parkinson-dementia complex and development of a new stable isotope dilution assay for BMAA detection in tissue

DOE Office of Scientific and Technical Information (OSTI.GOV)

Snyder, Laura R.; Cruz-Aguado, Reyniel; Sadilek, Martin

2009-10-15

{beta}-Methylamino-L-alanine (BMAA) has been proposed as a global contributor to neurodegenerative diseases, including Parkinson-dementia complex (PDC) of Guam and Alzheimer's disease (AD). The literature on the effects of BMAA is conflicting with some but not all in vitro data supporting a neurotoxic action, and experimental animal data failing to replicate the pattern of neurodegeneration of these human diseases, even at very high exposures. Recently, BMAA has been reported in human brain from individuals afflicted with PDC or AD. Some of the BMAA in human tissue reportedly is freely extractable (free) while some is protein-associated and liberated by techniques that hydrolyzemore » the peptide bond. The latter is especially intriguing since BMAA is a non-proteinogenic amino acid that has no known tRNA. We attempted to replicate these findings with techniques similar to those used by others; despite more than adequate sensitivity, we were unable to detect free BMAA. Recently, using a novel stable isotope dilution assay, we again were unable to detect free or protein-associated BMAA in human cerebrum. Here we review the development of our new assay for tissue detection of BMAA and show that we are able to detect free BMAA in liver but not cerebrum, nor do we detect any protein-associated BMAA in mice fed this amino acid. These studies demonstrate the importance of a sensitive and specific assay for tissue BMAA and seriously challenge the proposal that BMAA is accumulating in human brain.« less
Multi-Level, Multi Time-Scale Fluorescence Intermittency of Photosynthetic LH2 Complexes: A Precursor of Non-Photochemical Quenching?

PubMed

Schörner, Mario; Beyer, Sebastian Reinhardt; Southall, June; Cogdell, Richard J; Köhler, Jürgen

2015-11-05

The light harvesting complex LH2 is a chromoprotein that is an ideal system for studying protein dynamics via the spectral fluctuations of the emission of its intrinsic chromophores. We have immobilized these complexes in a polymer film and studied the fluctuations of the fluorescence intensity from individual complexes over 9 orders of magnitude in time. Combining time-tagged detection of single photons with a change-point analysis has allowed the unambigeous identification of the various intensity levels due to the huge statistical basis of the data set. We propose that the observed intensity level fluctuations reflect conformational changes of the protein backbone that might be a precursor of the mechanism from which nonphotochemical quenching of higher plants has evolved.
Reorganization of low-molecular-weight fraction of plasma proteins in the annual cycle of cyprinidae.

PubMed

Andreeva, A M; Lamas, N E; Serebryakova, M V; Ryabtseva, I P; Bolshakov, V V

2015-02-01

Reorganization of the low-molecular-weight fraction of cyprinid plasma was analyzed using various electrophoretic techniques (disc electrophoresis, electrophoresis in polyacrylamide concentration gradient, in polyacrylamide with urea, and in SDS-polyacrylamide). The study revealed coordinated changes in the low-molecular-weight protein fractions with seasonal dynamics and related reproductive rhythms of fishes. We used cultured species of the Cyprinidae family with sequenced genomes for the detection of these interrelations in fresh-water and anadromous cyprinid species. The common features of organization of fish low-molecular-weight plasma protein fractions made it possible to make reliable identification of their proteins. MALDI mass-spectrometry analysis revealed the presence of the same proteins (hemopexin, apolipoproteins, and serpins) in the low-molecular-weight plasma fraction in wild species and cultured species with sequenced genomes (carp, zebrafish). It is found that the proteins of the first two classes are organized as complexes made of protein oligomers. Stoichiometry of these complexes changes in concordance with the seasonal and reproductive rhythms.
Highly stable loading of Mcm proteins onto chromatin in living cells requires replication to unload

PubMed Central

Kuipers, Marjorie A.; Stasevich, Timothy J.; Sasaki, Takayo; Wilson, Korey A.; Hazelwood, Kristin L.; McNally, James G.; Davidson, Michael W.

2011-01-01

The heterohexameric minichromosome maintenance protein complex (Mcm2-7) functions as the eukaryotic helicase during DNA replication. Mcm2-7 loads onto chromatin during early G1 phase but is not converted into an active helicase until much later during S phase. Hence, inactive Mcm complexes are presumed to remain stably bound from early G1 through the completion of S phase. Here, we investigated Mcm protein dynamics in live mammalian cells. We demonstrate that Mcm proteins are irreversibly loaded onto chromatin cumulatively throughout G1 phase, showing no detectable exchange with a gradually diminishing soluble pool. Eviction of Mcm requires replication; during replication arrest, Mcm proteins remained bound indefinitely. Moreover, the density of immobile Mcms is reduced together with chromatin decondensation within sites of active replication, which provides an explanation for the lack of colocalization of Mcm with replication fork proteins. These results provide in vivo evidence for an exceptionally stable lockdown mechanism to retain all loaded Mcm proteins on chromatin throughout prolonged cell cycles. PMID:21220507
Recommended Mass Spectrometry-Based Strategies to Identify Ricin-Containing Samples

PubMed Central

Kalb, Suzanne R.; Schieltz, David M.; Becher, François; Astot, Crister; Fredriksson, Sten-Åke; Barr, John R.

2015-01-01

Ricin is a protein toxin produced by the castor bean plant (Ricinus communis) together with a related protein known as R. communis agglutinin (RCA120). Mass spectrometric (MS) assays have the capacity to unambiguously identify ricin and to detect ricin’s activity in samples with complex matrices. These qualitative and quantitative assays enable detection and differentiation of ricin from the less toxic RCA120 through determination of the amino acid sequence of the protein in question, and active ricin can be monitored by MS as the release of adenine from the depurination of a nucleic acid substrate. In this work, we describe the application of MS-based methods to detect, differentiate and quantify ricin and RCA120 in nine blinded samples supplied as part of the EQuATox proficiency test. Overall, MS-based assays successfully identified all samples containing ricin or RCA120 with the exception of the sample spiked with the lowest concentration (0.414 ng/mL). In fact, mass spectrometry was the most successful method for differentiation of ricin and RCA120 based on amino acid determination. Mass spectrometric methods were also successful at ranking the functional activities of the samples, successfully yielding semi-quantitative results. These results indicate that MS-based assays are excellent techniques to detect, differentiate, and quantify ricin and RCA120 in complex matrices. PMID:26610568
The SUFBC2 D complex is required for the biogenesis of all major classes of plastid Fe-S proteins.

PubMed

Hu, Xueyun; Kato, Yukako; Sumida, Akihiro; Tanaka, Ayumi; Tanaka, Ryouichi

2017-04-01

Iron-sulfur (Fe-S) proteins play crucial roles in plastids, participating in photosynthesis and other metabolic pathways. Fe-S clusters are thought to be assembled on a scaffold complex composed of SUFB, SUFC and SUFD proteins. However, several additional proteins provide putative scaffold functions in plastids, and, therefore, the contribution of SUFB, C and D proteins to overall Fe-S assembly still remains unclear. In order to gain insights regarding Fe-S cluster biosynthesis in plastids, we analyzed the complex composed of SUFB, C and D in Arabidopsis by blue native-polyacrylamide gel electrophoresis. Using this approach, a major complex of 170 kDa containing all subunits was detected, indicating that these proteins constitute a SUFBC 2 D complex similar to their well characterized bacterial counterparts. The functional effects of SUFB, SUFC or SUFD depletion were analyzed using an inducible RNAi silencing system to specifically target the aforementioned components; resulting in a decrease of various plastidic Fe-S proteins including the PsaA/B and PsaC subunits of photosystem I, ferredoxin and glutamine oxoglutarate aminotransferase. In contrast, the knockout of potential Fe-S scaffold proteins, NFU2 and HCF101, resulted in a specific decrease in the PsaA/B and PsaC levels. These results indicate that the functions of SUFB, SUFC and SUFD for Fe-S cluster biosynthesis cannot be replaced by other scaffold proteins and that SUFBC 2 D, NFU2 and HCF101 are involved in the same pathway for the biogenesis of PSI. Taken together, our results provide in vivo evidence supporting the hypothesis that SUFBC 2 D is the major, and possibly sole scaffold in plastids. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.
Proteins detection by polymer optical fibers sensitised with overlayers of block and random copolymers

NASA Astrophysics Data System (ADS)

El Sachat, Alexandros; Meristoudi, Anastasia; Markos, Christos; Pispas, Stergios; Riziotis, Christos

2014-03-01

A low cost and low complexity optical detection method of proteins is presented by employing a detection scheme based on electrostatic interactions, and implemented by sensitization of a polymer optical fibers' (POF) surface by thin overlayers of properly designed sensitive copolymer materials with predesigned charges. This method enables the fast detection of proteins having opposite charge to the overlayer, and also the effective discrimination of differently charged proteins like lysozyme (LYS) and bovine serum albumin (BSA). As sensitive materials the block and the random copolymers of the same monomers were employed, namely the block copolymer poly(styrene-b-2vinylpyridine) (PS-b- P2VP) and the corresponding random copolymer poly(styrene-r-2vinylpyridine) (PS-r-P2VP), of similar composition and molecular weights. Results show systematically different response between the block and the random copolymers, although of the same order of magnitude, drawing thus important conclusions on their applications' techno-economic aspects given that they have significantly different associated manufacturing method and costs. The use of the POF platform, in combination with those adaptable copolymer sensing materials could lead to efficient low cost bio-detection schemes.
Nucleic acid detection using BRET-beacons based on bioluminescent protein-DNA hybrids.

PubMed

Engelen, Wouter; van de Wiel, Kayleigh M; Meijer, Lenny H H; Saha, Bedabrata; Merkx, Maarten

2017-03-02

Bioluminescent molecular beacons have been developed using a modular design approach that relies on BRET between the bright luciferase NanoLuc and a Cy3 acceptor. While classical molecular beacons are hampered by background fluorescence and scattering, these BRET-beacons allow detection of low pM concentrations of nucleic acids directly in complex media.
Generation of amyloid-β is reduced by the interaction of calreticulin with amyloid precursor protein, presenilin and nicastrin.

PubMed

Stemmer, Nina; Strekalova, Elena; Djogo, Nevena; Plöger, Frank; Loers, Gabriele; Lutz, David; Buck, Friedrich; Michalak, Marek; Schachner, Melitta; Kleene, Ralf

2013-01-01

Dysregulation of the proteolytic processing of amyloid precursor protein by γ-secretase and the ensuing generation of amyloid-β is associated with the pathogenesis of Alzheimer's disease. Thus, the identification of amyloid precursor protein binding proteins involved in regulating processing of amyloid precursor protein by the γ-secretase complex is essential for understanding the mechanisms underlying the molecular pathology of the disease. We identified calreticulin as novel amyloid precursor protein interaction partner that binds to the γ-secretase cleavage site within amyloid precursor protein and showed that this Ca(2+)- and N-glycan-independent interaction is mediated by amino acids 330-344 in the C-terminal C-domain of calreticulin. Co-immunoprecipitation confirmed that calreticulin is not only associated with amyloid precursor protein but also with the γ-secretase complex members presenilin and nicastrin. Calreticulin was detected at the cell surface by surface biotinylation of cells overexpressing amyloid precursor protein and was co-localized by immunostaining with amyloid precursor protein and presenilin at the cell surface of hippocampal neurons. The P-domain of calreticulin located between the N-terminal N-domain and the C-domain interacts with presenilin, the catalytic subunit of the γ-secretase complex. The P- and C-domains also interact with nicastrin, another functionally important subunit of this complex. Transfection of amyloid precursor protein overexpressing cells with full-length calreticulin leads to a decrease in amyloid-β42 levels in culture supernatants, while transfection with the P-domain increases amyloid-β40 levels. Similarly, application of the recombinant P- or C-domains and of a synthetic calreticulin peptide comprising amino acid 330-344 to amyloid precursor protein overexpressing cells result in elevated amyloid-β40 and amyloid-β42 levels, respectively. These findings indicate that the interaction of calreticulin with amyloid precursor protein and the γ-secretase complex regulates the proteolytic processing of amyloid precursor protein by the γ-secretase complex, pointing to calreticulin as a potential target for therapy in Alzheimer's disease.

Reproducibility of combinatorial peptide ligand libraries for proteome capture evaluated by selected reaction monitoring.

PubMed

Di Girolamo, Francesco; Righetti, Pier Giorgio; Soste, Martin; Feng, Yuehan; Picotti, Paola

2013-08-26

Systems biology studies require the capability to quantify with high precision proteins spanning a broad range of abundances across multiple samples. However, the broad range of protein expression in cells often precludes the detection of low-abundance proteins. Different sample processing techniques can be applied to increase proteome coverage. Among these, combinatorial (hexa)peptide ligand libraries (CPLLs) bound to solid matrices have been used to specifically capture and detect low-abundance proteins in complex samples. To assess whether CPLL capture can be applied in systems biology studies involving the precise quantitation of proteins across a multitude of samples, we evaluated its performance across the whole range of protein abundances in Saccharomyces cerevisiae. We used selected reaction monitoring assays for a set of target proteins covering a broad abundance range to quantitatively evaluate the precision of the approach and its capability to detect low-abundance proteins. Replicated CPLL-isolates showed an average variability of ~10% in the amount of the isolated proteins. The high reproducibility of the technique was not dependent on the abundance of the protein or the amount of beads used for the capture. However, the protein-to-bead ratio affected the enrichment of specific proteins. We did not observe a normalization effect of CPLL beads on protein abundances. However, CPLLs enriched for and depleted specific sets of proteins and thus changed the abundances of proteins from a whole proteome extract. This allowed the identification of ~400 proteins otherwise undetected in an untreated sample, under the experimental conditions used. CPLL capture is thus a useful tool to increase protein identifications in proteomic experiments, but it should be coupled to the analysis of untreated samples, to maximize proteome coverage. Our data also confirms that CPLL capture is reproducible and can be confidently used in quantitative proteomic experiments. Combinatorial hexapeptide ligand libraries (CPLLs) bound to solid matrices have been proposed to specifically capture and detect low-abundance proteins in complex samples. To assess whether the CPLL capture can be confidently applied in systems biology studies involving the precise quantitation of proteins across a broad range of abundances and a multitude of samples, we evaluated its reproducibility and performance features. Using selected reaction monitoring assays for proteins covering the whole range of abundances we show that the technique is reproducible and compatible with quantitative proteomic studies. However, the protein-to-bead ratio affects the enrichment of specific proteins and CPLLs depleted specific sets of proteins from a whole proteome extract. Our results suggest that CPLL-based analyses should be coupled to the analysis of untreated samples, to maximize proteome coverage. Overall, our data confirms the applicability of CPLLs in systems biology research and guides the correct use of this technique. Copyright © 2013 Elsevier B.V. All rights reserved.
Hemolymph proteins of Anopheles gambiae larvae infected by Escherichia coli.

PubMed

He, Xuesong; Cao, Xiaolong; He, Yan; Bhattarai, Krishna; Rogers, Janet; Hartson, Steve; Jiang, Haobo

2017-09-01

Anopheles gambiae is a major vector of human malaria and its immune system in part determines the fate of ingested parasites. Proteins, hemocytes and fat body in hemolymph are critical components of this system, mediating both humoral and cellular defenses. Here we assessed differences in the hemolymph proteomes of water- and E. coli-pricked mosquito larvae by a gel-LC-MS approach. Among the 1756 proteins identified, 603 contained a signal peptide but accounted for two-third of the total protein amount on the quantitative basis. The sequence homology search indicated that 233 of the 1756 may be related to defense. In general, we did not detect substantial differences between the control and induced plasma samples in terms of protein numbers or levels. Protein distributions in the gel slices suggested post-translational modifications (e.g. proteolysis) and formation of serpin-protease complexes and high Mr immune complexes. Based on the twenty-five most abundant proteins, we further suggest that major functions of the larval hemolymph are storage, transport, and immunity. In summary, this study provided first data on constitution, levels, and possible functions of hemolymph proteins in the mosquito larvae, reflecting complex changes occurring in the fight against E. coli infection. Copyright © 2017 Elsevier Ltd. All rights reserved.
Synthetic Peptide Arrays for Pathway-Level Protein Monitoring by Liquid Chromatography-Tandem Mass Spectrometry*

PubMed Central

Hewel, Johannes A.; Liu, Jian; Onishi, Kento; Fong, Vincent; Chandran, Shamanta; Olsen, Jonathan B.; Pogoutse, Oxana; Schutkowski, Mike; Wenschuh, Holger; Winkler, Dirk F. H.; Eckler, Larry; Zandstra, Peter W.; Emili, Andrew

2010-01-01

Effective methods to detect and quantify functionally linked regulatory proteins in complex biological samples are essential for investigating mammalian signaling pathways. Traditional immunoassays depend on proprietary reagents that are difficult to generate and multiplex, whereas global proteomic profiling can be tedious and can miss low abundance proteins. Here, we report a target-driven liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategy for selectively examining the levels of multiple low abundance components of signaling pathways which are refractory to standard shotgun screening procedures and hence appear limited in current MS/MS repositories. Our stepwise approach consists of: (i) synthesizing microscale peptide arrays, including heavy isotope-labeled internal standards, for use as high quality references to (ii) build empirically validated high density LC-MS/MS detection assays with a retention time scheduling system that can be used to (iii) identify and quantify endogenous low abundance protein targets in complex biological mixtures with high accuracy by correlation to a spectral database using new software tools. The method offers a flexible, rapid, and cost-effective means for routine proteomic exploration of biological systems including “label-free” quantification, while minimizing spurious interferences. As proof-of-concept, we have examined the abundance of transcription factors and protein kinases mediating pluripotency and self-renewal in embryonic stem cell populations. PMID:20467045
The alternative complement pathway control protein H binds to immune complexes and serves their detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nydegger, U.E.; Corvetta, A.; Spaeth, P.J.

1983-01-01

During solubilization of immune complexes C3b becomes fixed to the immunoglobulin part and serves as a receptor for the alternative complement pathway control protein H. The H-C3b immune complex interaction can be made detectable using 4% polyethyleneglycol to separate free from bound /sup 125/I-H. Tetanus toxoid (Te)/anti-Te complexes kept soluble with fresh serum and containing 125 IU of specific antibody bound 18% of /sup 125/I-H; when fresh serum was chelated with 10 mM EDTA, /sup 125/I-H binding was only 5%. On sucrose density gradients, the H-binding material sedimented in the range of 12 to 30 S. In 36 serum samplesmore » from rheumatoid arthritis (RA) patients and in 12 serum samples from patients with systemic lupus erythematosus (SLE), /sup 125/I-H binding was significantly elevated to 9.5 +/- 4.7% (mean +/- 1 SD) and 13.3 +/- 5.6%, respectively, while /sup 125/I-H binding by 36 normal human sera was 4 +/- 2%. RA samples (17/36, 47%) and SLE samples (9/12, 75%) had H-binding values increased by more than 2 SD above the normal mean. The serum samples were also assessed for conglutinin- and C1q-binding activities; a significant correlation between H and C1q binding was observed (P less than 0.001); there was no correlation between H and conglutinin binding. Although binding to immune complexes through its interaction with C3b, H clearly detects a population of complexes other than conglutinin, thus expanding the possibilities of further characterizing pathological complexes.« less
XPC and human homologs of RAD23: intracellular localization and relationship to other nucleotide excision repair complexes.

PubMed Central

van der Spek, P J; Eker, A; Rademakers, S; Visser, C; Sugasawa, K; Masutani, C; Hanaoka, F; Bootsma, D; Hoeijmakers, J H

1996-01-01

The xeroderma pigmentosum syndrome complementation group C (XP-C) is due to a defect in the global genome repair subpathway of nucleotide excision repair (NER). The XPC protein is complexed with HHR23B, one of the two human homologs of the yeast NER protein, RAD23 (Masutani at al. (1994) EMBO J. 8, 1831-1843). Using heparin chromatography, gel filtration and native gel electrophoresis we demonstrate that the majority of HHR23B is in a free, non-complexed form, and that a minor fraction is tightly associated with XPC. In contrast, we cannot detect any bound HHR23A. Thus the HHR23 proteins may have an additional function independent of XPC. The fractionation behaviour suggests that the non-bound forms of the HHR23 proteins are not necessary for the core of the NER reaction. Although both HHR23 proteins share a high level of overall homology, they migrate very differently on native gels, pointing to a difference in conformation. Gel filtration suggests the XPC-HHR23B heterodimer resides in a high MW complex. However, immunodepletion studies starting from repair-competent Manley extracts fall to reveal a stable association of a significant fraction of the HHR23 proteins or the XPC-HHR23B complex with the basal transcription/repair factor TFIIH, or with the ERCC1 repair complex. Consistent with a function in repair or DNA/chromatin metabolism, immunofluorescence studies show all XPC, HHR23B and (the free) HHR23A to reside in the nucleus. PMID:8692695
Exploiting fluorescence for multiplex immunoassays on protein microarrays

NASA Astrophysics Data System (ADS)

Herbáth, Melinda; Papp, Krisztián; Balogh, Andrea; Matkó, János; Prechl, József

2014-09-01

Protein microarray technology is becoming the method of choice for identifying protein interaction partners, detecting specific proteins, carbohydrates and lipids, or for characterizing protein interactions and serum antibodies in a massively parallel manner. Availability of the well-established instrumentation of DNA arrays and development of new fluorescent detection instruments promoted the spread of this technique. Fluorescent detection has the advantage of high sensitivity, specificity, simplicity and wide dynamic range required by most measurements. Fluorescence through specifically designed probes and an increasing variety of detection modes offers an excellent tool for such microarray platforms. Measuring for example the level of antibodies, their isotypes and/or antigen specificity simultaneously can offer more complex and comprehensive information about the investigated biological phenomenon, especially if we take into consideration that hundreds of samples can be measured in a single assay. Not only body fluids, but also cell lysates, extracted cellular components, and intact living cells can be analyzed on protein arrays for monitoring functional responses to printed samples on the surface. As a rapidly evolving area, protein microarray technology offers a great bulk of information and new depth of knowledge. These are the features that endow protein arrays with wide applicability and robust sample analyzing capability. On the whole, protein arrays are emerging new tools not just in proteomics, but glycomics, lipidomics, and are also important for immunological research. In this review we attempt to summarize the technical aspects of planar fluorescent microarray technology along with the description of its main immunological applications.
The DnaJ-Like Zinc-Finger Protein HCF222 Is Required for Thylakoid Membrane Biogenesis in Plants1[OPEN

PubMed Central

Hartings, Stephanie; Paradies, Susanne; Karnuth, Bianca; Eisfeld, Sabrina; Mehsing, Jasmin; Wolff, Christian; Levey, Tatjana

2017-01-01

To understand the biogenesis of the thylakoid membrane in higher plants and to identify auxiliary proteins required to build up this highly complex membrane system, we have characterized the allelic nuclear mutants high chlorophyll fluorescence222-1 (hcf222-1) and hcf222-2 and isolated the causal gene by map-based cloning. In the ethyl methanesulfonate-induced mutant hcf222-1, the accumulation of the cytochrome b6f (Cytb6f) complex was reduced to 30% compared with the wild type. Other thylakoid membrane complexes accumulated to normal levels. The T-DNA knockout mutant hcf222-2 showed a more severe defect with respect to thylakoid membrane proteins and accumulated only 10% of the Cytb6f complex, accompanied by a reduction in photosystem II, the photosystem II light-harvesting complex, and photosystem I. HCF222 encodes a protein of 99 amino acids in Arabidopsis (Arabidopsis thaliana) that has similarities to the cysteine-rich zinc-binding domain of DnaJ chaperones. The insulin precipitation assay demonstrated that HCF222 has disulfide reductase activity in vitro. The protein is conserved in higher plants and bryophytes but absent in algae and cyanobacteria. Confocal fluorescence microscopy showed that a fraction of HCF222-green fluorescent protein was detectable in the endoplasmic reticulum but that it also could be recognized in chloroplasts. A fusion construct of HCF222 containing a plastid transit peptide targets the protein into chloroplasts and was able to complement the mutational defect. These findings indicate that the chloroplast-targeted HCF222 is indispensable for the maturation and/or assembly of the Cytb6f complex and is very likely involved in thiol-disulfide biochemistry at the thylakoid membrane. PMID:28572458
The DnaJ-Like Zinc-Finger Protein HCF222 Is Required for Thylakoid Membrane Biogenesis in Plants.

PubMed

Hartings, Stephanie; Paradies, Susanne; Karnuth, Bianca; Eisfeld, Sabrina; Mehsing, Jasmin; Wolff, Christian; Levey, Tatjana; Westhoff, Peter; Meierhoff, Karin

2017-07-01

To understand the biogenesis of the thylakoid membrane in higher plants and to identify auxiliary proteins required to build up this highly complex membrane system, we have characterized the allelic nuclear mutants high chlorophyll fluorescence222-1 ( hcf222-1 ) and hcf222-2 and isolated the causal gene by map-based cloning. In the ethyl methanesulfonate-induced mutant hcf222-1 , the accumulation of the cytochrome b 6 f (Cytb6f) complex was reduced to 30% compared with the wild type. Other thylakoid membrane complexes accumulated to normal levels. The T-DNA knockout mutant hcf222-2 showed a more severe defect with respect to thylakoid membrane proteins and accumulated only 10% of the Cytb6f complex, accompanied by a reduction in photosystem II, the photosystem II light-harvesting complex, and photosystem I. HCF222 encodes a protein of 99 amino acids in Arabidopsis ( Arabidopsis thaliana ) that has similarities to the cysteine-rich zinc-binding domain of DnaJ chaperones. The insulin precipitation assay demonstrated that HCF222 has disulfide reductase activity in vitro. The protein is conserved in higher plants and bryophytes but absent in algae and cyanobacteria. Confocal fluorescence microscopy showed that a fraction of HCF222-green fluorescent protein was detectable in the endoplasmic reticulum but that it also could be recognized in chloroplasts. A fusion construct of HCF222 containing a plastid transit peptide targets the protein into chloroplasts and was able to complement the mutational defect. These findings indicate that the chloroplast-targeted HCF222 is indispensable for the maturation and/or assembly of the Cytb6f complex and is very likely involved in thiol-disulfide biochemistry at the thylakoid membrane. © 2017 American Society of Plant Biologists. All Rights Reserved.
Selected reaction monitoring as an effective method for reliable quantification of disease-associated proteins in maple syrup urine disease.

PubMed

Fernández-Guerra, Paula; Birkler, Rune I D; Merinero, Begoña; Ugarte, Magdalena; Gregersen, Niels; Rodríguez-Pombo, Pilar; Bross, Peter; Palmfeldt, Johan

2014-09-01

Selected reaction monitoring (SRM) mass spectrometry can quantitatively measure proteins by specific targeting of peptide sequences, and allows the determination of multiple proteins in one single analysis. Here, we show the feasibility of simultaneous measurements of multiple proteins in mitochondria-enriched samples from cultured fibroblasts from healthy individuals and patients with mutations in branched-chain α-ketoacid dehydrogenase (BCKDH) complex. BCKDH is a mitochondrial multienzyme complex and its defective activity causes maple syrup urine disease (MSUD), a rare but severe inherited metabolic disorder. Four different genes encode the catalytic subunits of BCKDH: E1α (BCKDHA), E1β (BCKDHB), E2 (DBT), and E3 (DLD). All four proteins were successfully quantified in healthy individuals. However, the E1α and E1β proteins were not detected in patients carrying mutations in one of those genes, whereas mRNA levels were almost unaltered, indicating instability of E1α and E1β monomers. Using SRM we elucidated the protein effects of mutations generating premature termination codons or misfolded proteins. SRM is a complement to transcript level measurements and a valuable tool to shed light on molecular mechanisms and on effects of pharmacological therapies at protein level. SRM is particularly effective for inherited disorders caused by multiple proteins such as defects in multienzyme complexes.
Selected reaction monitoring as an effective method for reliable quantification of disease-associated proteins in maple syrup urine disease

PubMed Central

Fernández-Guerra, Paula; Birkler, Rune I D; Merinero, Begoña; Ugarte, Magdalena; Gregersen, Niels; Rodríguez-Pombo, Pilar; Bross, Peter; Palmfeldt, Johan

2014-01-01

Selected reaction monitoring (SRM) mass spectrometry can quantitatively measure proteins by specific targeting of peptide sequences, and allows the determination of multiple proteins in one single analysis. Here, we show the feasibility of simultaneous measurements of multiple proteins in mitochondria-enriched samples from cultured fibroblasts from healthy individuals and patients with mutations in branched-chain α-ketoacid dehydrogenase (BCKDH) complex. BCKDH is a mitochondrial multienzyme complex and its defective activity causes maple syrup urine disease (MSUD), a rare but severe inherited metabolic disorder. Four different genes encode the catalytic subunits of BCKDH: E1α (BCKDHA), E1β (BCKDHB), E2 (DBT), and E3 (DLD). All four proteins were successfully quantified in healthy individuals. However, the E1α and E1β proteins were not detected in patients carrying mutations in one of those genes, whereas mRNA levels were almost unaltered, indicating instability of E1α and E1β monomers. Using SRM we elucidated the protein effects of mutations generating premature termination codons or misfolded proteins. SRM is a complement to transcript level measurements and a valuable tool to shed light on molecular mechanisms and on effects of pharmacological therapies at protein level. SRM is particularly effective for inherited disorders caused by multiple proteins such as defects in multienzyme complexes. PMID:25333063
Force-Manipulation Single-Molecule Spectroscopy Studies of Enzymatic Dynamics

NASA Astrophysics Data System (ADS)

Lu, H. Peter; He, Yufan; Lu, Maolin; Cao, Jin; Guo, Qing

2014-03-01

Subtle conformational changes play a crucial role in protein functions, especially in enzymatic reactions involving complex substrate-enzyme interactions and chemical reactions. We applied AFM-enhanced and magnetic tweezers-correlated single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time by single-molecule FRET detections. Our single-molecule spectroscopy measurements of enzymatic conformational dynamics have revealed time bunching effect and intermittent coherence in conformational state change dynamics involving in enzymatic reaction cycles. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multi-step conformational motion along the coordinates of substrate-enzyme complex formation and product releasing. Our results support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation.
The Fanconi anemia protein FANCF forms a nuclear complex with FANCA, FANCC and FANCG.

PubMed

de Winter, J P; van der Weel, L; de Groot, J; Stone, S; Waisfisz, Q; Arwert, F; Scheper, R J; Kruyt, F A; Hoatlin, M E; Joenje, H

2000-11-01

Fanconi anemia (FA) is a chromosomal instability syndrome associated with a strong predisposition to cancer, particularly acute myeloid leukemia and squamous cell carcinoma. At the cellular level, FA is characterized by spontaneous chromosomal breakage and a unique hypersensitivity to DNA cross-linking agents. Complementation analysis has indicated that at least seven distinct genes are involved in the pathogenesis of FA. Despite the identification of four of these genes (FANCA, FANCC, FANCF and FANCG), the nature of the 'FA pathway' has remained enigmatic, as the FA proteins lack sequence homologies or motifs that could point to a molecular function. To further define this pathway, we studied the subcellular localizations and mutual interactions of the FA proteins, including the recently identified FANCF protein, in human lymphoblasts. FANCF was found predominantly in the nucleus, where it complexes with FANCA, FANCC and FANCG. These interactions were detected in wild-type and FA-D lymphoblasts, but not in lymphoblasts of other FA complementation groups. This implies that each of the FA proteins, except FANCD, is required for these complexes to form. Similarly, we show that the interaction between FANCA and FANCC is restricted to wild-type and FA-D cells. Furthermore, we document the subcellular localization of FANCA and the FANCA/FANCG complex in all FA complementation groups. Our results, along with published data, culminate in a model in which a multi-protein FA complex serves a nuclear function to maintain genomic integrity.
An NMR strategy to detect conformational differences in a protein complexed with highly analogous inhibitors in solution.

PubMed

Persons, John D; Khan, Shahid N; Ishima, Rieko

2018-04-12

This manuscript presents an NMR strategy to investigate conformational differences in protein-inhibitor complexes, when the inhibitors tightly bind to a protein at sub-nanomolar dissociation constants and are highly analogous to each other. Using HIV-1 protease (PR), we previously evaluated amide chemical shift differences, ΔCSPs, of PR bound to darunavir (DRV) compared to PR bound to several DRV analogue inhibitors, to investigate subtle but significant long-distance conformation changes caused by the inhibitor's chemical moiety variation [Khan, S. N., Persons, J. D. Paulsen, J. L., Guerrero, M., Schiffer, C. A., Kurt-Yilmaz, N., and Ishima, R., Biochemistry, (2018), 57, 1652-1662]. However, ΔCSPs are not ideal for investigating subtle PR-inhibitor interface differences because intrinsic differences in the electron shielding of the inhibitors affect protein ΔCSPs. NMR relaxation is also not suitable as it is not sensitive enough to detect small conformational differences in rigid regions among similar PR-inhibitor complexes. Thus, to gain insight into conformational differences at the inhibitor-protein interface, we recorded 15 N-half filtered NOESY spectra of PR bound to two highly analogous inhibitors and assessed NOEs between PR amide protons and inhibitor protons, between PR amide protons and hydroxyl side chains, and between PR amide protons and water protons. We also verified the PR amide-water NOEs using 2D water-NOE/ROE experiments. Differences in water-amide proton NOE peaks, possibly due to amide-protein hydrogen bonds, were observed between subunit A and subunit B, and between the DRV-bound form and an analogous inhibitor-bound form, which may contribute to remote conformational changes. Copyright © 2018 Elsevier Inc. All rights reserved.
DOE Office of Scientific and Technical Information (OSTI.GOV)

Galat, Andrzej, E-mail: galat@dsvidf.cea.fr; Thai, Robert

Highlights: • The hFKBP25 interacts with diverse components of macromolecular entities. • We show that the endogenous human FKBP25 is bound to polyribosomes. • The endogenous hFKBP25 co-immunoprecipitated with nucleosomal proteins. • FKBP25 could induce conformational switch in macromolecular complexes. - Abstract: In this paper, we show some evidence that a member of the FK506-binding proteins, FKBP25 is associated to diverse components that are part of several different intracellular large-molecular mass entities. The FKBP25 is a high-affinity rapamycin-binding immunophilin, which has nuclear translocation signals present in its PPIase domain but it was detected both in the cytoplasm compartment and inmore » the nuclear proteome. Analyses of antiFKBP25-immunoprecipitated proteins have revealed that the endogenous FKBP25 is associated to the core histones of the nucleosome, and with several proteins forming spliceosomal complexes and ribosomal subunits. Using polyclonal antiFKBP25 we have detected FKBP25 associated with polyribosomes. Added RNAs or 0.5 M NaCl release FKBP25 that was associated with the polyribosomes indicating that the immunophilin has an intrinsic capacity to form complexes with polyribonucleotides via its charged surface patches. Rapamycin or FK506 treatments of the polyribosomes isolated from porcine brain, HeLa and K568 cells caused a residual release of the endogenous FKBP25, which suggests that the immunophilin also binds to some proteins via its PPIase cavity. Our proteomics study indicates that the nuclear pool of the FKBP25 targets various nuclear proteins that are crucial for packaging of DNA, chromatin remodeling and pre-mRNA splicing whereas the cytosolic pool of this immunophilin is bound to some components of the ribosome.« less
Deep sequencing and proteomic analysis of the microRNA-induced silencing complex in human red blood cells.

PubMed

Azzouzi, Imane; Moest, Hansjoerg; Wollscheid, Bernd; Schmugge, Markus; Eekels, Julia J M; Speer, Oliver

2015-05-01

During maturation, erythropoietic cells extrude their nuclei but retain their ability to respond to oxidant stress by tightly regulating protein translation. Several studies have reported microRNA-mediated regulation of translation during terminal stages of erythropoiesis, even after enucleation. In the present study, we performed a detailed examination of the endogenous microRNA machinery in human red blood cells using a combination of deep sequencing analysis of microRNAs and proteomic analysis of the microRNA-induced silencing complex. Among the 197 different microRNAs detected, miR-451a was the most abundant, representing more than 60% of all read sequences. In addition, miR-451a and its known target, 14-3-3ζ mRNA, were bound to the microRNA-induced silencing complex, implying their direct interaction in red blood cells. The proteomic characterization of endogenous Argonaute 2-associated microRNA-induced silencing complex revealed 26 cofactor candidates. Among these cofactors, we identified several RNA-binding proteins, as well as motor proteins and vesicular trafficking proteins. Our results demonstrate that red blood cells contain complex microRNA machinery, which might enable immature red blood cells to control protein translation independent of de novo nuclei information. Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.
Proteogenomics: Integrating Next-Generation Sequencing and Mass Spectrometry to Characterize Human Proteomic Variation

NASA Astrophysics Data System (ADS)

Sheynkman, Gloria M.; Shortreed, Michael R.; Cesnik, Anthony J.; Smith, Lloyd M.

2016-06-01

Mass spectrometry-based proteomics has emerged as the leading method for detection, quantification, and characterization of proteins. Nearly all proteomic workflows rely on proteomic databases to identify peptides and proteins, but these databases typically contain a generic set of proteins that lack variations unique to a given sample, precluding their detection. Fortunately, proteogenomics enables the detection of such proteomic variations and can be defined, broadly, as the use of nucleotide sequences to generate candidate protein sequences for mass spectrometry database searching. Proteogenomics is experiencing heightened significance due to two developments: (a) advances in DNA sequencing technologies that have made complete sequencing of human genomes and transcriptomes routine, and (b) the unveiling of the tremendous complexity of the human proteome as expressed at the levels of genes, cells, tissues, individuals, and populations. We review here the field of human proteogenomics, with an emphasis on its history, current implementations, the types of proteomic variations it reveals, and several important applications.
Proteogenomics: Integrating Next-Generation Sequencing and Mass Spectrometry to Characterize Human Proteomic Variation

PubMed Central

Sheynkman, Gloria M.; Shortreed, Michael R.; Cesnik, Anthony J.; Smith, Lloyd M.

2016-01-01

Mass spectrometry–based proteomics has emerged as the leading method for detection, quantification, and characterization of proteins. Nearly all proteomic workflows rely on proteomic databases to identify peptides and proteins, but these databases typically contain a generic set of proteins that lack variations unique to a given sample, precluding their detection. Fortunately, proteogenomics enables the detection of such proteomic variations and can be defined, broadly, as the use of nucleotide sequences to generate candidate protein sequences for mass spectrometry database searching. Proteogenomics is experiencing heightened significance due to two developments: (a) advances in DNA sequencing technologies that have made complete sequencing of human genomes and transcriptomes routine, and (b) the unveiling of the tremendous complexity of the human proteome as expressed at the levels of genes, cells, tissues, individuals, and populations. We review here the field of human proteogenomics, with an emphasis on its history, current implementations, the types of proteomic variations it reveals, and several important applications. PMID:27049631
Parallel seed-based approach to multiple protein structure similarities detection

DOE PAGES

Chapuis, Guillaume; Le Boudic-Jamin, Mathilde; Andonov, Rumen; ...

2015-01-01

Finding similarities between protein structures is a crucial task in molecular biology. Most of the existing tools require proteins to be aligned in order-preserving way and only find single alignments even when multiple similar regions exist. We propose a new seed-based approach that discovers multiple pairs of similar regions. Its computational complexity is polynomial and it comes with a quality guarantee—the returned alignments have both root mean squared deviations (coordinate-based as well as internal-distances based) lower than a given threshold, if such exist. We do not require the alignments to be order preserving (i.e., we consider nonsequential alignments), which makesmore » our algorithm suitable for detecting similar domains when comparing multidomain proteins as well as to detect structural repetitions within a single protein. Because the search space for nonsequential alignments is much larger than for sequential ones, the computational burden is addressed by extensive use of parallel computing techniques: a coarse-grain level parallelism making use of available CPU cores for computation and a fine-grain level parallelism exploiting bit-level concurrency as well as vector instructions.« less
Structural basis of pesticide detection by enzymatic biosensing: a molecular docking and MD simulation study.

PubMed

Shahbaaz, Mohd; Kanchi, Suvardhan; Sabela, Myalowenkosi; Bisetty, Krishna

2018-05-01

Designing of rapid, facile, selective, and cost-effective biosensor technology is a growing area for the detection of various classes of pesticides. The biosensor with these features can be achieved only through the various bio-components using different transducers. This study, therefore, focuses on the usage of molecular docking, specificity tendencies, and capabilities of proteins for the detection of pesticides. Accordingly, the four transducers, acetylcholinesterase (ACH), cytochromes P450 (CYP), glutathione S-transferase (GST), and protein kinase C (PKC) were selected based on their applications including neurotransmitter, metabolism, detoxification enzyme, and protein phosphorylation. Then after molecular docking of the pesticides, fenobucarb, dichlorodiphenyltrichloroethane (DDT), and parathion onto each enzyme, the conformational behavior of the most stable complexes was further analyzed using 50 ns Molecular Dynamics (MD) simulations carried out under explicit water conditions. In the case of protein kinase C (PKC) and cytochrome P450 3A4 enzyme (CYP), the fenobucarb complex showed the most suitable combination of free energy of binding and inhibition constant -4.42 kcal/mol (573.73 μM) and -5.1 kcal/mol (183.49 μM), respectively. Parathion dominated for acetylcholinesterase (ACH) with -4.57 kcal/mol (448.09 μM) and lastly dichlorodiphenyltrichloroethane for glutathione S-transferase (GST), -5.43 kcal/mol (103.88 μM). The RMSD variations were critical for understanding the impact of pesticides as they distinctively influence the energetic attributes of the proteins. Overall, the outcomes from the extensive analysis provide an insight into the structural features of the proteins studied, thereby highlighting their potential use as a substrate in biorecognition sensing of pesticide compounds.
Structural changes of homodimers in the PDB.

PubMed

Koike, Ryotaro; Amemiya, Takayuki; Horii, Tatsuya; Ota, Motonori

2018-04-01

Protein complexes are involved in various biological phenomena. These complexes are intrinsically flexible, and structural changes are essential to their functions. To perform a large-scale automated analysis of the structural changes of complexes, we combined two original methods. An application, SCPC, compares two structures of protein complexes and decides the match of binding mode. Another application, Motion Tree, identifies rigid-body motions in various sizes and magnitude from the two structural complexes with the same binding mode. This approach was applied to all available homodimers in the Protein Data Bank (PDB). We defined two complex-specific motions: interface motion and subunit-spanning motion. In the former, each subunit of a complex constitutes a rigid body, and the relative movement between subunits occurs at the interface. In the latter, structural parts from distinct subunits constitute a rigid body, providing the relative movement spanning subunits. All structural changes were classified and examined. It was revealed that the complex-specific motions were common in the homodimers, detected in around 40% of families. The dimeric interfaces were likely to be small and flat for interface motion, while large and rugged for subunit-spanning motion. Interface motion was accompanied by a drastic change in contacts at the interface, while the change in the subunit-spanning motion was moderate. These results indicate that the interface properties of homodimers correlated with the type of complex-specific motion. The study demonstrates that the pipeline of SCPC and Motion Tree is useful for the massive analysis of structural change of protein complexes. Copyright © 2017 Elsevier Inc. All rights reserved.

Computational Methods to Predict Protein Interaction Partners

NASA Astrophysics Data System (ADS)

Valencia, Alfonso; Pazos, Florencio

In the new paradigm for studying biological phenomena represented by Systems Biology, cellular components are not considered in isolation but as forming complex networks of relationships. Protein interaction networks are among the first objects studied from this new point of view. Deciphering the interactome (the whole network of interactions for a given proteome) has been shown to be a very complex task. Computational techniques for detecting protein interactions have become standard tools for dealing with this problem, helping and complementing their experimental counterparts. Most of these techniques use genomic or sequence features intuitively related with protein interactions and are based on "first principles" in the sense that they do not involve training with examples. There are also other computational techniques that use other sources of information (i.e. structural information or even experimental data) or are based on training with examples.
Highly efficient purification of protein complexes from mammalian cells using a novel streptavidin-binding peptide and hexahistidine tandem tag system: Application to Bruton's tyrosine kinase

PubMed Central

Li, Yifeng; Franklin, Sarah; Zhang, Michael J; Vondriska, Thomas M

2011-01-01

Tandem affinity purification (TAP) is a generic approach for the purification of protein complexes. The key advantage of TAP is the engineering of dual affinity tags that, when attached to the protein of interest, allow purification of the target protein along with its binding partners through two consecutive purification steps. The tandem tag used in the original method consists of two IgG-binding units of protein A from Staphylococcus aureus (ProtA) and the calmodulin-binding peptide (CBP), and it allows for recovery of 20–30% of the bait protein in yeast. When applied to higher eukaryotes, however, this classical TAP tag suffers from low yields. To improve protein recovery in systems other than yeast, we describe herein the development of a three-tag system comprised of CBP, streptavidin-binding peptide (SBP) and hexa-histidine. We illustrate the application of this approach for the purification of human Bruton's tyrosine kinase (Btk), which results in highly efficient binding and elution of bait protein in both purification steps (>50% recovery). Combined with mass spectrometry for protein identification, this TAP strategy facilitated the first nonbiased analysis of Btk interacting proteins. The high efficiency of the SBP-His6 purification allows for efficient recovery of protein complexes formed with a target protein of interest from a small amount of starting material, enhancing the ability to detect low abundance and transient interactions in eukaryotic cell systems. PMID:21080425
CLMSVault: A Software Suite for Protein Cross-Linking Mass-Spectrometry Data Analysis and Visualization.

PubMed

Courcelles, Mathieu; Coulombe-Huntington, Jasmin; Cossette, Émilie; Gingras, Anne-Claude; Thibault, Pierre; Tyers, Mike

2017-07-07

Protein cross-linking mass spectrometry (CL-MS) enables the sensitive detection of protein interactions and the inference of protein complex topology. The detection of chemical cross-links between protein residues can identify intra- and interprotein contact sites or provide physical constraints for molecular modeling of protein structure. Recent innovations in cross-linker design, sample preparation, mass spectrometry, and software tools have significantly improved CL-MS approaches. Although a number of algorithms now exist for the identification of cross-linked peptides from mass spectral data, a dearth of user-friendly analysis tools represent a practical bottleneck to the broad adoption of the approach. To facilitate the analysis of CL-MS data, we developed CLMSVault, a software suite designed to leverage existing CL-MS algorithms and provide intuitive and flexible tools for cross-platform data interpretation. CLMSVault stores and combines complementary information obtained from different cross-linkers and search algorithms. CLMSVault provides filtering, comparison, and visualization tools to support CL-MS analyses and includes a workflow for label-free quantification of cross-linked peptides. An embedded 3D viewer enables the visualization of quantitative data and the mapping of cross-linked sites onto PDB structural models. We demonstrate the application of CLMSVault for the analysis of a noncovalent Cdc34-ubiquitin protein complex cross-linked under different conditions. CLMSVault is open-source software (available at https://gitlab.com/courcelm/clmsvault.git ), and a live demo is available at http://democlmsvault.tyerslab.com/ .
Method development of enantiomer separations by affinity capillary electrophoresis, cyclodextrin electrokinetic chromatography and capillary electrophoresis-mass spectrometry.

PubMed

Tanaka, Yoshihide

2002-07-01

Capillary electrophoresis (CE) has become a powerful tool for enantiomer separations during the last decade. Since 1993, the author has investigated enantiomer separations by affinity capillary electrophoresis (affinity CE) with some proteins and by cyclodextrin electrokinetic chromatography (CDEKC) with some charged cyclodextrins (CDs). Many successful enantiomer separations are demonstrated from our study in this review article. In the enantiomer separations by affinity CE, the deterioration of detection sensitivity was observed under high concentration of the protein in running solutions. The partial filling technique was practically useful to solve the serious problem. It allowed operation at high protein concentrations, such as 500 mumol/L, without the detection problem. Charged CDs had several advantages for the enantiomer separations over neutral ones. Strong electrostatic interactions between a charged CD and oppositely charged analytes should be effective for the formation of the complex. A large difference in electrophoretic mobility between the free analyte and the inclusion complex should also enhance the enantiomeric resolution. In CE-mass spectrometry (CE-MS), the partial filling technique was applied to avoid the introduction of nonvolatile chiral selectors into the CE-MS interface. By replacing the nonvolatile electrolytes in the running buffer by volatile ones, the separation conditions employed in CE with the UV detection method could be transferred to CE-MS.
The Crc and Hfq proteins of Pseudomonas putida cooperate in catabolite repression and formation of ribonucleic acid complexes with specific target motifs.

PubMed

Moreno, Renata; Hernández-Arranz, Sofía; La Rosa, Ruggero; Yuste, Luis; Madhushani, Anjana; Shingler, Victoria; Rojo, Fernando

2015-01-01

The Crc protein is a global regulator that has a key role in catabolite repression and optimization of metabolism in Pseudomonads. Crc inhibits gene expression post-transcriptionally, preventing translation of mRNAs bearing an AAnAAnAA motif [the catabolite activity (CA) motif] close to the translation start site. Although Crc was initially believed to bind RNA by itself, this idea was recently challenged by results suggesting that a protein co-purifying with Crc, presumably the Hfq protein, could account for the detected RNA-binding activity. Hfq is an abundant protein that has a central role in post-transcriptional gene regulation. Herein, we show that the Pseudomonas putida Hfq protein can recognize the CA motifs of RNAs through its distal face and that Crc facilitates formation of a more stable complex at these targets. Crc was unable to bind RNA in the absence of Hfq. However, pull-down assays showed that Crc and Hfq can form a co-complex with RNA containing a CA motif in vitro. Inactivation of the hfq or the crc gene impaired catabolite repression to a similar extent. We propose that Crc and Hfq cooperate in catabolite repression, probably through forming a stable co-complex with RNAs containing CA motifs to result in inhibition of translation initiation. © 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.
Selenoprotein K Binds Multiprotein Complexes and Is Involved in the Regulation of Endoplasmic Reticulum Homeostasis*

PubMed Central

Shchedrina, Valentina A.; Everley, Robert A.; Zhang, Yan; Gygi, Steven P.; Hatfield, Dolph L.; Gladyshev, Vadim N.

2011-01-01

Selenoprotein K (SelK) is an 11-kDa endoplasmic reticulum (ER) protein of unknown function. Herein, we defined a new eukaryotic protein family that includes SelK, selenoprotein S (SelS), and distantly related proteins. Comparative genomics analyses indicate that this family is the most widespread eukaryotic selenoprotein family. A biochemical search for proteins that interact with SelK revealed ER-associated degradation (ERAD) components (p97 ATPase, Derlins, and SelS). In this complex, SelK showed higher affinity for Derlin-1, whereas SelS had higher affinity for Derlin-2, suggesting that these selenoproteins could determine the nature of the substrate translocated through the Derlin channel. SelK co-precipitated with soluble glycosylated ERAD substrates and was involved in their degradation. Its gene contained a functional ER stress response element, and its expression was up-regulated by conditions that induce the accumulation of misfolded proteins in the ER. Components of the oligosaccharyltransferase complex (ribophorins, OST48, and STT3A) and an ER chaperone, calnexin, were found to bind SelK. A glycosylated form of SelK was also detected, reflecting its association with the oligosaccharyltransferase complex. These data suggest that SelK is involved in the Derlin-dependent ERAD of glycosylated misfolded proteins and that the function defined by the prototypic SelK is the widespread function of selenium in eukaryotes. PMID:22016385
Pathogen effectors target Arabidopsis EDS1 and alter its interactions with immune regulators.

PubMed

Bhattacharjee, Saikat; Halane, Morgan K; Kim, Sang Hee; Gassmann, Walter

2011-12-09

Plant resistance proteins detect the presence of specific pathogen effectors and initiate effector-triggered immunity. Few immune regulators downstream of resistance proteins have been identified, none of which are known virulence targets of effectors. We show that Arabidopsis ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1), a positive regulator of basal resistance and of effector-triggered immunity specifically mediated by Toll-interleukin-1 receptor-nucleotide binding-leucine-rich repeat (TIR-NB-LRR) resistance proteins, forms protein complexes with the TIR-NB-LRR disease resistance proteins RPS4 and RPS6 and with the negative immune regulator SRFR1 at a cytoplasmic membrane. Further, the cognate bacterial effectors AvrRps4 and HopA1 disrupt these EDS1 complexes. Tight association of EDS1 with TIR-NB-LRR-mediated immunity may therefore derive mainly from being guarded by TIR-NB-LRR proteins, and activation of this branch of effector-triggered immunity may directly connect to the basal resistance signaling pathway via EDS1.
Inhibition of the Membrane Attack Complex by Dengue Virus NS1 through Interaction with Vitronectin and Terminal Complement Proteins.

PubMed

Conde, Jonas Nascimento; da Silva, Emiliana Mandarano; Allonso, Diego; Coelho, Diego Rodrigues; Andrade, Iamara da Silva; de Medeiros, Luciano Neves; Menezes, Joice Lima; Barbosa, Angela Silva; Mohana-Borges, Ronaldo

2016-11-01

Dengue virus (DENV) infects millions of people worldwide and is a major public health problem. DENV nonstructural protein 1 (NS1) is a conserved glycoprotein that associates with membranes and is also secreted into the plasma in DENV-infected patients. The present study describes a novel mechanism by which NS1 inhibits the terminal complement pathway. We first identified the terminal complement regulator vitronectin (VN) as a novel DENV2 NS1 binding partner by using a yeast two-hybrid system. This interaction was further assessed by enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) assay. The NS1-VN complex was also detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the DENV2 NS1 protein, either by itself or by interacting with VN, hinders the formation of the membrane attack complex (MAC) and C9 polymerization. Finally, we showed that DENV2, West Nile virus (WNV), and Zika virus (ZIKV) NS1 proteins produced in mammalian cells inhibited C9 polymerization. Taken together, our results points to a role for NS1 as a terminal pathway inhibitor of the complement system. Dengue is the most important arthropod-borne viral disease nowadays and is caused by dengue virus (DENV). The flavivirus NS1 glycoprotein has been characterized functionally as a complement evasion protein that can attenuate the activation of the classical, lectin, and alternative pathways. The present study describes a novel mechanism by which DENV NS1 inhibits the terminal complement pathway. We identified the terminal complement regulator vitronectin (VN) as a novel DENV NS1 binding partner, and the NS1-VN complex was detected in plasmas from DENV-infected patients, suggesting that this interaction occurs during DENV infection. We also demonstrated that the NS1-VN complex inhibited membrane attack complex (MAC) formation, thus interfering with the complement terminal pathway. Interestingly, NS1 itself also inhibited MAC activity, suggesting a direct role of this protein in the inhibition process. Our findings imply a role for NS1 as a terminal pathway inhibitor of the complement system. Copyright © 2016, American Society for Microbiology. All Rights Reserved.
[VGKC-complex antibodies].

PubMed

Watanabe, Osamu

2013-04-01

Various antibodies are associated with voltage-gated potassium channels (VGKCs). Representative antibodies to VGKCs were first identified by radioimmunoassays using radioisotope-labeled alpha-dendrotoxin-VGKCs solubilized from rabbit brain. These antibodies were detected only in a proportion of patients with acquired neuromyotonia (Isaacs' syndrome). VGKC antibodies were also detected in patients with Morvan's syndrome and in those with a form of autoimmune limbic encephalitis. Recent studies indicated that the "VGKC" antibodies are mainly directed toward associated proteins (for example LGI-1 and CASPR-2) that complex with the VGKCs themselves. The "VGKC" antibodies are now commonly known as VGKC-complex antibodies. In general, LGI-1 antibodies are most commonly detected in patients with limbic encephalitis with syndrome of inappropriate secretion of antidiuretic hormone. CASPR-2 antibodies are present in the majority of patients with Morvan's syndrome. These patients develop combinations of CNS symptoms, autonomic dysfunction, and peripheral nerve hyperexcitability. Furthermore, VGKC-complex antibodies are tightly associated with chronic idiopathic pain. Hyperexcitability of nociceptive pathways has also been implicated. These antibodies may be detected in sera of some patients with neurodegenerative diseases (for example, amyotrophic lateral sclerosis and Creutzfeldt-Jakob disease).
Proteomic and computational analysis of bronchoalveolar proteins during the course of the acute respiratory distress syndrome.

PubMed

Chang, Dong W; Hayashi, Shinichi; Gharib, Sina A; Vaisar, Tomas; King, S Trevor; Tsuchiya, Mitsuhiro; Ruzinski, John T; Park, David R; Matute-Bello, Gustavo; Wurfel, Mark M; Bumgarner, Roger; Heinecke, Jay W; Martin, Thomas R

2008-10-01

Acute lung injury causes complex changes in protein expression in the lungs. Whereas most prior studies focused on single proteins, newer methods allowing the simultaneous study of many proteins could lead to a better understanding of pathogenesis and new targets for treatment. The purpose of this study was to examine the changes in protein expression in the bronchoalveolar lavage fluid (BALF) of patients during the course of the acute respiratory distress syndrome (ARDS). Using two-dimensional difference gel electrophoresis (DIGE), the expression of proteins in the BALF from patients on Days 1 (n = 7), 3 (n = 8), and 7 (n = 5) of ARDS were compared with findings in normal volunteers (n = 9). The patterns of protein expression were analyzed using principal component analysis (PCA). Biological processes that were enriched in the BALF proteins of patients with ARDS were identified using Gene Ontology (GO) analysis. Protein networks that model the protein interactions in the BALF were generated using Ingenuity Pathway Analysis. An average of 991 protein spots were detected using DIGE. Of these, 80 protein spots, representing 37 unique proteins in all of the fluids, were identified using mass spectrometry. PCA confirmed important differences between the proteins in the ARDS and normal samples. GO analysis showed that these differences are due to the enrichment of proteins involved in inflammation, infection, and injury. The protein network analysis showed that the protein interactions in ARDS are complex and redundant, and revealed unexpected central components in the protein networks. Proteomics and protein network analysis reveals the complex nature of lung protein interactions in ARDS. The results provide new insights about protein networks in injured lungs, and identify novel mediators that are likely to be involved in the pathogenesis and progression of acute lung injury.
Rapid Diagnostic Assay for Intact Influenza Virus Using a High Affinity Hemagglutinin Binding Protein.

PubMed

Anderson, Caitlin E; Holstein, Carly A; Strauch, Eva-Maria; Bennett, Steven; Chevalier, Aaron; Nelson, Jorgen; Fu, Elain; Baker, David; Yager, Paul

2017-06-20

Influenza is a ubiquitous and recurring infection that results in approximately 500 000 deaths globally each year. Commercially available rapid diagnostic tests are based upon detection of the influenza nucleoprotein, which are limited in that they are unable to differentiate by species and require an additional viral lysis step. Sample preprocessing can be minimized or eliminated by targeting the intact influenza virus, thereby reducing assay complexity and leveraging the large number of hemagglutinin proteins on the surface of each virus. Here, we report the development of a paper-based influenza assay that targets the hemagglutinin protein; the assay employs a combination of antibodies and novel computationally designed, recombinant affinity proteins as the capture and detection agents. This system leverages the customizability of recombinant protein design to target the conserved receptor-binding pocket of the hemagglutinin protein and to match the trimeric nature of hemagglutinin for improved avidity. Using this assay, we demonstrate the first instance of intact influenza virus detection using a combination of antibody and affinity proteins within a porous network. The recombinant head region binder based assays yield superior analytical sensitivity as compared to the antibody based assay, with lower limits of detection of 3.54 × 10 7 and 1.34 × 10 7 CEID 50 /mL for the mixed and all binder stacks, respectively. Not only does this work describe the development of a novel influenza assay, it also demonstrates the power of recombinant affinity proteins for use in rapid diagnostic assays.
DETECTION AND PURIFICATION OF TYROSINE-SULFATED PROTEINS USING A NOVEL ANTI-SULFOTYROSINE MONOCLONAL ANTIBODY*

PubMed Central

Hoffhines, Adam J.; Damoc, Eugen; Bridges, Kristie G.; Leary, Julie A.; Moore, Kevin L.

2006-01-01

Protein-tyrosine O-sulfation is a post-translational modification mediated by one of two Golgi tyrosylprotein sulfotransferases (TPST-1 & TPST-2) that catalyze the transfer of sulfate to tyrosine residues in secreted and transmembrane proteins. Tyrosine sulfation plays a role in protein-protein interactions in several well-defined systems. Although dozens of tyrosine-sulfated proteins are known, many more are likely to exist and await description. Advancing our understanding of the importance of tyrosine sulfation in biological systems requires the development of new tools for the detection and study of tyrosine-sulfated proteins. We have developed a novel anti-sulfotyrosine monoclonal antibody, called PSG2, that binds with high affinity and exquisite specificity to sulfotyrosine residues in peptides and proteins independent of sequence context. We show that it can detect tyrosinesulfated proteins in complex biological samples and can be used as a probe to assess the role of tyrosine sulfation in protein function. We also demonstrate the utility of PSG2 in the purification of tyrosine-sulfated proteins from crude tissue samples. Finally, Western blot analysis using PSG2 indicates that certain sperm/epididymal proteins are undersulfated in Tpst2−/− mice. This indicates that TPST-1 and TPST-2 have distinct macromolecular substrate specificities and provides clues as to the molecular mechanism of the infertility of Tpst2−/− males. PSG2 should be widely applicable for identification of tyrosine-sulfated proteins in other systems and organisms. PMID:17046811
Detection of human Dicer and Argonaute 2 catalytic activity

PubMed Central

Perron, Marjorie P.; Landry, Patricia; Plante, Isabelle; Provost, Patrick

2013-01-01

The microRNA (miRNA)-guided RNA silencing pathway is a central and well-defined cellular process involved in messenger RNA (mRNA) translational control. This complex regulatory process is achieved by a well orchestrated machinery composed of a relatively few protein components, among which the ribonuclease III (RNase III) Dicer and Argonaute 2 (Ago2) play a central role. These two proteins are essential and it is of particular interest to measure and detect their catalytic activity under various situations and/or conditions. In this chapter, we describe different protocols that aim to study and determine the catalytic activity of Dicer and Ago2 in cell extracts, immune complexes and size-fractionated cell extracts. Another protocol aimed at assessing miRNA binding to Ago2 is also described. These experimental approaches are likely to be useful to researchers investigating the main steps of miRNA biogenesis and function in human health and diseases. PMID:21528451
Detection and characterization of nonspecific, sparsely-populated binding modes in the early stages of complexation

PubMed Central

Cardone, A.; Bornstein, A.; Pant, H. C.; Brady, M.; Sriram, R.; Hassan, S. A.

2015-01-01

A method is proposed to study protein-ligand binding in a system governed by specific and non-specific interactions. Strong associations lead to narrow distributions in the proteins configuration space; weak and ultra-weak associations lead instead to broader distributions, a manifestation of non-specific, sparsely-populated binding modes with multiple interfaces. The method is based on the notion that a discrete set of preferential first-encounter modes are metastable states from which stable (pre-relaxation) complexes at equilibrium evolve. The method can be used to explore alternative pathways of complexation with statistical significance and can be integrated into a general algorithm to study protein interaction networks. The method is applied to a peptide-protein complex. The peptide adopts several low-population conformers and binds in a variety of modes with a broad range of affinities. The system is thus well suited to analyze general features of binding, including conformational selection, multiplicity of binding modes, and nonspecific interactions, and to illustrate how the method can be applied to study these problems systematically. The equilibrium distributions can be used to generate biasing functions for simulations of multiprotein systems from which bulk thermodynamic quantities can be calculated. PMID:25782918
Conformational heterogeneity in the Hsp70 chaperone-substrate ensemble identified from analysis of NMR-detected titration data.

PubMed

Sekhar, Ashok; Nagesh, Jayashree; Rosenzweig, Rina; Kay, Lewis E

2017-11-01

The Hsp70 chaperone system plays a critical role in cellular homeostasis by binding to client protein molecules. We have recently shown by methyl-TROSY NMR methods that the Escherichia coli Hsp70, DnaK, can form multiple bound complexes with a small client protein, hTRF1. In an effort to characterize the interactions further we report here the results of an NMR-based titration study of hTRF1 and DnaK, where both molecular components are monitored simultaneously, leading to a binding model. A central finding is the formation of a previously undetected 3:1 hTRF1-DnaK complex, suggesting that under heat shock conditions, DnaK might be able to protect cytosolic proteins whose net concentrations would exceed that of the chaperone. Moreover, these results provide new insight into the heterogeneous ensemble of complexes formed by DnaK chaperones and further emphasize the unique role of NMR spectroscopy in obtaining information about individual events in a complex binding scheme by exploiting a large number of probes that report uniquely on distinct binding processes. © 2017 The Protein Society.
Functional capabilities of an N-formyl peptide receptor-G(alpha)(i)(2) fusion protein: assemblies with G proteins and arrestins.

PubMed

Shi, Mei; Bennett, Teresa A; Cimino, Daniel F; Maestas, Diane C; Foutz, Terry D; Gurevich, Vsevolod V; Sklar, Larry A; Prossnitz, Eric R

2003-06-24

G protein-coupled receptors (GPCRs) must constantly compete for interactions with G proteins, kinases, and arrestins. To evaluate the interactions of these proteins with GPCRs in greater detail, we generated a fusion protein between the N-formyl peptide receptor and the G(alpha)(i2) protein. The functional capabilities of this chimeric protein were determined both in vivo, in stably transfected U937 cells, and in vitro, using a novel reconstitution system of solubilized components. The chimeric protein exhibited a cellular ligand binding affinity indistinguishable from that of the wild-type receptor and existed as a complex, when solubilized, containing betagamma subunits, as demonstrated by sucrose density sedimentation. The chimeric protein mobilized intracellular calcium and desensitized normally in response to agonist. Furthermore, the chimeric receptor was internalized and recycled at rates similar to those of the wild-type FPR. Confocal fluorescence microscopy revealed that internalized chimeric receptors, as identified with fluorescent ligand, colocalized with arrestin, as well as G protein, unlike wild-type receptors. Soluble reconstitution experiments demonstrated that the chimeric receptor, even in the phosphorylated state, existed as a high ligand affinity G protein complex, in the absence of exogenous G protein. This interaction was only partially prevented through the addition of arrestins. Furthermore, our results demonstrate that the GTP-bound state of the G protein alpha subunit displays no detectable affinity for the receptor. Together, these results indicate that complex interactions exist between GPCRs, in their unphosphorylated and phosphorylated states, G proteins, and arrestins, which result in the highly regulated control of GPCR function.
Simultaneous pre-concentration and separation on simple paper-based analytical device for protein analysis.

PubMed

Niu, Ji-Cheng; Zhou, Ting; Niu, Li-Li; Xie, Zhen-Sheng; Fang, Fang; Yang, Fu-Quan; Wu, Zhi-Yong

2018-02-01

In this work, fast isoelectric focusing (IEF) was successfully implemented on an open paper fluidic channel for simultaneous concentration and separation of proteins from complex matrix. With this simple device, IEF can be finished in 10 min with a resolution of 0.03 pH units and concentration factor of 10, as estimated by color model proteins by smartphone-based colorimetric detection. Fast detection of albumin from human serum and glycated hemoglobin (HBA1c) from blood cell was demonstrated. In addition, off-line identification of the model proteins from the IEF fractions with matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was also shown. This PAD IEF is potentially useful either for point of care test (POCT) or biomarker analysis as a cost-effective sample pretreatment method.
Detection and Immunolabeling of Peroxisomal Proteins.

PubMed

Schrader, Tina A; Islinger, Markus; Schrader, Michael

2017-01-01

Peroxisomes are essential organelles in mammals which contribute to cellular lipid metabolism and redox homeostasis. The spectrum of their functions in human health and disease is far from being complete, and unexpected and novel roles of peroxisomes are being discovered. To date, those include novel biological roles in antiviral defence, as intracellular signaling platforms and as protective organelles in sensory cells. Furthermore, peroxisomes are part of a complex network of interacting subcellular compartments which involves metabolic cooperation, cross-talk and membrane contacts. As potentially novel peroxisomal proteins are continuously discovered, there is great interest in the verification of their peroxisomal localization. Here, we present protocols used successfully in our laboratory for the detection and immunolabeling of peroxisomal proteins in cultured mammalian cells. We present immunofluorescence and fluorescence-based techniques as well as reagents to determine peroxisome-specific targeting and localization of candidate proteins.
Medium-Throughput Screen of Microbially Produced Serotonin via a G-Protein-Coupled Receptor-Based Sensor.

PubMed

Ehrenworth, Amy M; Claiborne, Tauris; Peralta-Yahya, Pamela

2017-10-17

Chemical biosensors, for which chemical detection triggers a fluorescent signal, have the potential to accelerate the screening of noncolorimetric chemicals produced by microbes, enabling the high-throughput engineering of enzymes and metabolic pathways. Here, we engineer a G-protein-coupled receptor (GPCR)-based sensor to detect serotonin produced by a producer microbe in the producer microbe's supernatant. Detecting a chemical in the producer microbe's supernatant is nontrivial because of the number of other metabolites and proteins present that could interfere with sensor performance. We validate the two-cell screening system for medium-throughput applications, opening the door to the rapid engineering of microbes for the increased production of serotonin. We focus on serotonin detection as serotonin levels limit the microbial production of hydroxystrictosidine, a modified alkaloid that could accelerate the semisynthesis of camptothecin-derived anticancer pharmaceuticals. This work shows the ease of generating GPCR-based chemical sensors and their ability to detect specific chemicals in complex aqueous solutions, such as microbial spent medium. In addition, this work sets the stage for the rapid engineering of serotonin-producing microbes.
Time-resolved vibrational spectroscopy detects protein-based intermediates in the photosynthetic oxygen-evolving cycle.

PubMed

Barry, Bridgette A; Cooper, Ian B; De Riso, Antonio; Brewer, Scott H; Vu, Dung M; Dyer, R Brian

2006-05-09

Photosynthetic oxygen production by photosystem II (PSII) is responsible for the maintenance of aerobic life on earth. The production of oxygen occurs at the PSII oxygen-evolving complex (OEC), which contains a tetranuclear manganese (Mn) cluster. Photo-induced electron transfer events in the reaction center lead to the accumulation of oxidizing equivalents on the OEC. Four sequential photooxidation reactions are required for oxygen production. The oxidizing complex cycles among five oxidation states, called the S(n) states, where n refers to the number of oxidizing equivalents stored. Oxygen release occurs during the S(3)-to-S(0) transition from an unstable intermediate, known as the S(4) state. In this report, we present data providing evidence for the production of an intermediate during each S state transition. These protein-derived intermediates are produced on the microsecond to millisecond time scale and are detected by time-resolved vibrational spectroscopy on the microsecond time scale. Our results suggest that a protein-derived conformational change or proton transfer reaction precedes Mn redox reactions during the S(2)-to-S(3) and S(3)-to-S(0) transitions.

Development of a homogeneous immunoassay system using protein A fusion fragmented Renilla luciferase.

PubMed

Mie, Masayasu; Thuy, Ngo Phan Bich; Kobatake, Eiry

2012-03-07

A homogeneous immunoassay system was developed using fragmented Renilla luciferase (Rluc). The B domain of protein A was fused to two Rluc fragments. When complexes between an antibody and fragmented Rluc fusion proteins bind to target molecules, the Rluc fragments come into close proximity and the luminescence activity of fragmented Rluc is restored by complementation. As proof-of-principle, this fragmented Rluc system was used to detect E. coli homogeneously using an anti-E. coli antibody.
Psp Stress Response Proteins Form a Complex with Mislocalized Secretins in the Yersinia enterocolitica Cytoplasmic Membrane.

PubMed

Srivastava, Disha; Moumene, Amal; Flores-Kim, Josué; Darwin, Andrew J

2017-09-12

The bacterial phage shock protein system (Psp) is a conserved extracytoplasmic stress response that is essential for the virulence of some pathogens, including Yersinia enterocolitica It is induced by events that can compromise inner membrane (IM) integrity, including the mislocalization of outer membrane pore-forming proteins called secretins. In the absence of the Psp system, secretin mislocalization permeabilizes the IM and causes rapid cell death. The Psp proteins PspB and PspC form an integral IM complex with two independent roles. First, the PspBC complex is required to activate the Psp response in response to some inducing triggers, including a mislocalized secretin. Second, PspBC are sufficient to counteract mislocalized secretin toxicity. Remarkably, secretin mislocalization into the IM induces psp gene expression without significantly affecting the expression of any other genes. Furthermore, psp null strains are killed by mislocalized secretins, whereas no other null mutants have been found to share this specific secretin sensitivity. This suggests an exquisitely specific relationship between secretins and the Psp system, but there has been no mechanism described to explain this. In this study, we addressed this deficiency by using a coimmunoprecipitation approach to show that the Psp proteins form a specific complex with mislocalized secretins in the Y. enterocolitica IM. Importantly, analysis of different secretin mutant proteins also revealed that this interaction is absolutely dependent on a secretin adopting a multimeric state. Therefore, the Psp system has evolved with the ability to detect and detoxify dangerous secretin multimers while ignoring the presence of innocuous monomers. IMPORTANCE The phage shock protein (Psp) response has been linked to important phenotypes in diverse bacteria, including those related to antibiotic resistance, biofilm formation, and virulence. This has generated widespread interest in understanding various aspects of its function. Outer membrane secretin proteins are essential components of export systems required for the virulence of many bacterial pathogens. However, secretins can mislocalize into the inner membrane, and this induces the Psp response in a highly specific manner and kills Psp-defective strains with similar specificity. There has been no mechanism described to explain this exquisitely specific relationship between secretins and the Psp system. Therefore, this study provides a critical advance by discovering that Psp effector proteins form a complex with secretins in the Yersinia enterocolitica inner membrane. Remarkably, this interaction is absolutely dependent on a secretin adopting its multimeric state. Therefore, the Psp system detects and detoxifies dangerous secretin multimers, while ignoring the presence of innocuous secretin monomers. Copyright © 2017 Srivastava et al.
Interaction of the Human Respiratory Syncytial Virus matrix protein with cellular adaptor protein complex 3 plays a critical role in trafficking.

PubMed

Ward, Casey; Maselko, Maciej; Lupfer, Christopher; Prescott, Meagan; Pastey, Manoj K

2017-01-01

Human Respiratory Syncytial Virus (HRSV) is a leading cause of bronchopneumonia in infants and the elderly. To date, knowledge of viral and host protein interactions within HRSV is limited and are critical areas of research. Here, we show that HRSV Matrix (M) protein interacts with the cellular adaptor protein complex 3 specifically via its medium subunit (AP-3Mu3A). This novel protein-protein interaction was first detected via yeast-two hybrid screen and was further confirmed in a mammalian system by immunofluorescence colocalization and co-immunoprecipitation. This novel interaction is further substantiated by the presence of a known tyrosine-based adaptor protein MU subunit sorting signal sequence, YXXФ: where Ф is a bulky hydrophobic residue, which is conserved across the related RSV M proteins. Analysis of point-mutated HRSV M derivatives indicated that AP-3Mu3A- mediated trafficking is contingent on the presence of the tyrosine residue within the YXXL sorting sequence at amino acids 197-200 of the M protein. AP-3Mu3A is up regulated at 24 hours post-infection in infected cells versus mock-infected HEp2 cells. Together, our data suggests that the AP-3 complex plays a critical role in the trafficking of HRSV proteins specifically matrix in epithelial cells. The results of this study add new insights and targets that may lead to the development of potential antivirals and attenuating mutations suitable for candidate vaccines in the future.
Evaluation of Gallium as a Tracer of Exogenous Hemoglobin-Haptoglobin Complexes for Targeted Drug Delivery Applications

NASA Astrophysics Data System (ADS)

Xu, Shengsheng; Kaltashov, Igor A.

2016-12-01

Haptoglobin (Hp) is a plasma glycoprotein that generates significant interest in the drug delivery community because of its potential for delivery of antiretroviral medicines with high selectivity to macrophages and monocytes, the latent reservoirs of human immunodeficiency virus. As is the case with other therapies that exploit transport networks for targeted drug delivery, the success of the design and optimization of Hp-based therapies will critically depend on the ability to accurately localize and quantitate Hp-drug conjugates on the varying and unpredictable background of endogenous proteins having identical structure. In this work, we introduce a new strategy for detecting and quantitating exogenous Hp and Hp-based drugs with high sensitivity in complex biological samples using gallium as a tracer of this protein and inductively coupled plasma mass spectrometry (ICP MS) as a method of detection. Metal label is introduced by reconstituting hemoglobin (Hb) with gallium(III)-protoporphyrin IX followed by its complexation with Hp. Formation of the Hp/Hb assembly and its stability are evaluated with native electrospray ionization mass spectrometry. Both stable isotopes of Ga give rise to an abundant signal in ICP MS of a human plasma sample spiked with the metal-labeled Hp/Hb complex. The metal label signal exceeds the spectral interferences' contributions by more than an order of magnitude even with the concentration of the exogenous protein below 10 nM, the level that is more than adequate for the planned pharmacokinetic studies of Hp-based therapeutics.
Bovine oocytes and early embryos express Staufen and ELAVL RNA-binding proteins.

PubMed

Calder, M D; Madan, P; Watson, A J

2008-05-01

RNA-binding proteins (RBP) influence RNA editing, localization, stability and translation and may contribute to oocyte developmental competence by regulating the stability and turnover of oogenetic mRNAs. The expression of Staufen 1 and 2 and ELAVL1, ELAVL2 RNA-binding proteins during cow early development was characterized. Cumulus-oocyte complexes were collected from slaughterhouse ovaries, matured, inseminated and subjected to embryo culture in vitro. Oocyte or preimplantation embryo pools were processed for RT-PCR and whole-mount immunofluorescence analysis of mRNA expression and protein distribution. STAU1 and STAU2 and ELAVL1 mRNAs and proteins were detected throughout cow preimplantation development from the germinal vesicle (GV) oocyte to the blastocyst stage. ELAVL2 mRNAs were detectable from the GV to the morula stage, whereas ELAVL2 protein was in all stages examined and localized to both cytoplasm and nuclei. The findings provide a foundation for investigating the role of RBPs during mammalian oocyte maturation and early embryogenesis.
Variations in immunohistochemical preservation of proteins in a mummification model

PubMed Central

Metcalfe, Ryan; Freemont, Tony

2012-01-01

Immunohistochemistry is an important tool in the investigation of ancient mummified remains because of its ability not only to detect proteins but also to isolate their location to specific tissues and thereby improve confidence that the results are genuine. A mouse model of Egyptian mummification has been used to demonstrate that the survival of proteins, judged by the retention of immunohistochemical staining, varies markedly. Some survive the process well, whereas others become barely detectable despite the morphology of the tissue being excellently preserved. The results obtained show that protein preservation is multi-factorial, with tissue type and degradation, and the properties of the protein itself all having significant effects. Proteins forming large, multi-subunit complexes such as collagen IV appear to be more resistant to degradation than those that do not, such as S-100. Although modern modelling studies cannot replicate the full extent of degradative processes and taphonomic changes experienced by real mummies, the results obtained can be useful for guiding research that requires ancient tissues. PMID:22050406
THz frequency spectrum of protein-solvent interaction energy using a recurrence plot-based Wiener-Khinchin method.

PubMed

Karain, Wael

2016-10-01

The dynamics of a protein and the water surrounding it are coupled via nonbonded energy interactions. This coupling can exhibit a complex, nonlinear, and nonstationary nature. The THz frequency spectrum for this interaction energy characterizes both the vibration spectrum of the water hydrogen bond network, and the frequency range of large amplitude modes of proteins. We use a Recurrence Plot based Wiener-Khinchin method RPWK to calculate this spectrum, and the results are compared to those determined using the classical auto-covariance-based Wiener-Khinchin method WK. The frequency spectra for the total nonbonded interaction energy extracted from molecular dynamics simulations between the β-Lactamase Inhibitory Protein BLIP, and water molecules within a 10 Å distance from the protein surface, are calculated at 150, 200, 250, and 310 K, respectively. Similar calculations are also performed for the nonbonded interaction energy between the residues 49ASP, 53TYR, and 142PHE in BLIP, with water molecules within 10 Å from each residue respectively at 150, 200, 250, and 310 K. A comparison of the results shows that RPWK performs better than WK, and is able to detect some frequency data points that WK fails to detect. This points to the importance of using methods capable of taking the complex nature of the protein-solvent energy landscape into consideration, and not to rely on standard linear methods. In general, RPWK can be a valuable addition to the analysis tools for protein molecular dynamics simulations. Proteins 2016; 84:1549-1557. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.
Cancer-cells on a chip for label-free optic detection of secreted molecules

NASA Astrophysics Data System (ADS)

Berthuy, Ophélie I.; Blum, Loïc. J.; Marquette, Christophe A.

2015-05-01

To unravel cell complexity, living-cell chips have been developed that allow delivery of experimental stimuli but also measurement of the resulting cellular responses. We have been developing a new concept for multiplexed detection of biomolecules secreted by different cancer cells. In the present report, we are making the proof of concept of cell small populations (from 1 to 100 cells) spotting, culture and secretion detection on a gold surface. For that purpose, antibodies and different cell lines were spotted using a piezoelectric spotter. In order to keep the cells in a hydrated environment during the robotized micropipetting and to address different cell lines on a single chip, a biocompatible alginate polymer was used. This approach enables the encapsulation of the cell in a very small volume (30 nL), directly on the substrate and permits a precise control of the number of cells in each alginate bead. After 24h of culture, the adherent cells are ready for surface plasmon resonance imaging (SPRi) experimentation. To enable the detection of secreted proteins, various antibodies are immobilized in an organized manner on a SPRi sensor and permitted the multiplex detection of different proteins secreted by the different cultured cell lines. Evidence of the real-time detection will be presented for Prostate Specific Antigen (PSA) and β-2-microglobulin (B2M) secreted by prostate cancer cells following induction by dihydrotestosterone (DHT). Different kinetics for the two secreted proteins were then demonstrated and precisely determined using the chip. There is no doubt that our chip will, in a near future, be applied to more multiplexed and complex biological secretion systems for which kinetic data are at the moment not reachable using standard cellular biology tools.
Multiplex detection of protein toxins using MALDI-TOF-TOF tandem mass spectrometry: application in unambiguous toxin detection from bioaerosol.

PubMed

Alam, Syed Imteyaz; Kumar, Bhoj; Kamboj, Dev Vrat

2012-12-04

Protein toxins, such as botulinum neurotoxins (BoNTs), Clostridium perfringens epsilon toxin (ETX), staphylococcal enterotoxin B (SEB), shiga toxin (STX), and plant toxin ricin, are involved in a number of diseases and are considered as potential agents for bioterrorism and warfare. From a bioterrorism and warfare perspective, these agents are likely to cause maximum damage to a civilian or military population through an inhalational route of exposure and aerosol is considered the envisaged mode of delivery. Unambiguous detection of toxin from aerosol is of paramount importance, both for bringing mitigation protocols into operation and for implementation of effective medical countermeasures, in case a "biological cloud" is seen over a population. A multiplex, unambiguous, and qualitative detection of protein toxins is reported here using tandem mass spectrometry with MALDI-TOF-TOF. The methodology involving simple sample processing steps was demonstrated to identify toxins (ETX, Clostridium perfringes phospholipase C, and SEB) from blind spiked samples. The novel directed search approach using a list of unique peptides was used to identify toxins from a complex protein mixture. The bioinformatic analysis of seven protein toxins for elucidation of unique peptides with conservation status across all known sequences provides a high confidence for detecting toxins originating from any geographical location and source organism. Use of tandem MS data with peptide sequence information increases the specificity of the method. A prototype for generation of aerosol using a nebulizer and collection using a cyclone collector was used to provide a proof of concept for unambiguous detection of toxin from aerosol using precursor directed tandem mass spectrometry combined with protein database searching. ETX prototoxin could be detected from aerosol at 0.2 ppb concentration in aerosol.
Antigen Retrieval to Improve the Immunocytochemistry Detection of Sigma-1 Receptors and ER Chaperones

PubMed Central

Hayashi, Teruo; Lewis, Abasha; Hayashi, Eri; Betenbaugh, Michael J.; Su, Tsung-Ping

2011-01-01

Molecular chaperones localized at the endoplasmic reticulum (ER) lumen constitutively or cellular stress-dependently associate with a variety of proteins to promote their proper folding or to inhibit protein misfolding. ER chaperones preferentially form large complexes with co-chaperones and/or misfolded proteins in a highly crowded cellular environment that often hampers their detection by immunocytochemistry (ICC). This study establishes an antigen retrieval (AR) protocol to improve the ICC detection of ER chaperones in cultured cells using widely available antibodies against synthetic peptides. Among ten different antigen retrieval/fixation conditions, only the AR with Tris-HCl (pH 9.5) containing 6 M urea (80 °C for 10 min) significantly improved the ICC detection of the novel ER chaperone sigma-1 receptor (Sig-1R) in Chinese hamster ovary cells. Extended fixation with 4% paraformaldehyde for 1 hr effectively preserved the morphology of the ER under the AR condition. This method greatly enhanced the signal-to-noise ratio in Sig-1R ICC, thus allowing for semi-quantitative detection of protein upregulation under ER stress. The AR similarly improved the ICC detection of a series of other major ER chaperones, including BiP/GRP78, GRP94, calnexin, calreticulin, ERp57, protein disulfide isomerase, and cyclophilin B. The improved ICC methodology using the urea AR at 80°C may improve ICC of ER molecules as well as visualization of ER structure and substructures. PMID:21573736
Detecting UV-lesions in the genome: The modular CRL4 ubiquitin ligase does it best!

PubMed

Scrima, Andrea; Fischer, Eric S; Lingaraju, Gondichatnahalli M; Böhm, Kerstin; Cavadini, Simone; Thomä, Nicolas H

2011-09-16

The DDB1-DDB2-CUL4-RBX1 complex serves as the primary detection device for UV-induced lesions in the genome. It simultaneously functions as a CUL4 type E3 ubiquitin ligase. We review the current understanding of this dual function ubiquitin ligase and damage detection complex. The DDB2 damage binding module is merely one of a large family of possible DDB1-CUL4 associated factors (DCAF), most of which are substrate receptors for other DDB1-CUL4 complexes. DDB2 and the Cockayne-syndrome A protein (CSA) function in nucleotide excision repair, whereas the remaining receptors operate in a wide range of other biological pathways. We will examine the modular architecture of DDB1-CUL4 in complex with DDB2, CSA and CDT2 focusing on shared architectural, targeting and regulatory principles. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
A Minimal Anaphase Promoting Complex/Cyclosome (APC/C) in Trypanosoma brucei

PubMed Central

Bessat, Mohamed; Knudsen, Giselle; Burlingame, Alma L.; Wang, Ching C.

2013-01-01

The anaphase-promoting complex/cyclosome (APC/C) is a multi-subunit E3 ubiquitin ligase that initiates chromosome segregation and mitotic exit by targeting critical cell-cycle regulators for proteolytic destruction. Previously, seven APC/C subunit homologues were identified in the genome of Trypanosoma brucei. In the present study, we tested five of them in yeast complementation studies and found none of them capable of complementing the yeast mutants lacking the corresponding subunits, suggesting significant discrepancies between the two APC/C’s. Subunit homologues of mitotic checkpoint complex (MCC) have not yet been identified in T. brucei, raising the possibility that a MCC-APC/C complex equivalent may not exist in T. brucei. We performed tandem affinity purification of the protein complex containing a APC1 fusion protein expressed in the cells enriched in different phases of the cell cycle of procyclic form T. brucei, and compared their protein profiles using LC-MS/MS analyses. The seven putative APC/C subunits were identified in the protein complex throughout the cell cycle together with three additional proteins designated the associated proteins (AP) AP1, AP2 and AP3. Abundance of the 10 proteins remained relatively unchanged throughout the cell cycle, suggesting that they are the core subunits of APC/C. AP1 turned out to be a homologue of APC4. An RNAi knockdown of APC4 and AP3 showed no detectable cellular phenotype, whereas an AP2 knockdown enriched the cells in G2/M phase. The AP2-depleted cells showed stabilized mitotic cyclin B. An accumulation of poly-ubiquitinated cyclin B was indicated in the cells treated with the proteasome inhibitor MG132, demonstrating the involvement of proteasome in degrading poly-ubiquitinated cyclin B. In all, a 10-subunit APC/C machinery with a conserved function is identified in T. brucei without linking to a MCC-like complex, thus indicating a unique T. brucei APC/C. PMID:23533609
Protein corona – from molecular adsorption to physiological complexity

PubMed Central

Docter, Dominic; Maskos, Michael

2015-01-01

Summary In biological environments, nanoparticles are enshrouded by a layer of biomolecules, predominantly proteins, mediating its subsequent interactions with cells. Detecting this protein corona, understanding its formation with regards to nanoparticle (NP) and protein properties, and elucidating its biological implications were central aims of bio-related nano-research throughout the past years. Here, we discuss the mechanistic parameters that are involved in the protein corona formation and the consequences of this corona formation for both, the particle, and the protein. We review consequences of corona formation for colloidal stability and discuss the role of functional groups and NP surface functionalities in shaping NP–protein interactions. We also elaborate the recent advances demonstrating the strong involvement of Coulomb-type interactions between NPs and charged patches on the protein surface. Moreover, we discuss novel aspects related to the complexity of the protein corona forming under physiological conditions in full serum. Specifically, we address the relation between particle size and corona composition and the latest findings that help to shed light on temporal evolution of the full serum corona for the first time. Finally, we discuss the most recent advances regarding the molecular-scale mechanistic role of the protein corona in cellular uptake of NPs. PMID:25977856
Protein corona - from molecular adsorption to physiological complexity.

PubMed

Treuel, Lennart; Docter, Dominic; Maskos, Michael; Stauber, Roland H

2015-01-01

In biological environments, nanoparticles are enshrouded by a layer of biomolecules, predominantly proteins, mediating its subsequent interactions with cells. Detecting this protein corona, understanding its formation with regards to nanoparticle (NP) and protein properties, and elucidating its biological implications were central aims of bio-related nano-research throughout the past years. Here, we discuss the mechanistic parameters that are involved in the protein corona formation and the consequences of this corona formation for both, the particle, and the protein. We review consequences of corona formation for colloidal stability and discuss the role of functional groups and NP surface functionalities in shaping NP-protein interactions. We also elaborate the recent advances demonstrating the strong involvement of Coulomb-type interactions between NPs and charged patches on the protein surface. Moreover, we discuss novel aspects related to the complexity of the protein corona forming under physiological conditions in full serum. Specifically, we address the relation between particle size and corona composition and the latest findings that help to shed light on temporal evolution of the full serum corona for the first time. Finally, we discuss the most recent advances regarding the molecular-scale mechanistic role of the protein corona in cellular uptake of NPs.
Protein Interactions and Localization of the Escherichia coli Accessory Protein HypA during Nickel Insertion to [NiFe] Hydrogenase*

PubMed Central

Chan Chung, Kim C.; Zamble, Deborah B.

2011-01-01

Nickel delivery during maturation of Escherichia coli [NiFe] hydrogenase 3 includes the accessory proteins HypA, HypB, and SlyD. Although the isolated proteins have been characterized, little is known about how they interact with each other and the hydrogenase 3 large subunit, HycE. In this study the complexes of HypA and HycE were investigated after modification with the Strep-tag II. Multiprotein complexes containing HypA, HypB, SlyD, and HycE were observed, consistent with the assembly of a single nickel insertion cluster. An interaction between HypA and HycE did not require the other nickel insertion proteins, but HypB was not found with the large subunit in the absence of HypA. The HypA-HycE complex was not detected in the absence of the HypC or HypD proteins, involved in the preceding iron insertion step, and this interaction is enhanced by nickel brought into the cell by the NikABCDE membrane transporter. Furthermore, without the hydrogenase 1, 2, and 3 large subunits, complexes between HypA, HypB, and SlyD were observed. These results support the hypothesis that HypA acts as a scaffold for assembly of the nickel insertion proteins with the hydrogenase precursor protein after delivery of the iron center. At different stages of the hydrogenase maturation process, HypA was observed at or near the cell membrane by using fluorescence confocal microscopy, as was HycE, suggesting membrane localization of the nickel insertion event. PMID:22016389
A parallelized binary search tree

USDA-ARS?s Scientific Manuscript database

PTTRNFNDR is an unsupervised statistical learning algorithm that detects patterns in DNA sequences, protein sequences, or any natural language texts that can be decomposed into letters of a finite alphabet. PTTRNFNDR performs complex mathematical computations and its processing time increases when i...
Characterization of molecular defects in xeroderma pigmentosum group F in relation to its clinically mild symptoms.

PubMed

Matsumura, Y; Nishigori, C; Yagi, T; Imamura, S; Takebe, H

1998-06-01

Xeroderma pigmentosum (XP) complementation group F was first reported in Japan and most XP-F patients reported to date are Japanese. The clinical features of XP-F patients are rather mild, including late onset of skin cancer. Recently a cDNA that corrects the repair deficiency of cultured XP-F cells was isolated. The XPF protein forms a tight complex with ERCC1 and this complex functions as a structure-specific endonuclease responsible for the 5' incision during DNA excision repair. Here we have identified XPF mRNA mutations and examined levels of the mRNA and protein expression in seven primary cell strains from Japanese XP-F patients. The XP-F cell strains were classified into three types in terms of the effect of the mutation on the predicted protein; (i) XPF proteins with amino acid substitutions; (ii) amino acid substituted and truncated XPF proteins; and (iii) truncated XPF protein only. A normal level of expression of XPF mRNA was observed in XP-F cells but XPF protein was extremely low. These results indicate that the detected mutations lead to unstable XPF protein, resulting in a decrease in formation of the ERCC1-XPF endonuclease complex. Slow excision repair of UV-induced DNA damage due to low residual endonuclease activity provides a plausible explanation for the typical mild phenotype of XP-F patients.
ABRF-PRG07: advanced quantitative proteomics study.

PubMed

Falick, Arnold M; Lane, William S; Lilley, Kathryn S; MacCoss, Michael J; Phinney, Brett S; Sherman, Nicholas E; Weintraub, Susan T; Witkowska, H Ewa; Yates, Nathan A

2011-04-01

A major challenge for core facilities is determining quantitative protein differences across complex biological samples. Although there are numerous techniques in the literature for relative and absolute protein quantification, the majority is nonroutine and can be challenging to carry out effectively. There are few studies comparing these technologies in terms of their reproducibility, accuracy, and precision, and no studies to date deal with performance across multiple laboratories with varied levels of expertise. Here, we describe an Association of Biomolecular Resource Facilities (ABRF) Proteomics Research Group (PRG) study based on samples composed of a complex protein mixture into which 12 known proteins were added at varying but defined ratios. All of the proteins were present at the same concentration in each of three tubes that were provided. The primary goal of this study was to allow each laboratory to evaluate its capabilities and approaches with regard to: detection and identification of proteins spiked into samples that also contain complex mixtures of background proteins and determination of relative quantities of the spiked proteins. The results returned by 43 participants were compiled by the PRG, which also collected information about the strategies used to assess overall performance and as an aid to development of optimized protocols for the methodologies used. The most accurate results were generally reported by the most experienced laboratories. Among laboratories that used the same technique, values that were closer to the expected ratio were obtained by more experienced groups.
Isolation and characterization of melanopsin and pinopsin expression within photoreceptive sites of reptiles

NASA Astrophysics Data System (ADS)

Frigato, Elena; Vallone, Daniela; Bertolucci, Cristiano; Foulkes, Nicholas S.

2006-08-01

Non-mammalian vertebrates have multiple extraocular photoreceptors, mainly localised in the pineal complex and the brain, to mediate irradiance detection. In this study, we report the full-length cDNA cloning of ruin lizard melanopsin and pinopsin. The high level of identity with opsins in both the transmembrane regions, where the chromophore binding site is located, and the intracellular loops, where the G-proteins interact, suggests that both melanopsin and pinopsin should be able to generate a stable photopigment, capable of triggering a transduction cascade mediated by G-proteins. Phylogenetic analysis showed that both opsins are located on the expected branches of the corresponding sequences of ortholog proteins. Subsequently, using RT-PCR and RPA analysis, we verified the expression of ruin lizard melanopsin and pinopsin in directly photosensitive organs, such as the lateral eye, brain, pineal gland and parietal eye. Melanopsin expression was detected in the lateral eye and all major regions of the brain. However, different from the situation in Xenopus and chicken, melanopsin is not expressed in the ruin lizard pineal. Pinopsin mRNA expression was only detected in the pineal complex. As a result of their phylogenetic position and ecology, reptiles provide the circadian field with some of the most interesting models for understanding the evolution of the vertebrate circadian timing system and its response to light. This characterization of melanopsin and pinopsin expression in the ruin lizard will be important for future studies aimed at understanding the molecular basis of circadian light detection in reptiles.
Structural Protein 4.1 in the Nucleus of Human Cells: Dynamic Rearrangements during Cell Division

PubMed Central

Krauss, Sharon Wald; Larabell, Carolyn A.; Lockett, Stephen; Gascard, Philippe; Penman, Sheldon; Mohandas, Narla; Chasis, Joel Anne

1997-01-01

Structural protein 4.1, first identified as a crucial 80-kD protein in the mature red cell membrane skeleton, is now known to be a diverse family of protein isoforms generated by complex alternative mRNA splicing, variable usage of translation initiation sites, and posttranslational modification. Protein 4.1 epitopes are detected at multiple intracellular sites in nucleated mammalian cells. We report here investigations of protein 4.1 in the nucleus. Reconstructions of optical sections of human diploid fibroblast nuclei using antibodies specific for 80-kD red cell 4.1 and for 4.1 peptides showed 4.1 immunofluorescent signals were intranuclear and distributed throughout the volume of the nucleus. After sequential extractions of cells in situ, 4.1 epitopes were detected in nuclear matrix both by immunofluorescence light microscopy and resinless section immunoelectron microscopy. Western blot analysis of fibroblast nuclear matrix protein fractions, isolated under identical extraction conditions as those for microscopy, revealed several polypeptide bands reactive to multiple 4.1 antibodies against different domains. Epitope-tagged protein 4.1 was detected in fibroblast nuclei after transient transfections using a construct encoding red cell 80-kD 4.1 fused to an epitope tag. Endogenous protein 4.1 epitopes were detected throughout the cell cycle but underwent dynamic spatial rearrangements during cell division. Protein 4.1 was observed in nucleoplasm and centrosomes at interphase, in the mitotic spindle during mitosis, in perichromatin during telophase, as well as in the midbody during cytokinesis. These results suggest that multiple protein 4.1 isoforms may contribute significantly to nuclear architecture and ultimately to nuclear function. PMID:9128242

Establishment of a protein frequency library and its application in the reliable identification of specific protein interaction partners.

PubMed

Boulon, Séverine; Ahmad, Yasmeen; Trinkle-Mulcahy, Laura; Verheggen, Céline; Cobley, Andy; Gregor, Peter; Bertrand, Edouard; Whitehorn, Mark; Lamond, Angus I

2010-05-01

The reliable identification of protein interaction partners and how such interactions change in response to physiological or pathological perturbations is a key goal in most areas of cell biology. Stable isotope labeling with amino acids in cell culture (SILAC)-based mass spectrometry has been shown to provide a powerful strategy for characterizing protein complexes and identifying specific interactions. Here, we show how SILAC can be combined with computational methods drawn from the business intelligence field for multidimensional data analysis to improve the discrimination between specific and nonspecific protein associations and to analyze dynamic protein complexes. A strategy is shown for developing a protein frequency library (PFL) that improves on previous use of static "bead proteomes." The PFL annotates the frequency of detection in co-immunoprecipitation and pulldown experiments for all proteins in the human proteome. It can provide a flexible and objective filter for discriminating between contaminants and specifically bound proteins and can be used to normalize data values and facilitate comparisons between data obtained in separate experiments. The PFL is a dynamic tool that can be filtered for specific experimental parameters to generate a customized library. It will be continuously updated as data from each new experiment are added to the library, thereby progressively enhancing its utility. The application of the PFL to pulldown experiments is especially helpful in identifying either lower abundance or less tightly bound specific components of protein complexes that are otherwise lost among the large, nonspecific background.
Uncoupling of transcription and translation of Fanconi anemia (FANC) complex proteins during spermatogenesis

PubMed Central

Jamsai, Duangporn; O’Connor, Anne E; O’Donnell, Liza; Lo, Jennifer Chi Yi; O’Bryan, Moira K

2015-01-01

Male germ cell genome integrity is critical for spermatogenesis, fertility and normal development of the offspring. Several DNA repair pathways exist in male germ cells. One such important pathway is the Fanconi anemia (FANC) pathway. Unlike in somatic cells, expression profiles and the role of the FANC pathway in germ cells remain largely unknown. In this study, we undertook an extensive expression analyses at both mRNA and protein levels of key components of the FANC pathway during spermatogenesis in the mouse. Herein we show that Fanc mRNAs and proteins displayed developmental enrichment within particular male germ cell types. Spermatogonia and pre-leptotene spermatocytes contained the majority of the FANC components examined i.e. complex I members FANCB, FANCG and FANCM, complex II members FANCD2 and FANCI, and complex III member FANCJ. Leptotene, zygotene and early pachytene spermatocytes contained FANCB, FANCG, FANCM and FANCD2. With the exception of FANCL, all FANC proteins examined were not detected in round spermatids. Elongating and elongated spermatids contained FANCB, FANCG, FANCL and FANCJ. qPCR analysis on isolated spermatocytes and round spermatids showed that Fancg, Fancl, Fancm, Fancd2, Fanci and Fancj mRNAs were expressed in both of these germ cell types, indicating that some degree of translational repression of these FANC proteins occurs during the transition from meiosis to spermiogenesis. Taken together, our findings raise the possibility that the assembly of FANC protein complexes in each of the male germ cell type is unique and may be distinct from the proposed model in mitotic cells. PMID:26413409
Gold nanoparticles-based electrochemical method for the detection of protein kinase with a peptide-like inhibitor as the bioreceptor

PubMed Central

Sun, Kai; Chang, Yong; Zhou, Binbin; Wang, Xiaojin; Liu, Lin

2017-01-01

This article presents a general method for the detection of protein kinase with a peptide-like kinase inhibitor as the bioreceptor, and it was done by converting gold nanoparticles (AuNPs)-based colorimetric assay into sensitive electrochemical analysis. In the colorimetric assay, the kinase-specific aptameric peptide triggered the aggregation of AuNPs in solution. However, the specific binding of peptide to the target protein (kinase) inhibited its ability to trigger the assembly of AuNPs. In the electrochemical analysis, peptides immobilized on a gold electrode and presented as solution triggered together the in situ formation of AuNPs-based network architecture on the electrode surface. Nevertheless, the formation of peptide–kinase complex on the electrode surface made the peptide-triggered AuNPs assembly difficult. Electrochemical impedance spectroscopy was used to measure the change in surface property in the binding events. When a ferrocene-labeled peptide (Fc-peptide) was used in this design, the network of AuNPs/Fc-peptide produced a good voltammetric signal. The competitive assay allowed for the detection of protein kinase A with a detection limit of 20 mU/mL. This work should be valuable for designing novel optical or electronic biosensors and likely lead to many detection applications. PMID:28331314
RNA–protein binding kinetics in an automated microfluidic reactor

PubMed Central

Ridgeway, William K.; Seitaridou, Effrosyni; Phillips, Rob; Williamson, James R.

2009-01-01

Microfluidic chips can automate biochemical assays on the nanoliter scale, which is of considerable utility for RNA–protein binding reactions that would otherwise require large quantities of proteins. Unfortunately, complex reactions involving multiple reactants cannot be prepared in current microfluidic mixer designs, nor is investigation of long-time scale reactions possible. Here, a microfluidic ‘Riboreactor’ has been designed and constructed to facilitate the study of kinetics of RNA–protein complex formation over long time scales. With computer automation, the reactor can prepare binding reactions from any combination of eight reagents, and is optimized to monitor long reaction times. By integrating a two-photon microscope into the microfluidic platform, 5-nl reactions can be observed for longer than 1000 s with single-molecule sensitivity and negligible photobleaching. Using the Riboreactor, RNA–protein binding reactions with a fragment of the bacterial 30S ribosome were prepared in a fully automated fashion and binding rates were consistent with rates obtained from conventional assays. The microfluidic chip successfully combines automation, low sample consumption, ultra-sensitive fluorescence detection and a high degree of reproducibility. The chip should be able to probe complex reaction networks describing the assembly of large multicomponent RNPs such as the ribosome. PMID:19759214
NMR structural studies of the supramolecular adducts between a liver cytosolic bile acid binding protein and gadolinium(III)-chelates bearing bile acids residues: molecular determinants of the binding of a hepatospecific magnetic resonance imaging contrast agent.

PubMed

Assfalg, Michael; Gianolio, Eliana; Zanzoni, Serena; Tomaselli, Simona; Russo, Vito Lo; Cabella, Claudia; Ragona, Laura; Aime, Silvio; Molinari, Henriette

2007-11-01

The binding affinities of a selected series of Gd(III) chelates bearing bile acid residues, potential hepatospecific MRI contrast agents, to a liver cytosolic bile acid transporter, have been determined through relaxivity measurements. The Ln(III) complexes of compound 1 were selected for further NMR structural analysis aimed at assessing the molecular determinants of binding. A number of NMR experiments have been carried out on the bile acid-like adduct, using both diamagnetic Y(III) and paramagnetic Gd(III) complexes, bound to a liver bile acid binding protein. The identified protein "hot spots" defined a single binding site located at the protein portal region. The presented findings will serve in a medicinal chemistry approach for the design of hepatocytes-selective gadolinium chelates for liver malignancies detection.
DETECTION OF TWO ISOMERIC BINDING CONFIGURATIONS IN A PROTEIN-APTAMER COMPLEX WITH A BIOLOGICAL NANOPORE

PubMed Central

Van Meervelt, Veerle; Soskine, Misha; Maglia, Giovanni

2015-01-01

Protein-DNA interactions play critical roles in biological systems, and they often involve complex mechanisms and dynamics that are not easily measured by ensemble experiments. Recently, we have shown that folded proteins can be internalised inside ClyA nanopores and studied by ionic current recordings at the single-molecule level. Here, we use ClyA nanopores to sample the interaction between the G-quadruplex fold of the thrombin binding aptamer (TBA) and human thrombin (HT). Surprisingly, the internalisation of the HT:TBA complex inside the nanopore induced two types of current blockades with distinguished residual current and lifetime. Using single nucleobase substitutions to TBA we showed that these two types of blockades originate from TBA binding to thrombin with two isomeric orientations. Voltage dependencies and the use of ClyA nanopores with two different diameters allowed assessing the effect of the applied potential and confinement, and revealed that the two binding configurations of TBA to HT display different lifetimes. These results show that the ClyA nanopores might provide a new approach to probe conformational heterogeneity in protein:DNA interactions. PMID:25493908
An SH2 domain-based tyrosine kinase assay using biotin ligase modified with a terbium(III) complex.

PubMed

Sueda, Shinji; Shinboku, Yuki; Kusaba, Takeshi

2013-01-01

Src homology 2 (SH2) domains are modules of approximately 100 amino acids and are known to bind phosphotyrosine-containing sequences with high affinity and specificity. In the present work, we developed an SH2 domain-based assay for Src tyrosine kinase using a unique biotinylation reaction from archaeon Sulfolobus tokodaii. S. tokodaii biotinylation has a unique property that biotin protein ligase (BPL) forms a stable complex with its biotinylated substrate protein (BCCP). Here, an SH2 domain from lymphocyte-specific tyrosine kinase was genetically fused to a truncated BCCP, and the resulting fusion protein was labeled through biotinylation with BPL carrying multiple copies of a luminescent Tb(3+) complex. The labeled SH2 fusion proteins were employed to detect a phosphorylated peptide immobilized on the surface of the microtiter plate, where the phosphorylated peptide was produced by phosphorylation to the substrate peptide by Src tyrosine kinase. Our assay allows for a reliable determination of the activity of Src kinase lower than 10 pg/μL by a simple procedure.
Protein docking by the interface structure similarity: how much structure is needed?

PubMed

Sinha, Rohita; Kundrotas, Petras J; Vakser, Ilya A

2012-01-01

The increasing availability of co-crystallized protein-protein complexes provides an opportunity to use template-based modeling for protein-protein docking. Structure alignment techniques are useful in detection of remote target-template similarities. The size of the structure involved in the alignment is important for the success in modeling. This paper describes a systematic large-scale study to find the optimal definition/size of the interfaces for the structure alignment-based docking applications. The results showed that structural areas corresponding to the cutoff values <12 Å across the interface inadequately represent structural details of the interfaces. With the increase of the cutoff beyond 12 Å, the success rate for the benchmark set of 99 protein complexes, did not increase significantly for higher accuracy models, and decreased for lower-accuracy models. The 12 Å cutoff was optimal in our interface alignment-based docking, and a likely best choice for the large-scale (e.g., on the scale of the entire genome) applications to protein interaction networks. The results provide guidelines for the docking approaches, including high-throughput applications to modeled structures.
Cyclin-dependent protein kinase and cyclin homologs SSN3 and SSN8 contribute to transcriptional control in yeast.

PubMed Central

Kuchin, S; Yeghiayan, P; Carlson, M

1995-01-01

The SSN3 and SSN8 genes of Saccharomyces cerevisiae were identified by mutations that suppress a defect in SNF1, a protein kinase required for release from glucose repression. Mutations in SSN3 and SSN8 also act synergistically with a mutation of the MIG1 repressor protein to relieve glucose repression. We have cloned the SSN3 and SSN8 genes. SSN3 encodes a cyclin-dependent protein kinase (cdk) homolog and is identical to UME5. SSN8 encodes a cyclin homolog 35% identical to human cyclin C. SSN3 and SSN8 fusion proteins interact in the two-hybrid system and coimmunoprecipitate from yeast cell extracts. Using an immune complex assay, we detected protein kinase activity that depends on both SSN3 and SSN8. Thus, the two SSN proteins are likely to function as a cdk-cyclin pair. Genetic analysis indicates that the SSN3-SSN8 complex contributes to transcriptional repression of diversely regulated genes and also affects induction of the GAL1 promoter. Images Fig. 3 Fig. 4 Fig. 5 PMID:7732022
DIGE compatible labelling of surface proteins on vital cells in vitro and in vivo.

PubMed

Mayrhofer, Corina; Krieger, Sigurd; Allmaier, Günter; Kerjaschki, Dontscho

2006-01-01

Efficient methods for profiling of the cell surface proteome are desirable to get a deeper insight in basic biological processes, to localise proteins and to uncover proteins differentially expressed in diseases. Here we present a strategy to target cell surface exposed proteins via fluorescence labelling using CyDye DIGE fluors. This method has been applied to human cell lines in vitro as well as to a complex biological system in vivo. It allows detection of fluorophore-tagged cell surface proteins and visualisation of the accessible proteome within a single 2-D gel, simplifying subsequent UV MALDI-MS analysis.
Modeling of protein binary complexes using structural mass spectrometry data

PubMed Central

Kamal, J.K. Amisha; Chance, Mark R.

2008-01-01

In this article, we describe a general approach to modeling the structure of binary protein complexes using structural mass spectrometry data combined with molecular docking. In the first step, hydroxyl radical mediated oxidative protein footprinting is used to identify residues that experience conformational reorganization due to binding or participate in the binding interface. In the second step, a three-dimensional atomic structure of the complex is derived by computational modeling. Homology modeling approaches are used to define the structures of the individual proteins if footprinting detects significant conformational reorganization as a function of complex formation. A three-dimensional model of the complex is constructed from these binary partners using the ClusPro program, which is composed of docking, energy filtering, and clustering steps. Footprinting data are used to incorporate constraints—positive and/or negative—in the docking step and are also used to decide the type of energy filter—electrostatics or desolvation—in the successive energy-filtering step. By using this approach, we examine the structure of a number of binary complexes of monomeric actin and compare the results to crystallographic data. Based on docking alone, a number of competing models with widely varying structures are observed, one of which is likely to agree with crystallographic data. When the docking steps are guided by footprinting data, accurate models emerge as top scoring. We demonstrate this method with the actin/gelsolin segment-1 complex. We also provide a structural model for the actin/cofilin complex using this approach which does not have a crystal or NMR structure. PMID:18042684
The Mini-Chromosome Maintenance (Mcm) Complexes Interact with DNA Polymerase α-Primase and Stimulate Its Ability to Synthesize RNA Primers

PubMed Central

You, Zhiying; De Falco, Mariarosaria; Kamada, Katsuhiko; Pisani, Francesca M.; Masai, Hisao

2013-01-01

The Mini-chromosome maintenance (Mcm) proteins are essential as central components for the DNA unwinding machinery during eukaryotic DNA replication. DNA primase activity is required at the DNA replication fork to synthesize short RNA primers for DNA chain elongation on the lagging strand. Although direct physical and functional interactions between helicase and primase have been known in many prokaryotic and viral systems, potential interactions between helicase and primase have not been explored in eukaryotes. Using purified Mcm and DNA primase complexes, a direct physical interaction is detected in pull-down assays between the Mcm2∼7 complex and the hetero-dimeric DNA primase composed of the p48 and p58 subunits. The Mcm4/6/7 complex co-sediments with the primase and the DNA polymerase α-primase complex in glycerol gradient centrifugation and forms a Mcm4/6/7-primase-DNA ternary complex in gel-shift assays. Both the Mcm4/6/7 and Mcm2∼7 complexes stimulate RNA primer synthesis by DNA primase in vitro. However, primase inhibits the Mcm4/6/7 helicase activity and this inhibition is abolished by the addition of competitor DNA. In contrast, the ATP hydrolysis activity of Mcm4/6/7 complex is not affected by primase. Mcm and primase proteins mutually stimulate their DNA-binding activities. Our findings indicate that a direct physical interaction between primase and Mcm proteins may facilitate priming reaction by the former protein, suggesting that efficient DNA synthesis through helicase-primase interactions may be conserved in eukaryotic chromosomes. PMID:23977294
Fabrication and characteristics of MOSFET protein chip for detection of ribosomal protein.

PubMed

Park, Keun-Yong; Kim, Min-Suk; Choi, Sie-Young

2005-04-15

A metal oxide silicon field effect transistor (MOSFET) protein chip for the easy detection of protein was fabricated and its characteristics were investigated. Generally, the drain current of the MOSFET is varied by the gate potential. It is expected that the formation of an antibody-antigen complex on the gate of MOSFET would lead to a detectable change in the charge distribution and thus, directly modulate the drain current of MOSFET. As such, the drain current of the MOSFET protein chip can be varied by ribosomal proteins absorbed by the self-assembled monolayer (SAM) immobilized on the gate (Au) surface, as ribosomal protein has positive charge, and these current variations then used as the response of the protein chip. The gate of MOSFET protein chip is not directly biased by an external voltage source, so called open gate or floating gate MOSFET, but rather chemically modified by immobilized molecular receptors called self-assembled monolayer (SAM). In our experiments, the current variation in the proposed protein chip was about 8% with a protein concentration of 0.7 mM. As the protein concentration increased, the drain current also gradually increased. In addition, there were some drift of the drain current in the device. It is considered that these drift might be caused by the drift from the MOSFET itself or protein absorption procedures that are relied on the facile attachment of thiol (-S) ligands to the gate (Au) surface. We verified the formation of SAM on the gold surface and the absorption of protein through the surface plasmon resonance (SPR) measurement.
Protein kinases responsible for the phosphorylation of the nuclear egress core complex of human cytomegalovirus.

PubMed

Sonntag, Eric; Milbradt, Jens; Svrlanska, Adriana; Strojan, Hanife; Häge, Sigrun; Kraut, Alexandra; Hesse, Anne-Marie; Amin, Bushra; Sonnewald, Uwe; Couté, Yohann; Marschall, Manfred

2017-10-01

Nuclear egress of herpesvirus capsids is mediated by a multi-component nuclear egress complex (NEC) assembled by a heterodimer of two essential viral core egress proteins. In the case of human cytomegalovirus (HCMV), this core NEC is defined by the interaction between the membrane-anchored pUL50 and its nuclear cofactor, pUL53. NEC protein phosphorylation is considered to be an important regulatory step, so this study focused on the respective role of viral and cellular protein kinases. Multiply phosphorylated pUL50 varieties were detected by Western blot and Phos-tag analyses as resulting from both viral and cellular kinase activities. In vitro kinase analyses demonstrated that pUL50 is a substrate of both PKCα and CDK1, while pUL53 can also be moderately phosphorylated by CDK1. The use of kinase inhibitors further illustrated the importance of distinct kinases for core NEC phosphorylation. Importantly, mass spectrometry-based proteomic analyses identified five major and nine minor sites of pUL50 phosphorylation. The functional relevance of core NEC phosphorylation was confirmed by various experimental settings, including kinase knock-down/knock-out and confocal imaging, in which it was found that (i) HCMV core NEC proteins are not phosphorylated solely by viral pUL97, but also by cellular kinases; (ii) both PKC and CDK1 phosphorylation are detectable for pUL50; (iii) no impact of PKC phosphorylation on NEC functionality has been identified so far; (iv) nonetheless, CDK1-specific phosphorylation appears to be required for functional core NEC interaction. In summary, our findings provide the first evidence that the HCMV core NEC is phosphorylated by cellular kinases, and that the complex pattern of NEC phosphorylation has functional relevance.
[Determination of cyclamate in complex matrix using HPLC after column derivatization with 4-fluoro-7-nitrobenzofurazan].

PubMed

Hauck, M; Köbler, H

1990-01-01

A method for the analysis of cyclamate in complex foodstuffs has been developed. This method is applicable in strongly coloured and protein-rich foodstuffs. The quantitative determination depends on oxidation of cyclamate to cyclohexylamine and derivatisation with 4-fluoro-7-nitrobenzofuran (NBD-F). The derivatives are analysed by HPLC on a C18: reversed-phase column, their minimal stability being 12 h. There are two possible methods of detection: (a) absorbance at 485 nm and (b) fluorescence with excitation at 485 nm and emission at 530 nm. The detection limit of cyclamate is 5 mg/kg foodstuff, with fluorescence detection 0.4 mg/kg. The recoveries are in the range of 88% to 104%.
Isofunctional Protein Subfamily Detection Using Data Integration and Spectral Clustering.

PubMed

Boari de Lima, Elisa; Meira, Wagner; Melo-Minardi, Raquel Cardoso de

2016-06-01

As increasingly more genomes are sequenced, the vast majority of proteins may only be annotated computationally, given experimental investigation is extremely costly. This highlights the need for computational methods to determine protein functions quickly and reliably. We believe dividing a protein family into subtypes which share specific functions uncommon to the whole family reduces the function annotation problem's complexity. Hence, this work's purpose is to detect isofunctional subfamilies inside a family of unknown function, while identifying differentiating residues. Similarity between protein pairs according to various properties is interpreted as functional similarity evidence. Data are integrated using genetic programming and provided to a spectral clustering algorithm, which creates clusters of similar proteins. The proposed framework was applied to well-known protein families and to a family of unknown function, then compared to ASMC. Results showed our fully automated technique obtained better clusters than ASMC for two families, besides equivalent results for other two, including one whose clusters were manually defined. Clusters produced by our framework showed great correspondence with the known subfamilies, besides being more contrasting than those produced by ASMC. Additionally, for the families whose specificity determining positions are known, such residues were among those our technique considered most important to differentiate a given group. When run with the crotonase and enolase SFLD superfamilies, the results showed great agreement with this gold-standard. Best results consistently involved multiple data types, thus confirming our hypothesis that similarities according to different knowledge domains may be used as functional similarity evidence. Our main contributions are the proposed strategy for selecting and integrating data types, along with the ability to work with noisy and incomplete data; domain knowledge usage for detecting subfamilies in a family with different specificities, thus reducing the complexity of the experimental function characterization problem; and the identification of residues responsible for specificity.
Isofunctional Protein Subfamily Detection Using Data Integration and Spectral Clustering

PubMed Central

Boari de Lima, Elisa; Meira, Wagner; de Melo-Minardi, Raquel Cardoso

2016-01-01

As increasingly more genomes are sequenced, the vast majority of proteins may only be annotated computationally, given experimental investigation is extremely costly. This highlights the need for computational methods to determine protein functions quickly and reliably. We believe dividing a protein family into subtypes which share specific functions uncommon to the whole family reduces the function annotation problem’s complexity. Hence, this work’s purpose is to detect isofunctional subfamilies inside a family of unknown function, while identifying differentiating residues. Similarity between protein pairs according to various properties is interpreted as functional similarity evidence. Data are integrated using genetic programming and provided to a spectral clustering algorithm, which creates clusters of similar proteins. The proposed framework was applied to well-known protein families and to a family of unknown function, then compared to ASMC. Results showed our fully automated technique obtained better clusters than ASMC for two families, besides equivalent results for other two, including one whose clusters were manually defined. Clusters produced by our framework showed great correspondence with the known subfamilies, besides being more contrasting than those produced by ASMC. Additionally, for the families whose specificity determining positions are known, such residues were among those our technique considered most important to differentiate a given group. When run with the crotonase and enolase SFLD superfamilies, the results showed great agreement with this gold-standard. Best results consistently involved multiple data types, thus confirming our hypothesis that similarities according to different knowledge domains may be used as functional similarity evidence. Our main contributions are the proposed strategy for selecting and integrating data types, along with the ability to work with noisy and incomplete data; domain knowledge usage for detecting subfamilies in a family with different specificities, thus reducing the complexity of the experimental function characterization problem; and the identification of residues responsible for specificity. PMID:27348631
Scanning confocal fluorescence microscopy for single molecule analysis of nucleotide excision repair complexes.

PubMed

Segers-Nolten, G M J; Wyman, C; Wijgers, N; Vermeulen, W; Lenferink, A T M; Hoeijmakers, J H J; Greve, J; Otto, C

2002-11-01

We used scanning confocal fluorescence microscopy to observe and analyze individual DNA- protein complexes formed between human nucleotide excision repair (NER) proteins and model DNA substrates. For this purpose human XPA protein was fused to EGFP, purified and shown to be functional. Binding of EGFP-labeled XPA protein to a Cy3.5-labeled DNA substrate, in the presence and absence of RPA, was assessed quantitatively by simultaneous excitation and emission detection of both fluorophores. Co-localization of Cy3.5 and EGFP signals within one diffraction limited spot indicated complexes of XPA with DNA. Measurements were performed on samples in a 1% agarose matrix in conditions that are compatible with protein activity and where reactions can be studied under equilibrium conditions. In these samples DNA alone was freely diffusing and protein-bound DNA was immobile, whereby they could be discriminated resulting in quantitative data on DNA binding. On the single molecule level approximately 10% of XPA co-localized with DNA; this increased to 32% in the presence of RPA. These results, especially the enhanced binding of XPA in the presence of RPA, are similar to those obtained in bulk experiments, validating the utility of scanning confocal fluorescence microscopy for investigating functional interactions at the single molecule level.
Robust co-regulation of tyrosine phosphorylation sites on proteins reveals novel protein interactions†

PubMed Central

Naegle, Kristen M.; White, Forest M.; Lauffenburger, Douglas A.; Yaffe, Michael B.

2012-01-01

Cell signaling networks propagate information from extracellular cues via dynamic modulation of protein–protein interactions in a context-dependent manner. Networks based on receptor tyrosine kinases (RTKs), for example, phosphorylate intracellular proteins in response to extracellular ligands, resulting in dynamic protein–protein interactions that drive phenotypic changes. Most commonly used methods for discovering these protein–protein interactions, however, are optimized for detecting stable, longer-lived complexes, rather than the type of transient interactions that are essential components of dynamic signaling networks such as those mediated by RTKs. Substrate phosphorylation downstream of RTK activation modifies substrate activity and induces phospho-specific binding interactions, resulting in the formation of large transient macromolecular signaling complexes. Since protein complex formation should follow the trajectory of events that drive it, we reasoned that mining phosphoproteomic datasets for highly similar dynamic behavior of measured phosphorylation sites on different proteins could be used to predict novel, transient protein–protein interactions that had not been previously identified. We applied this method to explore signaling events downstream of EGFR stimulation. Our computational analysis of robustly co-regulated phosphorylation sites, based on multiple clustering analysis of quantitative time-resolved mass-spectrometry phosphoproteomic data, not only identified known sitewise-specific recruitment of proteins to EGFR, but also predicted novel, a priori interactions. A particularly intriguing prediction of EGFR interaction with the cytoskeleton-associated protein PDLIM1 was verified within cells using co-immunoprecipitation and in situ proximity ligation assays. Our approach thus offers a new way to discover protein–protein interactions in a dynamic context- and phosphorylation site-specific manner. PMID:22851037
Detection of the Argonaute Protein Ago2 and microRNAs in the RNA Induced Silencing Complex (RISC) Using a Monoclonal Antibody

PubMed Central

Ikeda, Keigo; Satoh, Minoru; Pauley, Kaleb M.; Fritzler, Marvin J.; Reeves, Westley H.; Chan, Edward K.L.

2007-01-01

MicroRNAs (miRNAs) are short RNA molecules responsible for post-transcriptional gene silencing by the degradation or translational inhibition of their target messenger RNAs (mRNAs). This process of gene silencing, known as RNA interference (RNAi), is mediated by highly conserved Argonaute (Ago) proteins which are the key components of the RNA induced silencing complex (RISC). In humans, Ago2 is responsible for the endonuclease cleavage of targeted mRNA and it interacts with the mRNA-binding protein GW182, which is a marker for cytoplasmic foci referred to as GW bodies (GWBs). We demonstrated that the anti-Ago2 monoclonal antibody 4F9 recognized GWBs in a cell cycle dependent manner and was capable of capturing miRNAs associated with Ago2. Since Ago2 protein is the effector protein of RNAi, anti-Ago2 monoclonal antibody may be useful in capturing functional miRNAs. PMID:17054975

A combination of spin diffusion methods for the determination of protein-ligand complex structural ensembles.

PubMed

Pilger, Jens; Mazur, Adam; Monecke, Peter; Schreuder, Herman; Elshorst, Bettina; Bartoschek, Stefan; Langer, Thomas; Schiffer, Alexander; Krimm, Isabelle; Wegstroth, Melanie; Lee, Donghan; Hessler, Gerhard; Wendt, K-Ulrich; Becker, Stefan; Griesinger, Christian

2015-05-26

Structure-based drug design (SBDD) is a powerful and widely used approach to optimize affinity of drug candidates. With the recently introduced INPHARMA method, the binding mode of small molecules to their protein target can be characterized even if no spectroscopic information about the protein is known. Here, we show that the combination of the spin-diffusion-based NMR methods INPHARMA, trNOE, and STD results in an accurate scoring function for docking modes and therefore determination of protein-ligand complex structures. Applications are shown on the model system protein kinase A and the drug targets glycogen phosphorylase and soluble epoxide hydrolase (sEH). Multiplexing of several ligands improves the reliability of the scoring function further. The new score allows in the case of sEH detecting two binding modes of the ligand in its binding site, which was corroborated by X-ray analysis. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Detection of the argonaute protein Ago2 and microRNAs in the RNA induced silencing complex (RISC) using a monoclonal antibody.

PubMed

Ikeda, Keigo; Satoh, Minoru; Pauley, Kaleb M; Fritzler, Marvin J; Reeves, Westley H; Chan, Edward K L

2006-12-20

MicroRNAs (miRNAs) are short RNA molecules responsible for post-transcriptional gene silencing by the degradation or translational inhibition of their target messenger RNAs (mRNAs). This process of gene silencing, known as RNA interference (RNAi), is mediated by highly conserved Argonaute (Ago) proteins which are the key components of the RNA induced silencing complex (RISC). In humans, Ago2 is responsible for the endonuclease cleavage of targeted mRNA and it interacts with the mRNA-binding protein GW182, which is a marker for cytoplasmic foci referred to as GW bodies (GWBs). We demonstrated that the anti-Ago2 monoclonal antibody 4F9 recognized GWBs in a cell cycle dependent manner and was capable of capturing miRNAs associated with Ago2. Since Ago2 protein is the effector protein of RNAi, anti-Ago2 monoclonal antibody may be useful in capturing functional miRNAs.
Western blotting using capillary electrophoresis.

PubMed

Anderson, Gwendolyn J; M Cipolla, Cynthia; Kennedy, Robert T

2011-02-15

A microscale Western blotting system based on separating sodium-dodecyl sulfate protein complexes by capillary gel electrophoresis followed by deposition onto a blotting membrane for immunoassay is described. In the system, the separation capillary is grounded through a sheath capillary to a mobile X-Y translation stage which moves a blotting membrane past the capillary outlet for protein deposition. The blotting membrane is moistened with a methanol and buffer mixture to facilitate protein adsorption. Although discrete protein zones could be detected, bands were broadened by ∼1.7-fold by transfer to membrane. A complete Western blot for lysozyme was completed in about one hour with 50 pg mass detection limit from low microgram per milliliter samples. These results demonstrate substantial reduction in time requirements and improvement in mass sensitivity compared to conventional Western blots. Western blotting using capillary electrophoresis shows promise to analyze low volume samples with reduced reagents and time, while retaining the information content of a typical Western blot.
Mining protein-protein interaction networks: denoising effects

NASA Astrophysics Data System (ADS)

Marras, Elisabetta; Capobianco, Enrico

2009-01-01

A typical instrument to pursue analysis in complex network studies is the analysis of the statistical distributions. They are usually computed for measures which characterize network topology, and are aimed at capturing both structural and dynamics aspects. Protein-protein interaction networks (PPIN) have also been studied through several measures. It is in general observed that a power law is expected to characterize scale-free networks. However, mixing the original noise cover with outlying information and other system-dependent fluctuations makes the empirical detection of the power law a difficult task. As a result the uncertainty level increases when looking at the observed sample; in particular, one may wonder whether the computed features may be sufficient to explain the interactome. We then address noise problems by implementing both decomposition and denoising techniques that reduce the impact of factors known to affect the accuracy of power law detection.
Mapping multivalency and differential affinities within large intrinsically disordered protein complexes with segmental motion analysis.

PubMed

Milles, Sigrid; Lemke, Edward A

2014-07-07

Intrinsically disordered proteins (IDPs) can bind to multiple interaction partners. Numerous binding regions in the IDP that act in concert through complex cooperative effects facilitate such interactions, but complicate studying IDP complexes. To address this challenge we developed a combined fluorescence correlation and time-resolved polarization spectroscopy approach to study the binding properties of the IDP nucleoporin153 (Nup153) to nuclear transport receptors (NTRs). The detection of segmental backbone mobility of Nup153 within the unperturbed complex provided a readout of local, region-specific binding properties that are usually masked in measurements of the whole IDP. The binding affinities of functionally and structurally diverse NTRs to distinct regions of Nup153 can differ by orders of magnitudes-a result with implications for the diversity of transport routes in nucleocytoplasmic transport. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Interactions between the cyclic AMP receptor protein and the alpha subunit of RNA polymerase at the Escherichia coli galactose operon P1 promoter.

PubMed

Attey, A; Belyaeva, T; Savery, N; Hoggett, J; Fujita, N; Ishihama, A; Busby, S

1994-10-25

DNAase I footprinting has been used to study open complexes between Escherichia coli RNA polymerase and the galactose operon P1 promoter, both in the absence and the presence of CRP (the cyclic AMP receptor protein, a transcription activator). From the effects of deletion of the C-terminal part of the RNA polymerase alpha subunit, we deduce that alpha binds at the upstream end of both the binary RNA polymerase-galP1 and ternary RNA polymerase-CRP-galP1 complexes. Disruption of the alpha-upstream contact suppresses open complex formation at galP1 at lower temperatures. In ternary RNA polymerase-CRP-galP1 complexes, alpha appears to make direct contact with Activating Region 1 in CRP. DNAase I footprinting has been used to detect and quantify interactions between purified alpha and CRP bound at galP1.
Interactions between the cyclic AMP receptor protein and the alpha subunit of RNA polymerase at the Escherichia coli galactose operon P1 promoter.

PubMed Central

Attey, A; Belyaeva, T; Savery, N; Hoggett, J; Fujita, N; Ishihama, A; Busby, S

1994-01-01

DNAase I footprinting has been used to study open complexes between Escherichia coli RNA polymerase and the galactose operon P1 promoter, both in the absence and the presence of CRP (the cyclic AMP receptor protein, a transcription activator). From the effects of deletion of the C-terminal part of the RNA polymerase alpha subunit, we deduce that alpha binds at the upstream end of both the binary RNA polymerase-galP1 and ternary RNA polymerase-CRP-galP1 complexes. Disruption of the alpha-upstream contact suppresses open complex formation at galP1 at lower temperatures. In ternary RNA polymerase-CRP-galP1 complexes, alpha appears to make direct contact with Activating Region 1 in CRP. DNAase I footprinting has been used to detect and quantify interactions between purified alpha and CRP bound at galP1. Images PMID:7971267
Reassembly of a bioluminescent protein Renilla luciferase directed through DNA hybridization.

PubMed

Cissell, Kyle A; Rahimi, Yasmeen; Shrestha, Suresh; Deo, Sapna K

2009-01-01

Reassembly of split reporter proteins, also referred to as protein complementation, is utilized in the detection of protein-protein or protein-nucleic acid interactions. In this strategy, a reporter protein is fragmented into two inactive polypeptides to which interacting/binding partners are fused. The interaction between fused partners leads to the formation of a reassembled, active reporter. In this Communication, we have presented a proof-of-concept for the detection of a target nucleic acid sequence based on the reassembly of the bioluminescent reporter Renilla luciferase (Rluc), which is driven by DNA hybridization. Although, reassembly of Rluc though protein interactions has been demonstrated by others, the Rluc reassembly through DNA hybridization has not been shown yet, which is the novelty of this work. It is well established that bioluminescence detection offers significant advantages due to the absence of any background signal. In our study, two rationally designed fragments of Rluc were conjugated to complementary oligonucleotide probes. Hybridization of the two probes with fused Rluc fragments resulted in the reassembly of the fragments, generating active Rluc, measurable by the intensity of light given off upon addition of coelenterazine. Our study also shows that the reassembly of Rluc can be inhibited by an oligonucleotide probe that competes to bind to the hybridized probe-Rluc fragment complex, indicating a potential strategy for the quantitative detection of target nucleic acid. We were able to achieve the reassembly of Rluc fused to oligonucleotide probes using femtomole amounts of the probe-fragment protein conjugate. This concentration is approximately 4 orders of magnitude less than that reported using green fluorescent protein (GFP) as the reporter. A DNA-driven Rluc reassembly study performed in a cellular matrix did not show any interference from the matrix.
Generation of monoclonal antibodies and development of an immunofluorometric assay for the detection of CUZD1 in tissues and biological fluids.

PubMed

Farkona, Sofia; Soosaipillai, Antoninus; Filippou, Panagiota; Korbakis, Dimitrios; Serra, Stefano; Rückert, Felix; Diamandis, Eleftherios P; Blasutig, Ivan M

2017-12-01

CUB and zona pellucida-like domain-containing protein 1 (CUZD1) was identified as a pancreas-specific protein and was proposed as a candidate biomarker for pancreatic related disorders. CUZD1 protein levels in tissues and biological fluids have not been extensively examined. The purpose of the present study was to generate specific antibodies targeting CUZD1 to assess CUZD1 expression within tissues and biological fluids. Mouse monoclonal antibodies against CUZD1 were generated and used to perform immunohistochemical analyses and to develop a sensitive and specific enzyme-linked immunosorbent assay (ELISA). CUZD1 protein expression was assessed in various human tissue extracts and biological fluids and in gel filtration chromatography-derived fractions of pancreatic tissue extract, pancreatic juice and recombinant protein. Immunohistochemical staining of CUZD1 in pancreatic tissue showed that the protein is localized to the acinar cells and the lumen of the acini. Western blot analysis detected the protein in pancreatic tissue extract and pancreatic juice. The newly developed ELISA measured CUZD1 in high levels in pancreas and in much lower but detectable levels in several other tissues. In the biological fluids tested, CUZD1 expression was detected exclusively in pancreatic juice. The analysis of gel filtration chromatography-derived fractions of pancreatic tissue extract, pancreatic juice and recombinant CUZD1 suggested that the protein exists in high molecular weight protein complexes. This study describes the development of tools targeting CUZD1 protein, its tissue expression pattern and levels in several biological fluids. These new tools will facilitate future investigations aiming to delineate the role of CUZD1 in physiology and pathobiology. Copyright © 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
The Monitoring and Affinity Purification of Proteins Using Dual Tags with Tetracysteine Motifs

NASA Astrophysics Data System (ADS)

Giannone, Richard J.; Liu, Yie; Wang, Yisong

Identification and characterization of protein-protein interaction networks is essential for the elucidation of biochemical mechanisms and cellular function. Affinity purification in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a very powerful tactic for the identification of specific protein-protein interactions. In this chapter, we describe a comprehensive methodology that uses our recently developed dual-tag affinity purification system for the enrichment and identification of mammalian protein complexes. The protocol covers a series of separate but sequentially related techniques focused on the facile monitoring and purification of a dual-tagged protein of interest and its interacting partners via a system built with tetracysteine motifs and various combinations of affinity tags. Using human telomeric repeat binding factor 2 (TRF2) as an example, we demonstrate the power of the system in terms of bait protein recovery after dual-tag affinity purification, detection of bait protein subcellular localization and expression, and successful identification of known and potentially novel TRF2 interacting proteins. Although the protocol described here has been optimized for the identification and characterization of TRF2-associated proteins, it is, in principle, applicable to the study of any other mammalian protein complexes that may be of interest to the research community.
Evidence for the presence of key chlorophyll-biosynthesis-related proteins in the genus Rubrobacter (Phylum Actinobacteria) and its implications for the evolution and origin of photosynthesis.

PubMed

Gupta, Radhey S; Khadka, Bijendra

2016-02-01

Homologs showing high degree of sequence similarity to the three subunits of the protochlorophyllide oxidoreductase enzyme complex (viz. BchL, BchN, and BchB), which carries out a central role in chlorophyll-bacteriochlorophyll (Bchl) biosynthesis, are uniquely found in photosynthetic organisms. The results of BLAST searches and homology modeling presented here show that proteins exhibiting a high degree of sequence and structural similarity to the BchB and BchN proteins are also present in organisms from the high G+C Gram-positive phylum of Actinobacteria, specifically in members of the genus Rubrobacter (R. x ylanophilus and R. r adiotolerans). The results presented exclude the possibility that the observed BLAST hits are for subunits of the nitrogenase complex or the chlorin reductase complex. The branching in phylogenetic trees and the sequence characteristics of the Rubrobacter BchB/BchN homologs indicate that these homologs are distinct from those found in other photosynthetic bacteria and that they may represent ancestral forms of the BchB/BchN proteins. Although a homolog showing high degree of sequence similarity to the BchL protein was not detected in Rubrobacter, another protein, belonging to the ParA/Soj/MinD family, present in these bacteria, exhibits high degree of structural similarity to the BchL. In addition to the BchB/BchN homologs, Rubrobacter species also contain homologs showing high degree of sequence similarity to different subunits of magnesium chelatase (BchD, BchH, and BchI) as well as proteins showing significant similarity to the BchP and BchG proteins. Interestingly, no homologs corresponding to the BchX, BchY, and BchZ proteins were detected in the Rubrobacter species. These results provide the first suggestive evidence that some form of photosynthesis either exists or was anciently present within the phylum Actinobacteria (high G+C Gram-positive) in members of the genus Rubrobacter. The significance of these results concerning the origin of the Bchl-based photosynthesis is also discussed.
The split Renilla luciferase complementation assay is useful for identifying the interaction of Epstein-Barr virus protein kinase BGLF4 and a heat shock protein Hsp90.

PubMed

Wang, J; Guo, W; Long, C; Zhou, H; Wang, H; Sun, X

2016-03-01

Protein-protein interactions can regulate different cellular processes, such as transcription, translation, and oncogenic transformation. The split Renilla luciferase complementation assay (SRLCA) is one of the techniques that detect protein-protein interactions. The SRLCA is based on the complementation of the LN and LC non-functional halves of Renilla luciferase fused to possibly interacting proteins which after interaction form a functional enzyme and emit luminescence. The BGLF4 of Epstein-Barr virus (EBV) is a viral protein kinase that is expressed during the early and late stages of lytic cycles, which can regulate multiple cellular and viral substrates to optimize the DNA replication environment. The heat shock protein Hsp90 is a molecular chaperone that maintains the integrity of structure and function of various interacting proteins, which can form a complex with BGLF4 and stabilize its expression in cells. The interaction between BGLF4 and Hsp90 could be specifically detected through the SRLCA. The region of aa 250-295 of BGLF4 is essential for the BGLF4/Hsp90 interaction and the mutation of Phe-254, Leu-266, and Leu-267 can disrupt this interaction. These results suggest that the SRLCA can specifically detect the BGLF4/Hsp90 interaction and provide a reference to develop inhibitors that disrupt the BGLF4/Hsp90 interaction.
Finding trans-regulatory genes and protein complexes modulating meiotic recombination hotspots of human, mouse and yeast.

PubMed

Wu, Min; Kwoh, Chee-Keong; Li, Xiaoli; Zheng, Jie

2014-09-11

The regulatory mechanism of recombination is one of the most fundamental problems in genomics, with wide applications in genome wide association studies (GWAS), birth-defect diseases, molecular evolution, cancer research, etc. Recombination events cluster into short genomic regions called "recombination hotspots". Recently, a zinc finger protein PRDM9 was reported to regulate recombination hotspots in human and mouse genomes. In addition, a 13-mer motif contained in the binding sites of PRDM9 is found to be enriched in human hotspots. However, this 13-mer motif only covers a fraction of hotspots, indicating that PRDM9 is not the only regulator of recombination hotspots. Therefore, the challenge of discovering other regulators of recombination hotspots becomes significant. Furthermore, recombination is a complex process. Hence, multiple proteins acting as machinery, rather than individual proteins, are more likely to carry out this process in a precise and stable manner. Therefore, the extension of the prediction of individual trans-regulators to protein complexes is also highly desired. In this paper, we introduce a pipeline to identify genes and protein complexes associated with recombination hotspots. First, we prioritize proteins associated with hotspots based on their preference of binding to hotspots and coldspots. Second, using the above identified genes as seeds, we apply the Random Walk with Restart algorithm (RWR) to propagate their influences to other proteins in protein-protein interaction (PPI) networks. Hence, many proteins without DNA-binding information will also be assigned a score to implicate their roles in recombination hotspots. Third, we construct sub-PPI networks induced by top genes ranked by RWR for various species (e.g., yeast, human and mouse) and detect protein complexes in those sub-PPI networks. The GO term analysis show that our prioritizing methods and the RWR algorithm are capable of identifying novel genes associated with recombination hotspots. The trans-regulators predicted by our pipeline are enriched with epigenetic functions (e.g., histone modifications), demonstrating the epigenetic regulatory mechanisms of recombination hotspots. The identified protein complexes also provide us with candidates to further investigate the molecular machineries for recombination hotspots. Moreover, the experimental data and results are available on our web site http://www.ntu.edu.sg/home/zhengjie/data/RecombinationHotspot/NetPipe/.
Detection of Antibodies in Blood Plasma Using Bioluminescent Sensor Proteins and a Smartphone.

PubMed

Arts, Remco; den Hartog, Ilona; Zijlema, Stefan E; Thijssen, Vito; van der Beelen, Stan H E; Merkx, Maarten

2016-04-19

Antibody detection is of fundamental importance in many diagnostic and bioanalytical assays, yet current detection techniques tend to be laborious and/or expensive. We present a new sensor platform (LUMABS) based on bioluminescence resonance energy transfer (BRET) that allows detection of antibodies directly in solution using a smartphone as the sole piece of equipment. LUMABS are single-protein sensors that consist of the blue-light emitting luciferase NanoLuc connected via a semiflexible linker to the green fluorescent acceptor protein mNeonGreen, which are kept close together using helper domains. Binding of an antibody to epitope sequences flanking the linker disrupts the interaction between the helper domains, resulting in a large decrease in BRET efficiency. The resulting change in color of the emitted light from green-blue to blue can be detected directly in blood plasma, even at picomolar concentrations of antibody. Moreover, the modular architecture of LUMABS allows changing of target specificity by simple exchange of epitope sequences, as demonstrated here for antibodies against HIV1-p17, hemagglutinin (HA), and dengue virus type I. The combination of sensitive ratiometric bioluminescent detection and the intrinsic modularity of the LUMABS design provides an attractive generic platform for point-of-care antibody detection that avoids the complex liquid handling steps associated with conventional immunoassays.
Development of a Dehalogenase-Based Protein Fusion Tag Capable of Rapid, Selective and Covalent Attachment to Customizable Ligands

PubMed Central

Encell, Lance P; Friedman Ohana, Rachel; Zimmerman, Kris; Otto, Paul; Vidugiris, Gediminas; Wood, Monika G; Los, Georgyi V; McDougall, Mark G; Zimprich, Chad; Karassina, Natasha; Learish, Randall D; Hurst, Robin; Hartnett, James; Wheeler, Sarah; Stecha, Pete; English, Jami; Zhao, Kate; Mendez, Jacqui; Benink, Hélène A; Murphy, Nancy; Daniels, Danette L; Slater, Michael R; Urh, Marjeta; Darzins, Aldis; Klaubert, Dieter H; Bulleit, Robert F; Wood, Keith V

2012-01-01

Our fundamental understanding of proteins and their biological significance has been enhanced by genetic fusion tags, as they provide a convenient method for introducing unique properties to proteins so that they can be examinedin isolation. Commonly used tags satisfy many of the requirements for applications relating to the detection and isolation of proteins from complex samples. However, their utility at low concentration becomes compromised if the binding affinity for a detection or capture reagent is not adequate to produce a stable interaction. Here, we describe HaloTag® (HT7), a genetic fusion tag based on a modified haloalkane dehalogenase designed and engineered to overcome the limitation of affinity tags by forming a high affinity, covalent attachment to a binding ligand. HT7 and its ligand have additional desirable features. The tag is relatively small, monomeric, and structurally compatible with fusion partners, while the ligand is specific, chemically simple, and amenable to modular synthetic design. Taken together, the design features and molecular evolution of HT7 have resulted in a superior alternative to common tags for the overexpression, detection, and isolation of target proteins. PMID:23248739
Structural Analysis of N- and O-glycans Using ZIC-HILIC/Dialysis Coupled to NMR Detection

DOE Office of Scientific and Technical Information (OSTI.GOV)

Qu, Yi; Feng, Ju; Deng, Shuang

2014-11-19

Protein glycosylation, an important and complex post-translational modification (PTM), is involved in various biological processes including the receptor-ligand and cell-cell interaction, and plays a crucial role in many biological functions. However, little is known about the glycan structures of important biological complex samples, and the conventional glycan enrichment strategy (i.e., size-exclusion column [SEC] separation,) prior to nuclear magnetic resonance (NMR) detection is time-consuming and tedious. In this study, we employed SEC, Zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC), and ZIC-HILIC coupled with dialysis strategies to enrich the glycopeptides from the pronase E digests of RNase B, followed by NMR analysis ofmore » the glycoconjugate. Our results suggest that the ZIC-HILIC enrichment coupled with dialysis is the most efficient, which was thus applied to the analysis of biological complex sample, the pronase E digest of the secreted proteins from the fungi Aspergillus niger. The NMR spectra revealed that the secreted proteins from A. niger contain both N-linked glycans with a high-mannose core and O-linked glycans bearing mannose and glucose with 1->3 and 1->6 linkages. In all, our study provides compelling evidence that ZIC-HILIC separation coupled to dialysis is superior to the commonly used SEC separation to prepare glycopeptides for the downstream NMR analysis, which could greatly facilitate the future NMR-based glycoproteomics research.« less
Sulfo-NHS-SS-biotin derivatization: a versatile tool for MALDI mass analysis of PTMs in lysine-rich proteins.

PubMed

Markoutsa, Stavroula; Bahr, Ute; Papasotiriou, Dimitrios G; Häfner, Ann-Kathrin; Karas, Michael; Sorg, Bernd L

2014-03-01

The discovery of PTMs in proteins by MS requires nearly complete sequence coverage of the detected proteolytic peptides. Unfortunately, mass spectrometric analysis of the desired sequence fragments is often impeded due to low ionization efficiency and/or signal suppression in complex samples. When several lysine residues are in close proximity tryptic peptides may be too short for mass analysis. Moreover, modified peptides often appear in low stoichiometry and need to be enriched before analysis. We present here how the use of sulfo-NHS-SS-biotin derivatization of lysine side chain can help to detect PTMs in lysine-rich proteins. This label leads to a mass shift which can be adjusted by reduction of the SS bridge and alkylation with different reagents. Low intensity peptides can be enriched by use of streptavidin beads. Using this method, the functionally relevant protein kinase A phosphorylation site in 5-lipoxygenase was detected for the first time by MS. Additionally, methylation and acetylation could be unambiguously determined in histones. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Distinct functions for IFT140 and IFT20 in opsin transport.

PubMed Central

Crouse, Jacquelin A.; Lopes, Vanda S.; SanAgustin, Jovenal T.; Keady, Brian T.; Williams, David S.; Pazour, Gregory J.

2014-01-01

In the vertebrate retina, light is detected by the outer segments of photoreceptor rods and cones, which are highly modified cilia. Like other cilia, outer segments have no protein synthetic capacity and depend on proteins made in the cell body for their formation and maintenance. The mechanism of transport into the outer segment is not fully understood but intraflagellar transport (IFT) is thought to be a major mechanism for moving protein from the cell body into the cilium. In the case of photoreceptor cells, the high density of receptors and the disk turnover that occurs daily necessitates much higher rates of transport than would be required in other cilia. In this work, we show that the IFT complex A protein IFT140 is required for development and maintenance of outer segments. In earlier work we found that acute deletion of Ift20 caused opsin to accumulate at the Golgi complex. In this work we find that acute deletion of Ift140 does not cause opsin to accumulate at the Golgi complex but rather it accumulates in the plasma membrane of the inner segments. This work is strong support of a model of opsin transport where IFT20 is involved in the movement from the Golgi complex to the base of the cilium. Then, once at the base, the opsin is carried through the connecting cilium by an IFT complex that includes IFT140. PMID:24619649
An inhibitor of eIF2 activity in the sRNA pool of eukaryotic cells.

PubMed

Centrella, Michael; Porter, David L; McCarthy, Thomas L

2011-08-15

Eukaryotic protein synthesis is a multi-step and highly controlled process that includes an early initiation complex containing eukaryotic initiation factor 2 (eIF2), GTP, and methionine-charged initiator methionyl-tRNA (met-tRNAi). During studies to reconstruct formation of the ternary complex containing these molecules, we detected a potent inhibitor in low molecular mass RNA (sRNA) preparations of eukaryotic tRNA. The ternary complex inhibitor (TCI) was retained in the total sRNA pool after met-tRNAi was charged by aminoacyl tRNA synthetase, co-eluted with sRNA by size exclusion chromatography, but resolved from met-tRNAi by ion exchange chromatography. The adverse effect of TCI was not overcome by high GTP or magnesium omission and was independent of GTP regeneration. Rather, TCI suppressed the rate of ternary complex formation, and disrupted protein synthesis and the accumulation of heavy polymeric ribosomes in reticulocyte lysates in vitro. Lastly, a component or components in ribosome depleted cell lysate significantly reversed TCI activity. Since assembly of the met-tRNAi/eIF2/GTP ternary complex is integral to protein synthesis, awareness of TCI is important to avoid confusion in studies of translation initiation. A clear definition of TCI may also allow a better appreciation of physiologic or pathologic situations, factors, and events that control protein synthesis in vivo. Copyright © 2011 Elsevier B.V. All rights reserved.
The nuclear import of ribosomal proteins is regulated by mTOR

PubMed Central

Kazyken, Dubek; Kaz, Yelimbek; Kiyan, Vladimir; Zhylkibayev, Assylbek A.; Chen, Chien-Hung; Agarwal, Nitin K.; Sarbassov, Dos D.

2014-01-01

Mechanistic target of rapamycin (mTOR) is a central component of the essential signaling pathway that regulates cell growth and proliferation by controlling anabolic processes in cells. mTOR exists in two distinct mTOR complexes known as mTORC1 and mTORC2 that reside mostly in cytoplasm. In our study, the biochemical characterization of mTOR led to discovery of its novel localization on nuclear envelope where it associates with a critical regulator of nuclear import Ran Binding Protein 2 (RanBP2). We show that association of mTOR with RanBP2 is dependent on the mTOR kinase activity that regulates the nuclear import of ribosomal proteins. The mTOR kinase inhibitors within thirty minutes caused a substantial decrease of ribosomal proteins in the nuclear but not cytoplasmic fraction. Detection of a nuclear accumulation of the GFP-tagged ribosomal protein rpL7a also indicated its dependence on the mTOR kinase activity. The nuclear abundance of ribosomal proteins was not affected by inhibition of mTOR Complex 1 (mTORC1) by rapamycin or deficiency of mTORC2, suggesting a distinctive role of the nuclear envelope mTOR complex in the nuclear import. Thus, we identified that mTOR in association with RanBP2 mediates the active nuclear import of ribosomal proteins. PMID:25294810

Engineering the cell surface display of cohesins for assembly of cellulosome-inspired enzyme complexes on Lactococcus lactis

PubMed Central

2010-01-01

Background The assembly and spatial organization of enzymes in naturally occurring multi-protein complexes is of paramount importance for the efficient degradation of complex polymers and biosynthesis of valuable products. The degradation of cellulose into fermentable sugars by Clostridium thermocellum is achieved by means of a multi-protein "cellulosome" complex. Assembled via dockerin-cohesin interactions, the cellulosome is associated with the cell surface during cellulose hydrolysis, forming ternary cellulose-enzyme-microbe complexes for enhanced activity and synergy. The assembly of recombinant cell surface displayed cellulosome-inspired complexes in surrogate microbes is highly desirable. The model organism Lactococcus lactis is of particular interest as it has been metabolically engineered to produce a variety of commodity chemicals including lactic acid and bioactive compounds, and can efficiently secrete an array of recombinant proteins and enzymes of varying sizes. Results Fragments of the scaffoldin protein CipA were functionally displayed on the cell surface of Lactococcus lactis. Scaffolds were engineered to contain a single cohesin module, two cohesin modules, one cohesin and a cellulose-binding module, or only a cellulose-binding module. Cell toxicity from over-expression of the proteins was circumvented by use of the nisA inducible promoter, and incorporation of the C-terminal anchor motif of the streptococcal M6 protein resulted in the successful surface-display of the scaffolds. The facilitated detection of successfully secreted scaffolds was achieved by fusion with the export-specific reporter staphylococcal nuclease (NucA). Scaffolds retained their ability to associate in vivo with an engineered hybrid reporter enzyme, E. coli β-glucuronidase fused to the type 1 dockerin motif of the cellulosomal enzyme CelS. Surface-anchored complexes exhibited dual enzyme activities (nuclease and β-glucuronidase), and were displayed with efficiencies approaching 104 complexes/cell. Conclusions We report the successful display of cellulosome-inspired recombinant complexes on the surface of Lactococcus lactis. Significant differences in display efficiency among constructs were observed and attributed to their structural characteristics including protein conformation and solubility, scaffold size, and the inclusion and exclusion of non-cohesin modules. The surface-display of functional scaffold proteins described here represents a key step in the development of recombinant microorganisms capable of carrying out a variety of metabolic processes including the direct conversion of cellulosic substrates into fuels and chemicals. PMID:20840763
Cytoskeleton changes following differentiation of N1E-115 neuroblastoma cell line.

PubMed

Oh, J-E; Karlmark Raja, K; Shin, J-H; Pollak, A; Hengstschläger, M; Lubec, G

2006-10-01

No systematic approach to detect expression of differentiation-related elements was published so far. The undifferentiated N1E-115 neuroblastoma cell line was switched into a neuronal phenotype by DMSO treatment and used for proteomic experiments. We used two-dimensional gel electrophoresis followed by unambiguous mass spectrometrical identification of proteins to generate a map of cytoskeleton proteins (CPs), i.e., to search for differentiation-related structures. Alpha-actin, actin-like protein 6A, gamma-tubulin complex component 2, tubulin alpha 3/alpha 7, CLIP associating protein 2, B4 integrin interactor homolog were detectable in the undifferentiated cell line exclusively and neuron-specific CPs drebrin and presynaptic density protein 95, actin-related protein 2/3, alpha and beta-centractin, PDZ-domain actin binding protein, actinin alpha 1, profilin II, ezrin, coactosin-like protein, transgelin 2, myosin light polypeptide 6, tubulin alpha 2, 6 and 7, beta tubulin (94% similar with tubulin beta-2), tubulin beta 3, tubulin tyrosine ligase-like protein 1, lamin B1 and keratin 20 were observed in the differentiated cell line only. We herein identified differentiation-related expressional patterns thus providing new evidence for the role of CPs in the process of neuronal differentiation.
Grb-IR: A SH2-Domain-Containing Protein that Binds to the Insulin Receptor and Inhibits Its Function

NASA Astrophysics Data System (ADS)

Liu, Feng; Roth, Richard A.

1995-10-01

To identify potential signaling molecules involved in mediating insulin-induced biological responses, a yeast two-hybrid screen was performed with the cytoplasmic domain of the human insulin receptor (IR) as bait to trap high-affinity interacting proteins encoded by human liver or HeLa cDNA libraries. A SH2-domain-containing protein was identified that binds with high affinity in vitro to the autophosphorylated IR. The mRNA for this protein was found by Northern blot analyses to be highest in skeletal muscle and was also detected in fat by PCR. To study the role of this protein in insulin signaling, a full-length cDNA encoding this protein (called Grb-IR) was isolated and stably expressed in Chinese hamster ovary cells overexpressing the human IR. Insulin treatment of these cells resulted in the in situ formation of a complex of the IR and the 60-kDa Grb-IR. Although almost 75% of the Grb-IR protein was bound to the IR, it was only weakly tyrosine-phosphorylated. The formation of this complex appeared to inhibit the insulin-induced increase in tyrosine phosphorylation of two endogenous substrates, a 60-kDa GTPase-activating-protein-associated protein and, to a lesser extent, IR substrate 1. The subsequent association of this latter protein with phosphatidylinositol 3-kinase also appeared to be inhibited. These findings raise the possibility that Grb-IR is a SH2-domain-containing protein that directly complexes with the IR and serves to inhibit signaling or redirect the IR signaling pathway.
Surface Induced Dissociation Yields Quaternary Substructure of Refractory Noncovalent Phosphorylase B and Glutamate Dehydrogenase Complexes

NASA Astrophysics Data System (ADS)

Ma, Xin; Zhou, Mowei; Wysocki, Vicki H.

2014-03-01

Ion mobility (IM) and tandem mass spectrometry (MS/MS) coupled with native MS are useful for studying noncovalent protein complexes. Collision induced dissociation (CID) is the most common MS/MS dissociation method. However, some protein complexes, including glycogen phosphorylase B kinase (PHB) and L-glutamate dehydrogenase (GDH) examined in this study, are resistant to dissociation by CID at the maximum collision energy available in the instrument. Surface induced dissociation (SID) was applied to dissociate the two refractory protein complexes. Different charge state precursor ions of the two complexes were examined by CID and SID. The PHB dimer was successfully dissociated to monomers and the GDH hexamer formed trimeric subcomplexes that are informative of its quaternary structure. The unfolding of the precursor and the percentages of the distinct products suggest that the dissociation pathways vary for different charge states. The precursors at lower charge states (+21 for PHB dimer and +27 for GDH hexamer) produce a higher percentage of folded fragments and dissociate more symmetrically than the precusors at higher charge states (+29 for PHB dimer and +39 for GDH hexamer). The precursors at lower charge state may be more native-like than the higher charge state because a higher percentage of folded fragments and a lower percentage of highly charged unfolded fragments are detected. The combination of SID and charge reduction is shown to be a powerful tool for quaternary structure analysis of refractory noncovalent protein complexes, as illustrated by the data for PHB dimer and GDH hexamer.
Development of automated high throughput single molecular microfluidic detection platform for signal transduction analysis

NASA Astrophysics Data System (ADS)

Huang, Po-Jung; Baghbani Kordmahale, Sina; Chou, Chao-Kai; Yamaguchi, Hirohito; Hung, Mien-Chie; Kameoka, Jun

2016-03-01

Signal transductions including multiple protein post-translational modifications (PTM), protein-protein interactions (PPI), and protein-nucleic acid interaction (PNI) play critical roles for cell proliferation and differentiation that are directly related to the cancer biology. Traditional methods, like mass spectrometry, immunoprecipitation, fluorescence resonance energy transfer, and fluorescence correlation spectroscopy require a large amount of sample and long processing time. "microchannel for multiple-parameter analysis of proteins in single-complex (mMAPS)"we proposed can reduce the process time and sample volume because this system is composed by microfluidic channels, fluorescence microscopy, and computerized data analysis. In this paper, we will present an automated mMAPS including integrated microfluidic device, automated stage and electrical relay for high-throughput clinical screening. Based on this result, we estimated that this automated detection system will be able to screen approximately 150 patient samples in a 24-hour period, providing a practical application to analyze tissue samples in a clinical setting.
The Gag Cleavage Product, p12, is a Functional Constituent of the Murine Leukemia Virus Pre-Integration Complex

PubMed Central

Laham-Karam, Nihay; Selig, Sara; Ehrlich, Marcelo; Bacharach, Eran

2010-01-01

The p12 protein is a cleavage product of the Gag precursor of the murine leukemia virus (MLV). Specific mutations in p12 have been described that affect early stages of infection, rendering the virus replication-defective. Such mutants showed normal generation of genomic DNA but no formation of circular forms, which are markers of nuclear entry by the viral DNA. This suggested that p12 may function in early stages of infection but the precise mechanism of p12 action is not known. To address the function and follow the intracellular localization of the wt p12 protein, we generated tagged p12 proteins in the context of a replication-competent virus, which allowed for the detection of p12 at early stages of infection by immunofluorescence. p12 was found to be distributed to discrete puncta, indicative of macromolecular complexes. These complexes were localized to the cytoplasm early after infection, and thereafter accumulated adjacent to mitotic chromosomes. This chromosomal accumulation was impaired for p12 proteins with a mutation that rendered the virus integration-defective. Immunofluorescence demonstrated that intracellular p12 complexes co-localized with capsid, a known constituent of the MLV pre-integration complex (PIC), and immunofluorescence combined with fluorescent in situ hybridization (FISH) revealed co-localization of the p12 proteins with the incoming reverse transcribed viral DNA. Interactions of p12 with the capsid and with the viral DNA were also demonstrated by co-immunoprecipitation. These results imply that p12 proteins are components of the MLV PIC. Furthermore, a large excess of wt PICs did not rescue the defect in integration of PICs derived from mutant p12 particles, demonstrating that p12 exerts its function as part of this complex. Altogether, these results imply that p12 proteins are constituent of the MLV PIC and function in directing the PIC from the cytoplasm towards integration. PMID:21085616
Phosphatidylinositol 3,4,5-trisphosphate activity probes for the labeling and proteomic characterization of protein binding partners.

PubMed

Rowland, Meng M; Bostic, Heidi E; Gong, Denghuang; Speers, Anna E; Lucas, Nathan; Cho, Wonhwa; Cravatt, Benjamin F; Best, Michael D

2011-12-27

Phosphatidylinositol polyphosphate lipids, such as phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P₃], regulate critical biological processes, many of which are aberrant in disease. These lipids often act as site-specific ligands in interactions that enforce membrane association of protein binding partners. Herein, we describe the development of bifunctional activity probes corresponding to the headgroup of PI(3,4,5)P₃ that are effective for identifying and characterizing protein binding partners from complex samples, namely cancer cell extracts. These probes contain both a photoaffinity tag for covalent labeling of target proteins and a secondary handle for subsequent detection or manipulation of labeled proteins. Probes bearing different secondary tags were exploited, either by direct attachment of a fluorescent dye for optical detection or by using an alkyne that can be derivatized after protein labeling via click chemistry. First, we describe the design and modular synthetic strategy used to generate multiple probes with different reporter tags of use for characterizing probe-labeled proteins. Next, we report initial labeling studies using purified protein, the PH domain of Akt, in which probes were found to label this target, as judged by in-gel detection. Furthermore, protein labeling was abrogated by controls including competition with an unlabeled PI(3,4,5)P₃ headgroup analogue as well as through protein denaturation, indicating specific labeling. In addition, probes featuring linkers of different lengths between the PI(3,4,5)P₃ headgroup and photoaffinity tag led to variations in protein labeling, indicating that a shorter linker was more effective in this case. Finally, proteomic labeling studies were performed using cell extracts; labeled proteins were observed by in-gel detection and characterized using postlabeling with biotin, affinity chromatography, and identification via tandem mass spectrometry. These studies yielded a total of 265 proteins, including both known and novel candidate PI(3,4,5)P₃-binding proteins.
Phosphatidylinositol (3,4,5)-Trisphosphate Activity Probes for the Labeling and Proteomic Characterization of Protein Binding Partners

PubMed Central

Rowland, Meng M.; Bostic, Heidi E.; Gong, Denghuang; Speers, Anna E.; Lucas, Nathan; Cho, Wonhwa; Cravatt, Benjamin F.; Best, Michael D.

2013-01-01

Phosphatidylinositol polyphosphate lipids, such as phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3), regulate critical biological processes, many of which are aberrant in disease. These lipids often act as site-specific ligands in interactions that enforce membrane-association of protein binding partners. Herein, we describe the development of bifunctional activity probes corresponding to the headgroup of PI(3,4,5)P3 that are effective for identifying and characterizing protein binding partners from complex samples, namely cancer cell extracts. These probes contain both a photoaffinity tag for covalent labeling of target proteins as well as a secondary handle for subsequent detection or manipulation of labeled proteins. Probes bearing different secondary tags were exploited, either by direct attachment of a fluorescent dye for optical detection or by using an alkyne that can be derivatized after protein labeling via click chemistry. First, we describe the design and modular synthetic strategy used to generate multiple probes with different reporter tags of use for characterizing probe-labeled proteins. Next, we report initial labeling studies using purified protein, the PH domain of Akt, in which probes were found to label this target, as judged by on-gel detection. Furthermore, protein labeling was abrogated by controls including competition with an unlabeled PI(3,4,5)P3 headgroup analog as well as through protein denaturation, indicating specific labeling. In addition, probes featuring different linker lengths between the PI(3,4,5)P3 headgroup and photoaffinity tag led to variations in protein labeling, indicating that a shorter linker was more effective in this case. Finally, proteomic labeling studies were performed using cell extracts, labeled proteins were observed by in-gel detection and characterized using post-labeling with biotin, affinity chromatography and identification via tandem mass spectrometry. These studies yielded a total of 265 proteins, including both known and novel candidate PI(3,4,5)P3-binding proteins. PMID:22074223
The salivary secretome of the tsetse fly Glossina pallidipes (Diptera: Glossinidae) infected by salivary gland hypertrophy virus.

PubMed

Kariithi, Henry M; Ince, Ikbal A; Boeren, Sjef; Abd-Alla, Adly M M; Parker, Andrew G; Aksoy, Serap; Vlak, Just M; Oers, Monique M van

2011-11-01

The competence of the tsetse fly Glossina pallidipes (Diptera; Glossinidae) to acquire salivary gland hypertrophy virus (SGHV), to support virus replication and successfully transmit the virus depends on complex interactions between Glossina and SGHV macromolecules. Critical requisites to SGHV transmission are its replication and secretion of mature virions into the fly's salivary gland (SG) lumen. However, secretion of host proteins is of equal importance for successful transmission and requires cataloging of G. pallidipes secretome proteins from hypertrophied and non-hypertrophied SGs. After electrophoretic profiling and in-gel trypsin digestion, saliva proteins were analyzed by nano-LC-MS/MS. MaxQuant/Andromeda search of the MS data against the non-redundant (nr) GenBank database and a G. morsitans morsitans SG EST database, yielded a total of 521 hits, 31 of which were SGHV-encoded. On a false discovery rate limit of 1% and detection threshold of least 2 unique peptides per protein, the analysis resulted in 292 Glossina and 25 SGHV MS-supported proteins. When annotated by the Blast2GO suite, at least one gene ontology (GO) term could be assigned to 89.9% (285/317) of the detected proteins. Five (∼1.8%) Glossina and three (∼12%) SGHV proteins remained without a predicted function after blast searches against the nr database. Sixty-five of the 292 detected Glossina proteins contained an N-terminal signal/secretion peptide sequence. Eight of the SGHV proteins were predicted to be non-structural (NS), and fourteen are known structural (VP) proteins. SGHV alters the protein expression pattern in Glossina. The G. pallidipes SG secretome encompasses a spectrum of proteins that may be required during the SGHV infection cycle. These detected proteins have putative interactions with at least 21 of the 25 SGHV-encoded proteins. Our findings opens venues for developing novel SGHV mitigation strategies to block SGHV infections in tsetse production facilities such as using SGHV-specific antibodies and phage display-selected gut epithelia-binding peptides.
A probabilistic model for detecting rigid domains in protein structures.

PubMed

Nguyen, Thach; Habeck, Michael

2016-09-01

Large-scale conformational changes in proteins are implicated in many important biological functions. These structural transitions can often be rationalized in terms of relative movements of rigid domains. There is a need for objective and automated methods that identify rigid domains in sets of protein structures showing alternative conformational states. We present a probabilistic model for detecting rigid-body movements in protein structures. Our model aims to approximate alternative conformational states by a few structural parts that are rigidly transformed under the action of a rotation and a translation. By using Bayesian inference and Markov chain Monte Carlo sampling, we estimate all parameters of the model, including a segmentation of the protein into rigid domains, the structures of the domains themselves, and the rigid transformations that generate the observed structures. We find that our Gibbs sampling algorithm can also estimate the optimal number of rigid domains with high efficiency and accuracy. We assess the power of our method on several thousand entries of the DynDom database and discuss applications to various complex biomolecular systems. The Python source code for protein ensemble analysis is available at: https://github.com/thachnguyen/motion_detection : mhabeck@gwdg.de. © The Author 2016. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
Visualizing repetitive diffusion activity of double-strand RNA binding proteins by single molecule fluorescence assays.

PubMed

Koh, Hye Ran; Wang, Xinlei; Myong, Sua

2016-08-01

TRBP, one of double strand RNA binding proteins (dsRBPs), is an essential cofactor of Dicer in the RNA interference pathway. Previously we reported that TRBP exhibits repetitive diffusion activity on double strand (ds)RNA in an ATP independent manner. In the TRBP-Dicer complex, the diffusion mobility of TRBP facilitates Dicer-mediated RNA cleavage. Such repetitive diffusion of dsRBPs on a nucleic acid at the nanometer scale can be appropriately captured by several single molecule detection techniques. Here, we provide a step-by-step guide to four different single molecule fluorescence assays by which the diffusion activity of dsRBPs on dsRNA can be detected. One color assay, termed protein induced fluorescence enhancement enables detection of unlabeled protein binding and diffusion on a singly labeled RNA. Two-color Fluorescence Resonance Energy Transfer (FRET) in which labeled dsRBPs is applied to labeled RNA, allows for probing the motion of protein along the RNA axis. Three color FRET reports on the diffusion movement of dsRBPs from one to the other end of RNA. The single molecule pull down assay provides an opportunity to collect dsRBPs from mammalian cells and examine the protein-RNA interaction at single molecule platform. Copyright © 2016 Elsevier Inc. All rights reserved.
Solving the Secondary Structure Matching Problem in Cryo-EM De Novo Modeling Using a Constrained K-Shortest Path Graph Algorithm.

PubMed

Al Nasr, Kamal; Ranjan, Desh; Zubair, Mohammad; Chen, Lin; He, Jing

2014-01-01

Electron cryomicroscopy is becoming a major experimental technique in solving the structures of large molecular assemblies. More and more three-dimensional images have been obtained at the medium resolutions between 5 and 10 Å. At this resolution range, major α-helices can be detected as cylindrical sticks and β-sheets can be detected as plain-like regions. A critical question in de novo modeling from cryo-EM images is to determine the match between the detected secondary structures from the image and those on the protein sequence. We formulate this matching problem into a constrained graph problem and present an O(Δ(2)N(2)2(N)) algorithm to this NP-Hard problem. The algorithm incorporates the dynamic programming approach into a constrained K-shortest path algorithm. Our method, DP-TOSS, has been tested using α-proteins with maximum 33 helices and α-β proteins up to five helices and 12 β-strands. The correct match was ranked within the top 35 for 19 of the 20 α-proteins and all nine α-β proteins tested. The results demonstrate that DP-TOSS improves accuracy, time and memory space in deriving the topologies of the secondary structure elements for proteins with a large number of secondary structures and a complex skeleton.
Phospho-ubiquitin-PARK2 complex as a marker for mitophagy defects.

PubMed

Callegari, Sylvie; Oeljeklaus, Silke; Warscheid, Bettina; Dennerlein, Sven; Thumm, Michael; Rehling, Peter; Dudek, Jan

2017-01-02

The E3 ubiquitin ligase PARK2 and the mitochondrial protein kinase PINK1 are required for the initiation of mitochondrial damage-induced mitophagy. Together, PARK2 and PINK1 generate a phospho-ubiquitin signal on outer mitochondrial membrane proteins that triggers recruitment of the autophagy machinery. This paper describes the detection of a defined 500-kDa phospho-ubiquitin-rich PARK2 complex that accumulates on mitochondria upon treatment with the membrane uncoupler CCCP. Formation of this complex is dependent on the presence of PINK1 and is absent in mutant forms of PARK2, whereby mitophagy is also arrested. These results signify a functional signaling complex that is essential for the progression of mitophagy. The visualization of the PARK2 signaling complex represents a novel marker for this critical step in mitophagy and can be used to monitor mitophagy progression in PARK2 mutants and to uncover additional upstream factors required for PARK2-mediated mitophagy signaling.
Microtubule actin crosslinking factor 1b: a novel plakin that localizes to the Golgi complex.

PubMed

Lin, Chung-Ming; Chen, Hui-Jye; Leung, Conrad L; Parry, David A D; Liem, Ronald K H

2005-08-15

MACF1 (microtubule actin crosslinking factor), also called ACF7 (actin crosslinking family 7) is a cytoskeletal linker protein that can associate with both actin filaments and microtubules. We have identified a novel alternatively spliced isoform of MACF1. We named this isoform MACF1b and renamed the original isoform MACF1a. MACF1b is identical to MACF1a, except that it has a region containing plakin (or plectin) repeats in the middle of the molecule. MACF1b is ubiquitously expressed in adult tissues with especially high levels in the lung. We studied the subcellular localization of MACF1b proteins in mammalian cell lines. In two lung cell lines, MACF1b was chiefly localized to the Golgi complex. Upon treatments that disrupt the Golgi complex, MACF1b redistributed into the cytosol, but remained co-localized with the dispersed Golgi ministacks. MACF1b proteins can be detected in the enriched Golgi fraction by western blotting. The domain of MACF1b that targets it to the Golgi was found at the N-terminal part of the region that contains the plakin repeats. Reducing the level of MACF1 proteins by small-interfering RNA resulted in the dispersal of the Golgi complex.
Losses, Expansions, and Novel Subunit Discovery of Adaptor Protein Complexes in Haptophyte Algae.

PubMed

Lee, Laura J Y; Klute, Mary J; Herman, Emily K; Read, Betsy; Dacks, Joel B

2015-11-01

The phylum Haptophyta (Diaphoratickes) contains marine algae that perform biomineralization, extruding large, distinctive calcium carbonate scales (coccoliths) that completely cover the cell. Coccolith production is an important part of global carbon cycling; however, the membrane trafficking pathway by which they are secreted has not yet been elucidated. In most eukaryotes, post-Golgi membrane trafficking involves five heterotetrameric adaptor protein (AP) complexes, which impart cargo selection specificity. To better understand coccolith secretion, we performed comparative genomic, phylogenetic, and transcriptomic analyses of the AP complexes in Emiliania huxleyi strains 92A, Van556, EH2, and CCMP1516, and related haptophytes Gephyrocapsa oceanica and Isochrysis galbana; the latter has lost the ability to biomineralize. We show that haptophytes have a modified membrane trafficking system (MTS), as we found both AP subunit losses and duplications. Additionally, we identified a single conserved subunit of the AP-related TSET complex, whose expression suggests a functional role in membrane trafficking. Finally, we detected novel alpha adaptin ear and gamma adaptin ear proteins, the first of their kind to be described outside of opisthokonts. These novel ear proteins and the sculpting of the MTS may support the capacity for biomineralization in haptophytes, enhancing their ability to perform this highly specialized form of secretion. Copyright © 2015 Elsevier GmbH. All rights reserved.
Development of optical immunosensors for detection of proteins in serum.

PubMed

Kyprianou, Dimitris; Chianella, Iva; Guerreiro, Antonio; Piletska, Elena V; Piletsky, Sergey A

2013-01-15

The detection of proteins in biological samples such as blood, serum or plasma by biosensors is very challenging due to the complex nature of the matrix, which contains a high level of many interfering compounds. Here we show the application of a novel polymeric immobilisation matrix that helps in the detection of specific protein analytes in biological samples by surface plasmon resonance (SPR) immunosensors. This polymer matrix contains thioacetal functional groups included in the network, and these groups do not require any further activation in order to react with proteins, making it attractive for sensor fabrication. The protein prostate specific antigen (PSA) was selected as a model target analyte. A sandwich format with two primary antibodies recognising different parts (epitopes) of the analyte was used for the detection of PSA in serum. The efficiency of the reduction of non-specific binding achieved with novel polymer was compared with those of other techniques such as coating of sensor surface with polyethylene glycol (PEG), use of charged hydrophilic aspartic acid and surfactants such as Tween20. The detection limit of the polymer based immunosensor was 0.1 ng ml(-1) for free form PSA (f-PSA) in buffer and 5 ng ml(-1) in 20% serum. This is an improvement compared with similar devices reported on literature, indicating the potential of the immunosensor developed here for the analysis of real samples. Copyright © 2012 Elsevier B.V. All rights reserved.
ICE1 promotes the link between splicing and nonsense-mediated mRNA decay

PubMed Central

Baird, Thomas D; Cheng, Ken Chih-Chien; Chen, Yu-Chi; Buehler, Eugen; Martin, Scott E; Inglese, James

2018-01-01

The nonsense-mediated mRNA decay (NMD) pathway detects aberrant transcripts containing premature termination codons (PTCs) and regulates expression of 5–10% of non-aberrant human mRNAs. To date, most proteins involved in NMD have been identified by genetic screens in model organisms; however, the increased complexity of gene expression regulation in human cells suggests that additional proteins may participate in the human NMD pathway. To identify proteins required for NMD, we performed a genome-wide RNAi screen against >21,000 genes. Canonical members of the NMD pathway were highly enriched as top hits in the siRNA screen, along with numerous candidate NMD factors, including the conserved ICE1/KIAA0947 protein. RNAseq studies reveal that depletion of ICE1 globally enhances accumulation and stability of NMD-target mRNAs. Further, our data suggest that ICE1 uses a putative MIF4G domain to interact with exon junction complex (EJC) proteins and promotes the association of the NMD protein UPF3B with the EJC. PMID:29528287
Identification of novel extracellular protein for PCB/biphenyl metabolism in Rhodococcus jostii RHA1.

PubMed

Atago, Yuki; Shimodaira, Jun; Araki, Naoto; Bin Othman, Nor'azizi; Zakaria, Zuriati; Fukuda, Masao; Futami, Junichiro; Hara, Hirofumi

2016-05-01

Rhodococcus jostii RHA1 (RHA1) degrades polychlorinated biphenyl (PCB) via co-metabolism with biphenyl. To identify the novel open reading frames (ORFs) that contribute to PCB/biphenyl metabolism in RHA1, we compared chromatin immunoprecipitation chip and transcriptomic data. Six novel ORFs involved in PCB/biphenyl metabolism were identified. Gene deletion mutants of these 6 ORFs were made and were tested for their ability to grow on biphenyl. Interestingly, only the ro10225 deletion mutant showed deficient growth on biphenyl. Analysis of Ro10225 protein function showed that growth of the ro10225 deletion mutant on biphenyl was recovered when exogenous recombinant Ro10225 protein was added to the culture medium. Although Ro10225 protein has no putative secretion signal sequence, partially degraded Ro10225 protein was detected in conditioned medium from wild-type RHA1 grown on biphenyl. This Ro10225 fragment appeared to form a complex with another PCB/biphenyl oxidation enzyme. These results indicated that Ro10225 protein is essential for the formation of the PCB/biphenyl dioxygenase complex in RHA1.
Application of “omics” to Prion Biomarker Discovery

PubMed Central

Huzarewich, Rhiannon L. C. H.; Siemens, Christine G.; Booth, Stephanie A.

2010-01-01

The advent of genomics and proteomics has been a catalyst for the discovery of biomarkers able to discriminate biological processes such as the pathogenesis of complex diseases. Prompt detection of prion diseases is particularly desirable given their transmissibility, which is responsible for a number of human health risks stemming from exogenous sources of prion protein. Diagnosis relies on the ability to detect the biomarker PrPSc, a pathological isoform of the host protein PrPC, which is an essential component of the infectious prion. Immunochemical detection of PrPSc is specific and sensitive enough for antemortem testing of brain tissue, however, this is not the case in accessible biological fluids or for the detection of recently identified novel prions with unique biochemical properties. A complementary approach to the detection of PrPSc itself is to identify alternative, “surrogate” gene or protein biomarkers indicative of disease. Biomarkers are also useful to track the progress of disease, especially important in the assessment of therapies, or to identify individuals “at risk”. In this review we provide perspective on current progress and pitfalls in the use of “omics” technologies to screen body fluids and tissues for biomarker discovery in prion diseases. PMID:20224650
Subunit mass fingerprinting of mitochondrial complex I.

PubMed

Morgner, Nina; Zickermann, Volker; Kerscher, Stefan; Wittig, Ilka; Abdrakhmanova, Albina; Barth, Hans-Dieter; Brutschy, Bernhard; Brandt, Ulrich

2008-10-01

We have employed laser induced liquid bead ion desorption (LILBID) mass spectrometry to determine the total mass and to study the subunit composition of respiratory chain complex I from Yarrowia lipolytica. Using 5-10 pmol of purified complex I, we could assign all 40 known subunits of this membrane bound multiprotein complex to peaks in LILBID subunit fingerprint spectra by comparing predicted protein masses to observed ion masses. Notably, even the highly hydrophobic subunits encoded by the mitochondrial genome were easily detectable. Moreover, the LILBID approach allowed us to spot and correct several errors in the genome-derived protein sequences of complex I subunits. Typically, the masses of the individual subunits as determined by LILBID mass spectrometry were within 100 Da of the predicted values. For the first time, we demonstrate that LILBID spectrometry can be successfully applied to a complex I band eluted from a blue-native polyacrylamide gel, making small amounts of large multiprotein complexes accessible for subunit mass fingerprint analysis even if they are membrane bound. Thus, the LILBID subunit mass fingerprint method will be of great value for efficient proteomic analysis of complex I and its assembly intermediates, as well as of other water soluble and membrane bound multiprotein complexes.

Protein lipoxidation: Detection strategies and challenges

PubMed Central

Aldini, Giancarlo; Domingues, M. Rosário; Spickett, Corinne M.; Domingues, Pedro; Altomare, Alessandra; Sánchez-Gómez, Francisco J.; Oeste, Clara L.; Pérez-Sala, Dolores

2015-01-01

Enzymatic and non-enzymatic lipid metabolism can give rise to reactive species that may covalently modify cellular or plasma proteins through a process known as lipoxidation. Under basal conditions, protein lipoxidation can contribute to normal cell homeostasis and participate in signaling or adaptive mechanisms, as exemplified by lipoxidation of Ras proteins or of the cytoskeletal protein vimentin, both of which behave as sensors of electrophilic species. Nevertheless, increased lipoxidation under pathological conditions may lead to deleterious effects on protein structure or aggregation. This can result in impaired degradation and accumulation of abnormally folded proteins contributing to pathophysiology, as may occur in neurodegenerative diseases. Identification of the protein targets of lipoxidation and its functional consequences under pathophysiological situations can unveil the modification patterns associated with the various outcomes, as well as preventive strategies or potential therapeutic targets. Given the wide structural variability of lipid moieties involved in lipoxidation, highly sensitive and specific methods for its detection are required. Derivatization of reactive carbonyl species is instrumental in the detection of adducts retaining carbonyl groups. In addition, use of tagged derivatives of electrophilic lipids enables enrichment of lipoxidized proteins or peptides. Ultimate confirmation of lipoxidation requires high resolution mass spectrometry approaches to unequivocally identify the adduct and the targeted residue. Moreover, rigorous validation of the targets identified and assessment of the functional consequences of these modifications are essential. Here we present an update on methods to approach the complex field of lipoxidation along with validation strategies and functional assays illustrated with well-studied lipoxidation targets. PMID:26072467
Selective detection of target proteins by peptide-enabled graphene biosensor.

PubMed

Khatayevich, Dmitriy; Page, Tamon; Gresswell, Carolyn; Hayamizu, Yuhei; Grady, William; Sarikaya, Mehmet

2014-04-24

Direct molecular detection of biomarkers is a promising approach for diagnosis and monitoring of numerous diseases, as well as a cornerstone of modern molecular medicine and drug discovery. Currently, clinical applications of biomarkers are limited by the sensitivity, complexity and low selectivity of available indirect detection methods. Electronic 1D and 2D nano-materials such as carbon nanotubes and graphene, respectively, offer unique advantages as sensing substrates for simple, fast and ultrasensitive detection of biomolecular binding. Versatile methods, however, have yet to be developed for simultaneous functionalization and passivation of the sensor surface to allow for enhanced detection and selectivity of the device. Herein, we demonstrate selective detection of a model protein against a background of serum protein using a graphene sensor functionalized via self-assembling multifunctional short peptides. The two peptides are engineered to bind to graphene and undergo co-assembly in the form of an ordered monomolecular film on the substrate. While the probe peptide displays the bioactive molecule, the passivating peptide prevents non-specific protein adsorption onto the device surface, ensuring target selectivity. In particular, we demonstrate a graphene field effect transistor (gFET) biosensor which can detect streptavidin against a background of serum bovine albumin at less than 50 ng/ml. Our nano-sensor design, allows us to restore the graphene surface and utilize each sensor in multiple experiments. The peptide-enabled gFET device has great potential to address a variety of bio-sensing problems, such as studying ligand-receptor interactions, or detection of biomarkers in a clinical setting. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Biosynthesis of edeine: II. Localization of edeine synthetase within Bacillus brevis Vm4.

PubMed

Kurylo-Borowska, Z

1975-07-14

Edeine-synthesizing polyenzymes, associated with a complex of sytoplasmic membrane and DNA, were obtained from gently lysed cells of Bacillus brevis Vm4. The polyenzymes-membrane-DNA complex, isolated from dells intensively synthesizing edeines (18--20 h culture) contained edeine B. Edeine B was found to be bound covalently t o the edeine synthetase. The amount of edeine bound to polyenzymes was 0.1--0.3 mumol/mg protein, depending on the age of cells. Detachment of deeine synthetase with a covalently bound edeine B from the membrane-DNA complex was accomplished by a treatment with (NH4)2-SO4 at 45--55% saturation or by DEAE-cellulose column fractionation. In contrast to other components of the complex, the edeine-polyenzymes fragment was not adsorbed to the DEAE-cellulose. Sephadex G-200 column chromatography separated the edeine-polyenzymes complex into 3 fractions. Edeine-polyenzymes complex, obtained from lysozyme-Brij-58-DNAase treated cells, contained edeine B bound to two protein fractions of mol. wt 210 000 and 160 000. Edeine-polyenzymes complex detached from the complex with the membrane and DNA contained edeine B, bound only to protein fraction of mol. wt 210 000. Edeine A was not found in the edeine-polyenzymes complex. No accumulation of free antibiotics within 16--22 h old cells of B. brevis Vm4 was detected. The edeine-polyenzymes complex associated with the DNA-membrane complex has shown no antimicrobial activity. By treating of above with alkali, edeine B of specific activity: 80 units/mjmol was released. The complex of DNA-membrane associated with edeine-polyenzymes complex was able to synthesize DNA, under the conditions described for synthesis, directed by a DNA-membrane complex. Edeine when associated with this complex did not effect the DNA-synthesizing activity.
Structural insights into NHEJ: building up an integrated picture of the dynamic DSB repair super complex, one component and interaction at a time

PubMed Central

Williams, Gareth J.; Hammel, Michal; Radhakrishnan, Sarvan Kumar; Ramsden, Dale; Lees-Miller, Susan P.; Tainer, John A.

2014-01-01

Non-homologous end joining (NHEJ) is the major pathway for repair of DNA double-strand breaks (DSBs) in human cells. NHEJ is also needed for V(D)J recombination and the development of T and B cells in vertebrate immune systems, and acts in both the generation and prevention of non-homologous chromosomal translocations, a hallmark of genomic instability and many human cancers. X-ray crystal structures, cryo-electron microscopy envelopes, and small angle X-ray scattering (SAXS) solution conformations and assemblies are defining most of the core protein components for NHEJ: Ku70/Ku80 heterodimer; the DNA dependent protein kinase catalytic subunit (DNA-PKcs); the structure-specific endonuclease Artemis along with polynucleotide kinase/phosphatase (PNKP), aprataxin and PNKP related protein (APLF); the scaffolding proteins XRCC4 and XLF (XRCC4-like factor); DNA polymerases, and DNA ligase IV (Lig IV). The dynamic assembly of multi-protein NHEJ complexes at DSBs is regulated in part by protein phosphorylation. The basic steps of NHEJ have been biochemically defined to require: 1) DSB detection by the Ku heterodimer with subsequent DNA-PKcs tethering to form the DNA-PKcs-Ku-DNA complex (termed DNA-PK), 2) lesion processing, and 3) DNA end ligation by Lig IV, which functions in complex with XRCC4 and XLF. The current integration of structures by combined methods is resolving puzzles regarding the mechanisms, coordination and regulation of these three basic steps. Overall, structural results suggest the NHEJ system forms a flexing scaffold with the DNA-PKcs HEAT repeats acting as compressible macromolecular springs suitable to store and release conformational energy to apply forces to regulate NHEJ complexes and the DNA substrate for DNA end protection, processing, and ligation. PMID:24656613
Expanding the bovine milk proteome through extensive fractionation.

PubMed

Nissen, Asger; Bendixen, Emøke; Ingvartsen, Klaus Lønne; Røntved, Christine Maria

2013-01-01

Bovine milk is an agricultural product of tremendous value worldwide. It contains proteins, fat, lactose, vitamins, and minerals. It provides nutrition and immunological protection (e.g., in the gastrointestinal tract) to the newborn and young calf. It also forms an important part of human nutrition. The repertoire of proteins in milk (i.e., its proteome) is vast and complex. The milk proteome can be described in detail by mass spectrometry-based proteomics. However, the high concentration of dominating proteins in milk reduces mass spectrometry detection sensitivity and limits detection of low abundant proteins. Further, the general health and udder health of the dairy cows delivering the milk may influence the composition of the milk proteome. To gain a more exhaustive and true picture of the milk proteome, we performed an extensive preanalysis fractionation of raw composite milk collected from documented healthy cows in early lactation. Four simple and industrially applicable techniques exploring the physical and chemical properties of milk, including acidification, filtration, and centrifugation, were used for separation of the proteins. This resulted in 5 different fractions, whose content of proteins were compared with the proteins of nonfractionated milk using 2-dimensional liquid chromatography tandem mass spectrometry analysis. To validate the proteome analysis, spectral counts and ELISA were performed on 7 proteins using the ELISA for estimation of the detection sensitivity limit of the 2-dimensional liquid chromatography tandem mass spectrometry analysis. Each fractionation technique resulted in identification of a unique subset of proteins. However, high-speed centrifugation of milk to whey was by far the best method to achieve high and repeatable proteome coverage. The total number of milk proteins initially detected in nonfractionated milk and the fractions were 635 in 2 replicates. Removal of dominant proteins and filtering for redundancy across the different fractions reduced the number to 376 unique proteins in 2 replicates. In addition, 366 proteins were detected by this process in 1 replicate. Hence, by applying different fractionation techniques to milk, we expanded the milk proteome. The milk proteome map may serve as a reference for scientists working in the dairy sector. Copyright © 2013 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.
Interaction of the alpha-subunit of Escherichia coli RNA polymerase with DNA: rigid body nature of the protein-DNA contact.

PubMed

Heyduk, E; Baichoo, N; Heyduk, T

2001-11-30

The alpha-subunit of Escherichia coli RNA polymerase plays an important role in the activity of many promoters by providing a direct protein-DNA contact with a specific sequence (UP element) located upstream of the core promoter sequence. To obtain insight into the nature of thermodynamic forces involved in the formation of this protein-DNA contact, the binding of the alpha-subunit of E. coli RNA polymerase to a fluorochrome-labeled DNA fragment containing the rrnB P1 promoter UP element sequence was quantitatively studied using fluorescence polarization. The alpha dimer and DNA formed a 1:1 complex in solution. Complex formation at 25 degrees C was enthalpy-driven, the binding was accompanied by a net release of 1-2 ions, and no significant specific ion effects were observed. The van't Hoff plot of temperature dependence of binding was linear suggesting that the heat capacity change (Deltac(p)) was close to zero. Protein footprinting with hydroxyradicals showed that the protein did not change its conformation upon protein-DNA contact formation. No conformational changes in the DNA molecule were detected by CD spectroscopy upon protein-DNA complex formation. The thermodynamic characteristics of the binding together with the lack of significant conformational changes in the protein and in the DNA suggested that the alpha-subunit formed a rigid body-like contact with the DNA in which a tight complementary recognition interface between alpha-subunit and DNA was not formed.
Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio-layer interferometry.

PubMed

Naik, Subhashchandra; Kumru, Ozan S; Cullom, Melissa; Telikepalli, Srivalli N; Lindboe, Elizabeth; Roop, Taylor L; Joshi, Sangeeta B; Amin, Divya; Gao, Phillip; Middaugh, C Russell; Volkin, David B; Fisher, Mark T

2014-10-01

The ability of a GroEL-based bio-layer interferometry (BLI) assay to detect structurally altered and/or aggregated species of pharmaceutically relevant proteins is demonstrated. Assay development included optimizing biotinylated-GroEL immobilization to streptavidin biosensors, combined with biophysical and activity measurements showing native and biotinylated GroEL are both stable and active. First, acidic fibroblast growth factor (FGF-1) was incubated under conditions known to promote (40°C) and inhibit (heparin addition) molten globule formation. Heat exposed (40°C) FGF-1 exhibited binding to GroEL-biosensors, which was significantly diminished in the presence of heparin. Second, a polyclonal human IgG solution containing 6-8% non-native dimer showed an increase in higher molecular weight aggregates upon heating by size exclusion chromatography (SEC). The poly IgG solution displayed binding to GroEL-biosensors initially with progressively increased binding upon heating. Enriched preparations of the IgG dimers or monomers showed significant binding to GroEL-biosensors. Finally, a thermally treated IgG1 monoclonal antibody (mAb) solution also demonstrated increased GroEL-biosensor binding, but with different kinetics. The bound complexes could be partially to fully dissociated after ATP addition (i.e., specific GroEL binding) depending on the protein, environmental stress, and the assay's experimental conditions. Transmission electron microscopy (TEM) images of GroEL-mAb complexes, released from the biosensor, also confirmed interaction of bound complexes at the GroEL binding site with heat-stressed mAb. Results indicate that the GroEL-biosensor-BLI method can detect conformationally altered and/or early aggregation states of proteins, and may potentially be useful as a rapid, stability-indicating biosensor assay for monitoring the structural integrity and physical stability of therapeutic protein candidates. © 2014 The Protein Society.
Microfluidic molecular assay platform for the detection of miRNAs, mRNAs, proteins, and post-translational modifications at single-cell resolution

DOE PAGES

Wu, Meiye; Singh, Anup K.

2014-07-15

In this study, cell signaling is a dynamic and complex process. A typical signaling pathway may begin with activation of cell surface receptors, leading to activation kinase cascade that culminates in induction of mRNA and non-coding miRNA production in the nucleus, followed by modulation of mRNA expression by miRNAs in the cytosol, and end with production of proteins in response to the signaling pathway. Signaling pathways involve proteins, miRNA, and mRNAs, along with various forms of transient post-translational modifications, and detecting each type of signaling molecule requires categorically different sample preparation methods such as Western blotting for proteins, PCR formore » nucleic acids, and flow cytometry for post-translational modifications. Since we know that cells in populations behave heterogeneously1, especially in the cases of stem cells, cancer, and hematopoiesis, there is need for a new technology that provides capability to detect and quantify multiple categories of signaling molecules in intact single cells to provide a comprehensive view of the cell’s physiological state. In this technical brief, we describe our microfluidic platform with a portfolio of customized molecular assays that can detect nucleic acids, proteins, and post-translational modifications in single intact cells with >95% reduction in reagent requirement in under 8 hours.« less
Microfluidic molecular assay platform for the detection of miRNAs, mRNAs, proteins, and posttranslational modifications at single-cell resolution.

PubMed

Wu, Meiye; Singh, Anup K

2014-12-01

Cell signaling is a dynamic and complex process. A typical signaling pathway may begin with activation of cell surface receptors, leading to activation of a kinase cascade that culminates in induction of messenger RNA (mRNA) and noncoding microRNA (miRNA) production in the nucleus, followed by modulation of mRNA expression by miRNAs in the cytosol, and end with production of proteins in response to the signaling pathway. Signaling pathways involve proteins, miRNA, and mRNAs, along with various forms of transient posttranslational modifications, and detecting each type of signaling molecule requires categorically different sample preparation methods such as Western blotting for proteins, PCR for nucleic acids, and flow cytometry for posttranslational modifications. Since we know that cells in populations behave heterogeneously,(1) especially in the cases of stem cells, cancer, and hematopoiesis, there is need for a new technology that provides capability to detect and quantify multiple categories of signaling molecules in intact single cells to provide a comprehensive view of the cell's physiological state. In this Technology Brief, we describe our automated microfluidic platform with a portfolio of customized molecular assays that can detect nucleic acids, proteins, and posttranslational modifications in single intact cells with >95% reduction in reagent requirement in under 8 h. © 2014 Society for Laboratory Automation and Screening.
Adaptation of Tri-molecular fluorescence complementation allows assaying of regulatory Csr RNA-protein interactions in bacteria.

PubMed

Gelderman, Grant; Sivakumar, Anusha; Lipp, Sarah; Contreras, Lydia

2015-02-01

sRNAs play a significant role in controlling and regulating cellular metabolism. One of the more interesting aspects of certain sRNAs is their ability to make global changes in the cell by interacting with regulatory proteins. In this work, we demonstrate the use of an in vivo Tri-molecular Fluorescence Complementation assay to detect and visualize the central regulatory sRNA-protein interaction of the Carbon Storage Regulatory system in E. coli. The Carbon Storage Regulator consists primarily of an RNA binding protein, CsrA, that alters the activity of mRNA targets and of an sRNA, CsrB, that modulates the activity of CsrA. We describe the construction of a fluorescence complementation system that detects the interactions between CsrB and CsrA. Additionally, we demonstrate that the intensity of the fluorescence of this system is able to detect changes in the affinity of the CsrB-CsrA interaction, as caused by mutations in the protein sequence of CsrA. While previous methods have adopted this technique to study mRNA or RNA localization, this is the first attempt to use this technique to study the sRNA-protein interaction directly in bacteria. This method presents a potentially powerful tool to study complex bacterial RNA protein interactions in vivo. © 2014 Wiley Periodicals, Inc.
Positive Darwinian Selection in the Piston That Powers Proton Pumps in Complex I of the Mitochondria of Pacific Salmon

PubMed Central

Garvin, Michael R.; Bielawski, Joseph P.; Gharrett, Anthony J.

2011-01-01

The mechanism of oxidative phosphorylation is well understood, but evolution of the proteins involved is not. We combined phylogenetic, genomic, and structural biology analyses to examine the evolution of twelve mitochondrial encoded proteins of closely related, yet phenotypically diverse, Pacific salmon. Two separate analyses identified the same seven positively selected sites in ND5. A strong signal was also detected at three sites of ND2. An energetic coupling analysis revealed several structures in the ND5 protein that may have co-evolved with the selected sites. These data implicate Complex I, specifically the piston arm of ND5 where it connects the proton pumps, as important in the evolution of Pacific salmon. Lastly, the lineage to Chinook experienced rapid evolution at the piston arm. PMID:21969854
Positive Darwinian selection in the piston that powers proton pumps in complex I of the mitochondria of Pacific salmon.

PubMed

Garvin, Michael R; Bielawski, Joseph P; Gharrett, Anthony J

2011-01-01

The mechanism of oxidative phosphorylation is well understood, but evolution of the proteins involved is not. We combined phylogenetic, genomic, and structural biology analyses to examine the evolution of twelve mitochondrial encoded proteins of closely related, yet phenotypically diverse, Pacific salmon. Two separate analyses identified the same seven positively selected sites in ND5. A strong signal was also detected at three sites of ND2. An energetic coupling analysis revealed several structures in the ND5 protein that may have co-evolved with the selected sites. These data implicate Complex I, specifically the piston arm of ND5 where it connects the proton pumps, as important in the evolution of Pacific salmon. Lastly, the lineage to Chinook experienced rapid evolution at the piston arm.
RNA stores tau reversibly in complex coacervates

PubMed Central

Lin, Yanxian; Eschmann, Neil A.; Zhou, Hongjun; Rauch, Jennifer N.; Hernandez, Israel; Guzman, Elmer; Kosik, Kenneth S.; Han, Songi

2017-01-01

Nonmembrane-bound organelles that behave like liquid droplets are widespread among eukaryotic cells. Their dysregulation appears to be a critical step in several neurodegenerative conditions. Here, we report that tau protein, the primary constituent of Alzheimer neurofibrillary tangles, can form liquid droplets and therefore has the necessary biophysical properties to undergo liquid-liquid phase separation (LLPS) in cells. Consonant with the factors that induce LLPS, tau is an intrinsically disordered protein that complexes with RNA to form droplets. Uniquely, the pool of RNAs to which tau binds in living cells are tRNAs. This phase state of tau is held in an approximately 1:1 charge balance across the protein and the nucleic acid constituents, and can thus be maximal at different RNA:tau mass ratios, depending on the biopolymer constituents involved. This feature is characteristic of complex coacervation. We furthermore show that the LLPS process is directly and sensitively tuned by salt concentration and temperature, implying it is modulated by both electrostatic interactions between the involved protein and nucleic acid constituents, as well as net changes in entropy. Despite the high protein concentration within the complex coacervate phase, tau is locally freely tumbling and capable of diffusing through the droplet interior. In fact, tau in the condensed phase state does not reveal any immediate changes in local protein packing, local conformations and local protein dynamics from that of tau in the dilute solution state. In contrast, the population of aggregation-prone tau as induced by the complexation with heparin is accompanied by large changes in local tau conformations and irreversible aggregation. However, prolonged residency within the droplet state eventually results in the emergence of detectable β-sheet structures according to thioflavin-T assay. These findings suggest that the droplet state can incubate tau and predispose the protein toward the formation of insoluble fibrils. PMID:28683104
Detection of specific protein-protein interactions in nanocages by engineering bipartite FlAsH binding sites.

PubMed

Cornell, Thomas A; Fu, Jing; Newland, Stephanie H; Orner, Brendan P

2013-11-06

Proteins that form cage-like structures have been of much recent cross-disciplinary interest due to their application to bioconjugate and materials chemistry, their biological functions spanning multiple essential cellular processes, and their complex structure, often defined by highly symmetric protein–protein interactions. Thus, establishing the fundamentals of their formation, through detecting and quantifying important protein–protein interactions, could be crucial to understanding essential cellular machinery, and for further development of protein-based technologies. Herein we describe a method to monitor the assembly of protein cages by detecting specific, oligomerization state dependent, protein–protein interactions. Our strategy relies on engineering protein monomers to include cysteine pairs that are presented proximally if the cage state assembles. These assembled pairs of cysteines act as binding sites for the fluorescent reagent FlAsH, which, once bound, provides a readout for successful oligomerization. As a proof of principle, we applied this technique to the iron storage protein, DNA-binding protein from starved cells from E. coli. Several linker lengths and conformations for the presentation of the cysteine pairs were screened to optimize the engineered binding sites. We confirmed that our designs were successful in both lysates and with purified proteins, and that FlAsH binding was dependent upon cage assembly. Following successful characterization of the assay, its throughput was expanded. A two-dimension matrix of pH and denaturing buffer conditions was screened to optimize nanocage stability. We intend to use this method for the high throughput screening of protein cage libraries and of conditions for the generation of inorganic nanoparticles within the cavity of these and other cage proteins.
Evaluation of Nonferrous Metals as Potential In Vivo Tracers of Transferrin-Based Therapeutics

NASA Astrophysics Data System (ADS)

Zhao, Hanwei; Wang, Shunhai; Nguyen, Son N.; Elci, S. Gokhan; Kaltashov, Igor A.

2016-02-01

Transferrin (Tf) is a promising candidate for targeted drug delivery. While development of such products is impossible without the ability to monitor biodistribution of Tf-drug conjugates in tissues and reliable measurements of their levels in blood and other biological fluids, the presence of very abundant endogenous Tf presents a significant impediment to such efforts. Several noncognate metals have been evaluated in this work as possible tracers of exogenous transferrin in complex biological matrices using inductively coupled plasma mass spectrometry (ICP MS) as a detection tool. Placing Ni(II) on a His-tag of recombinant Tf resulted in formation of a marginally stable protein-metal complex, which readily transfers the metal to ubiquitous physiological scavengers, such as serum albumin. An alternative strategy targeted iron-binding pockets of Tf, where cognate Fe(III) was replaced by metal ions known to bind this protein. Both Ga(III) and In(III) were evaluated, with the latter being vastly superior as a tracer (stronger binding to Tf unaffected by the presence of metal scavengers and the retained ability to associate with Tf receptor). Spiking serum with indium-loaded Tf followed by ICP MS detection demonstrated that protein quantities as low as 0.04 nM can be readily detected in animal blood. Combining laser ablation with ICP MS detection allows distribution of exogenous Tf to be mapped within animal tissue cross-sections with spatial resolution exceeding 100 μm. The method can be readily extended to a range of other therapeutics where metalloproteins are used as either carriers or payloads.
Dimethylformamide interferes with Coomassie dye staining of proteins on blue native gel electrophoresis.

PubMed

Raghupathy, V; Oommen, Anna; Ramachandran, Anup

2014-06-15

Blue native gel electrophoresis (BN-PAGE) is used extensively for characterization of mitochondrial respiratory complexes and uses the binding of Coomassie brilliant blue G-250 to visualize proteins. Oxidative modification of sulfhydryl groups of such proteins can be evaluated by labeling with iodoacetamide conjugated to biotin (BIAM) and detected with streptavidin peroxidase on Western blots following BN-PAGE. However, dissolving BIAM in dimethylformamide, a recommended solvent, reduces Coomassie blue G staining to proteins during BN-PAGE. This interference is prevented by dissolving BIAM in dimethyl sulfoxide. Precautions in the use of the dye for protein staining subsequent to BIAM labeling are discussed. Copyright © 2014 Elsevier Inc. All rights reserved.
Selective staining of proteins with hydrophobic surface sites on a native electrophoretic gel.

PubMed

Bertsch, Martina; Kassner, Richard J

2003-01-01

Chemical proteomics aims to characterize all of the proteins in the proteome with respect to their function, which is associated with their interaction with other molecules. We propose the identification of a subproteomic library of expressed proteins whose native structures are typified by the presence of hydrophobic surface sites, which are often involved in interactions with small molecules, membrane lipids, and other proteins, pertaining to their functions. We demonstrate that soluble globular proteins with hydrophobic surface sites can be detected selectively by staining on an electrophoretic gel run under nondenaturing conditions. The application of these staining techniques may help elucidate new catalytic, transport, and regulatory functionalities in complex proteomic screenings.
Profiling protein function with small molecule microarrays

PubMed Central

Winssinger, Nicolas; Ficarro, Scott; Schultz, Peter G.; Harris, Jennifer L.

2002-01-01

The regulation of protein function through posttranslational modification, local environment, and protein–protein interaction is critical to cellular function. The ability to analyze on a genome-wide scale protein functional activity rather than changes in protein abundance or structure would provide important new insights into complex biological processes. Herein, we report the application of a spatially addressable small molecule microarray to an activity-based profile of proteases in crude cell lysates. The potential of this small molecule-based profiling technology is demonstrated by the detection of caspase activation upon induction of apoptosis, characterization of the activated caspase, and inhibition of the caspase-executed apoptotic phenotype using the small molecule inhibitor identified in the microarray-based profile. PMID:12167675
Protein 3-Nitrotyrosine in Complex Biological Samples: Quantification by High-Pressure Liquid Chromatography/Electrochemical Detection and Emergence of Proteomic Approaches for Unbiased Identification of Modification Sites

PubMed Central

Nuriel, Tal; Deeb, Ruba S.; Hajjar, David P.; Gross, Steven S.

2008-01-01

Nitration of tyrosine residues by nitric oxide (NO)-derived species results in the accumulation of 3-nitrotyrosine in proteins, a hallmark of nitrosative stress in cells and tissues. Tyrosine nitration is recognized as one of the multiple signaling modalities used by NO-derived species for the regulation of protein structure and function in health and disease. Various methods have been described for the quantification of protein 3-nitrotyrosine residues, and several strategies have been presented toward the goal of proteome-wide identification of protein tyrosine modification sites. This chapter details a useful protocol for the quantification of 3-nitrotyrosine in cells and tissues using high-pressure liquid chromatography with electrochemical detection. Additionally, this chapter describes a novel biotin-tagging strategy for specific enrichment of 3-nitrotyrosine-containing peptides. Application of this strategy, in conjunction with high-throughput MS/MS-based peptide sequencing, is anticipated to fuel efforts in developing comprehensive inventories of nitrosative stress-induced protein-tyrosine modification sites in cells and tissues. PMID:18554526
Evidence of insulin-like growth factor binding protein-3 proteolysis during growth hormone stimulation testing.

PubMed

Nwosu, Benjamin U; Soyka, Leslie A; Angelescu, Amanda; Lee, Mary M

2011-01-01

The ternary complex is composed of insulin-like growth factor (IGF)-I, IGF binding protein (IGFBP)-3 and acid labile subunit (ALS). Growth hormone (GH) promotes IGFBP-3 proteolysis to release free IGF-I, ALS, and IGFBP-3 fragments. Our aim was to determine whether elevated GH levels during GH stimulation testing would trigger IGFBP-3 proteolysis. This prospective study of 10 short prepubertal children (height standard deviation score -2.37 +/- 0.31) used arginine and GH releasing hormone stimulation to study dynamic changes in the ternary complex moieties. IGFBP-3 was measured in two assays: a radioimmunoassay (RIA) that detects both cleaved and intact IGFBP-3; and an immunochemiluminescence assay (ICMA) that detects only intact IGFBP-3. IGFBP-3 measured by RIA increased by 19% (p < 0.05), while IGFBP-3 measured by ICMA did not significantly increase (6.1%). The significant increase in IGFBP-3 measured by RIA, but not ICMA, provides evidence of IGFBP-3 proteolysis during acute GH stimulation.

Crosslinking transcription factors to their recognition sequences with PtII complexes

NASA Technical Reports Server (NTRS)

Chu, B. C.; Orgel, L. E.

1992-01-01

We have prepared phosphorothioate-containing cyclic oligodeoxynucleotides that fold into 'dumbbells' containing CRE and TRE sequences, the binding sequences for the CREB and JUN proteins, respectively. Six phosphorothioate residues were introduced into each of the recognition sequences. K2PtCl4 crosslinks CRE to CREB and TRE to JUN. The extent of crosslinking is about eight times greater than that observed with standard oligodeoxynucleotides and amounts to 30-50% of the efficiency of non-covalent association as estimated by gel-shift assays. Crosslinking is reversed by incubation with NaCN. The crosslinking reaction is specific--a dumbbell oligonucleotide with six phosphorothioate groups introduced into the Sp1 recognition sequence could not be crosslinked efficiently to CREB or JUN proteins with K2PtCl4. The binding of TRE to CREB is not strong enough for effective detection by gel-shift assays, but the TRE-CREB complex is crosslinked efficiently by K2PtCl4 and can then readily be detected.
Recognition of dengue virus protein using epitope-mediated molecularly imprinted film.

PubMed

Tai, Dar-Fu; Lin, Chung-Yin; Wu, Tzong-Zeng; Chen, Li-Kuang

2005-08-15

Molecularly imprinted film was fabricated in the presence of a pentadecapeptide onto a quartz crystal microbalance (QCM) chip. This 15-mer peptide has been known as the linear epitope of the dengue virus NS1 protein. Imprinting resulted in an increased polymer affinity toward the corresponding templates but also to the virus protein. Direct detection of the dengue virus protein was achieved quantitatively. The QCM chip response to the NS1 protein was obtained using epitope-mediated imprinting demonstrating a comparable frequency shift in chips immobilized with monoclonal antibodies. The binding effect was further enhanced and confirmed using a monoclonal antibody to form a sandwich with the MIP-NS1 protein complex on the chip. No pretreatment was required.
The Yeast Saccharomyces cerevisiae: a versatile model system for the identification and characterization of bacterial virulence proteins.

PubMed

Siggers, Keri A; Lesser, Cammie F

2008-07-17

Microbial pathogens utilize complex secretion systems to deliver proteins into host cells. These effector proteins target and usurp host cell processes to promote infection and cause disease. While secretion systems are conserved, each pathogen delivers its own unique set of effectors. The identification and characterization of these effector proteins has been difficult, often limited by the lack of detectable signal sequences and functional redundancy. Model systems including yeast, worms, flies, and fish are being used to circumvent these issues. This technical review details the versatility and utility of yeast Saccharomyces cerevisiae as a system to identify and characterize bacterial effectors.
A stochastic context free grammar based framework for analysis of protein sequences

PubMed Central

Dyrka, Witold; Nebel, Jean-Christophe

2009-01-01

Background In the last decade, there have been many applications of formal language theory in bioinformatics such as RNA structure prediction and detection of patterns in DNA. However, in the field of proteomics, the size of the protein alphabet and the complexity of relationship between amino acids have mainly limited the application of formal language theory to the production of grammars whose expressive power is not higher than stochastic regular grammars. However, these grammars, like other state of the art methods, cannot cover any higher-order dependencies such as nested and crossing relationships that are common in proteins. In order to overcome some of these limitations, we propose a Stochastic Context Free Grammar based framework for the analysis of protein sequences where grammars are induced using a genetic algorithm. Results This framework was implemented in a system aiming at the production of binding site descriptors. These descriptors not only allow detection of protein regions that are involved in these sites, but also provide insight in their structure. Grammars were induced using quantitative properties of amino acids to deal with the size of the protein alphabet. Moreover, we imposed some structural constraints on grammars to reduce the extent of the rule search space. Finally, grammars based on different properties were combined to convey as much information as possible. Evaluation was performed on sites of various sizes and complexity described either by PROSITE patterns, domain profiles or a set of patterns. Results show the produced binding site descriptors are human-readable and, hence, highlight biologically meaningful features. Moreover, they achieve good accuracy in both annotation and detection. In addition, findings suggest that, unlike current state-of-the-art methods, our system may be particularly suited to deal with patterns shared by non-homologous proteins. Conclusion A new Stochastic Context Free Grammar based framework has been introduced allowing the production of binding site descriptors for analysis of protein sequences. Experiments have shown that not only is this new approach valid, but produces human-readable descriptors for binding sites which have been beyond the capability of current machine learning techniques. PMID:19814800
Posttranslational processing of the prohormone-cleaving Kex2 protease in the Saccharomyces cerevisiae secretory pathway.

PubMed

Wilcox, C A; Fuller, R S

1991-10-01

The Kex2 protease of the yeast Saccharomyces cerevisiae is a prototypical eukaryotic prohormone-processing enzyme that cleaves precursors of secreted peptides at pairs of basic residues. Here we have established the pathway of posttranslational modification of Kex2 protein using immunoprecipitation of the biosynthetically pulse-labeled protein from a variety of wild-type and mutant yeast strains as the principal methodology. Kex2 protein is initially synthesized as a prepro-enzyme that undergoes cotranslational signal peptide cleavage and addition of Asn-linked core oligosaccharide and Ser/Thr-linked mannose in the ER. The earliest detectable species, I1 (approximately 129 kD), undergoes rapid amino-terminal proteolytic removal of a approximately 9-kD pro-segment yielding species I2 (approximately 120 kD) before arrival at the Golgi complex. Transport to the Golgi complex is marked by extensive elaboration of Ser/Thr-linked chains and minor modification of Asn-linked oligosaccharide. During the latter phase of its lifetime, Kex2 protein undergoes a gradual increase in apparent molecular weight. This final modification serves as a marker for association of Kex2 protease with a late compartment of the yeast Golgi complex in which it is concentrated about 27-fold relative to other secretory proteins.
A holistic approach to dissecting SPARC family protein complexity reveals FSTL-1 as an inhibitor of pancreatic cancer cell growth.

PubMed

Viloria, Katrina; Munasinghe, Amanda; Asher, Sharan; Bogyere, Roberto; Jones, Lucy; Hill, Natasha J

2016-11-25

SPARC is a matricellular protein that is involved in both pancreatic cancer and diabetes. It belongs to a wider family of proteins that share structural and functional similarities. Relatively little is known about this extended family, but evidence of regulatory interactions suggests the importance of a holistic approach to their study. We show that Hevin, SPOCKs, and SMOCs are strongly expressed within islets, ducts, and blood vessels, suggesting important roles for these proteins in the normal pancreas, while FSTL-1 expression is localised to the stromal compartment reminiscent of SPARC. In direct contrast to SPARC, however, FSTL-1 expression is reduced in pancreatic cancer. Consistent with this, FSTL-1 inhibited pancreatic cancer cell proliferation. The complexity of SPARC family proteins is further revealed by the detection of multiple cell-type specific isoforms that arise due to a combination of post-translational modification and alternative splicing. Identification of splice variants lacking a signal peptide suggests the existence of novel intracellular isoforms. This study underlines the importance of addressing the complexity of the SPARC family and provides a new framework to explain their controversial and contradictory effects. We also demonstrate for the first time that FSTL-1 suppresses pancreatic cancer cell growth.
Pulse Detecting Genetic Circuit – A New Design Approach

PubMed Central

Inniss, Mara; Iba, Hitoshi; Way, Jeffrey C.

2016-01-01

A robust cellular counter could enable synthetic biologists to design complex circuits with diverse behaviors. The existing synthetic-biological counters, responsive to the beginning of the pulse, are sensitive to the pulse duration. Here we present a pulse detecting circuit that responds only at the falling edge of a pulse–analogous to negative edge triggered electric circuits. As biological events do not follow precise timing, use of such a pulse detector would enable the design of robust asynchronous counters which can count the completion of events. This transcription-based pulse detecting circuit depends on the interaction of two co-expressed lambdoid phage-derived proteins: the first is unstable and inhibits the regulatory activity of the second, stable protein. At the end of the pulse the unstable inhibitor protein disappears from the cell and the second protein triggers the recording of the event completion. Using stochastic simulation we showed that the proposed design can detect the completion of the pulse irrespective to the pulse duration. In our simulation we also showed that fusing the pulse detector with a phage lambda memory element we can construct a counter which can be extended to count larger numbers. The proposed design principle is a new control mechanism for synthetic biology which can be integrated in different circuits for identifying the completion of an event. PMID:27907045
Pulse Detecting Genetic Circuit - A New Design Approach.

PubMed

Noman, Nasimul; Inniss, Mara; Iba, Hitoshi; Way, Jeffrey C

2016-01-01

A robust cellular counter could enable synthetic biologists to design complex circuits with diverse behaviors. The existing synthetic-biological counters, responsive to the beginning of the pulse, are sensitive to the pulse duration. Here we present a pulse detecting circuit that responds only at the falling edge of a pulse-analogous to negative edge triggered electric circuits. As biological events do not follow precise timing, use of such a pulse detector would enable the design of robust asynchronous counters which can count the completion of events. This transcription-based pulse detecting circuit depends on the interaction of two co-expressed lambdoid phage-derived proteins: the first is unstable and inhibits the regulatory activity of the second, stable protein. At the end of the pulse the unstable inhibitor protein disappears from the cell and the second protein triggers the recording of the event completion. Using stochastic simulation we showed that the proposed design can detect the completion of the pulse irrespective to the pulse duration. In our simulation we also showed that fusing the pulse detector with a phage lambda memory element we can construct a counter which can be extended to count larger numbers. The proposed design principle is a new control mechanism for synthetic biology which can be integrated in different circuits for identifying the completion of an event.
Identification of proteins from tuberculin purified protein derivative (PPD) by LC-MS/MS.

PubMed

Borsuk, Sibele; Newcombe, Jane; Mendum, Tom A; Dellagostin, Odir A; McFadden, Johnjoe

2009-11-01

The tuberculin purified protein derivative (PPD) is a widely used diagnostic antigen for tuberculosis, however it is poorly defined. Most mycobacterial proteins are extensively denatured by the procedure employed in its preparation, which explains previous difficulties in identifying constituents from PPD to characterize their behaviour in B- and T-cell reactions. We here described a proteomics-based characterization of PPD from several different sources by LC-MS/MS, which combines the solute separation power of HPLC, with the detection power of a mass spectrometer. The technique is able to identify proteins from complex mixtures of peptide fragments. A total of 171 different proteins were identified among the four PPD samples (two bovine PPD and two avium PPD) from Brazil and UK. The majority of the proteins were cytoplasmic (77.9%) and involved in intermediary metabolism and respiration (24.25%) but there was a preponderance of proteins involved in lipid metabolism. We identified a group of 21 proteins that are present in both bovine PPD but were not detected in avium PPD preparation. In addition, four proteins found in bovine PPD are absent in Mycobacterium bovis BCG vaccine strain. This study provides a better understanding of the tuberculin PPD components leading to the identification of additional antigens useful as reagents for specific diagnosis of tuberculosis.
Demonstration of protein-fragment complementation assay using purified firefly luciferase fragments

PubMed Central

2013-01-01

Background Human interactome is predicted to contain 150,000 to 300,000 protein-protein interactions, (PPIs). Protein-fragment complementation assay (PCA) is one of the most widely used methods to detect PPI, as well as Förster resonance energy transfer (FRET). To date, successful applications of firefly luciferase (Fluc)-based PCA have been reported in vivo, in cultured cells and in cell-free lysate, owing to its high sensitivity, high signal-to-background (S/B) ratio, and reversible response. Here we show the assay also works with purified proteins with unexpectedly rapid kinetics. Results Split Fluc fragments both fused with a rapamycin-dependently interacting protein pair were made and expressed in E. coli system, and purified to homogeneity. When the proteins were used for PCA to detect rapamycin-dependent PPI, they enabled a rapid detection (~1 s) of PPI with high S/B ratio. When Fn7-8 domains (7 nm in length) that was shown to abrogate GFP mutant-based FRET was inserted between split Fluc and FKBP12 as a rigid linker, it still showed some response, suggesting less limitation in interacting partner’s size. Finally, the stability of the probe was investigated. Preincubation of the probes at 37 degreeC up to 1 h showed marked decrease of the luminescent signal to 1.5%, showing the limited stability of this system. Conclusion Fluc PCA using purified components will enable a rapid and handy detection of PPIs with high S/B ratio, avoiding the effects of concomitant components. Although the system might not be suitable for large-scale screening due to its limited stability, it can detect an interaction over larger distance than by FRET. This would be the first demonstration of Fluc PCA in vitro, which has a distinct advantage over other PPI assays. Our system enables detection of direct PPIs without risk of perturbation by PPI mediators in the complex cellular milieu. PMID:23536995
Analysis of splicing complexes on native gels.

PubMed

Ares, Manuel

2013-10-01

Splicing requires a complex set of ATP-dependent macromolecular associations that lead to the rearrangement of just a few covalent bonds in the pre-mRNA substrate. Seeing only the covalent bonds breaking and forming is seeing only a very small part of the process. Analysis of native splicing complexes into which the radiolabeled substrate has been assembled, but not necessarily completely reacted, has provided a good understanding of the process. This protocol describes a gel method for detecting and analyzing yeast splicing complexes formed in vitro on a radiolabeled pre-mRNA substrate. Complexes formed during the splicing reaction are quenched by dilution and addition of an excess of RNA, which is thought to strip nonspecifically bound proteins from the labeled substrate RNA. After loading on a low-percentage, low-cross-linking ratio composite agarose-acrylamide gel (in 10% glycerol), labeled bands are detected. These can be extracted and shown to contain small nuclear RNAs (snRNAs) and partly reacted pre-mRNA.
Domain similarity based orthology detection.

PubMed

Bitard-Feildel, Tristan; Kemena, Carsten; Greenwood, Jenny M; Bornberg-Bauer, Erich

2015-05-13

Orthologous protein detection software mostly uses pairwise comparisons of amino-acid sequences to assert whether two proteins are orthologous or not. Accordingly, when the number of sequences for comparison increases, the number of comparisons to compute grows in a quadratic order. A current challenge of bioinformatic research, especially when taking into account the increasing number of sequenced organisms available, is to make this ever-growing number of comparisons computationally feasible in a reasonable amount of time. We propose to speed up the detection of orthologous proteins by using strings of domains to characterize the proteins. We present two new protein similarity measures, a cosine and a maximal weight matching score based on domain content similarity, and new software, named porthoDom. The qualities of the cosine and the maximal weight matching similarity measures are compared against curated datasets. The measures show that domain content similarities are able to correctly group proteins into their families. Accordingly, the cosine similarity measure is used inside porthoDom, the wrapper developed for proteinortho. porthoDom makes use of domain content similarity measures to group proteins together before searching for orthologs. By using domains instead of amino acid sequences, the reduction of the search space decreases the computational complexity of an all-against-all sequence comparison. We demonstrate that representing and comparing proteins as strings of discrete domains, i.e. as a concatenation of their unique identifiers, allows a drastic simplification of search space. porthoDom has the advantage of speeding up orthology detection while maintaining a degree of accuracy similar to proteinortho. The implementation of porthoDom is released using python and C++ languages and is available under the GNU GPL licence 3 at http://www.bornberglab.org/pages/porthoda .
Boundaries of mass resolution in native mass spectrometry.

PubMed

Lössl, Philip; Snijder, Joost; Heck, Albert J R

2014-06-01

Over the last two decades, native mass spectrometry (MS) has emerged as a valuable tool to study intact proteins and noncovalent protein complexes. Studied experimental systems range from small-molecule (drug)-protein interactions, to nanomachineries such as the proteasome and ribosome, to even virus assembly. In native MS, ions attain high m/z values, requiring special mass analyzers for their detection. Depending on the particular mass analyzer used, instrumental mass resolution does often decrease at higher m/z but can still be above a couple of thousand at m/z 5000. However, the mass resolving power obtained on charge states of protein complexes in this m/z region is experimentally found to remain well below the inherent instrument resolution of the mass analyzers employed. Here, we inquire into reasons for this discrepancy and ask how native MS would benefit from higher instrumental mass resolution. To answer this question, we discuss advantages and shortcomings of mass analyzers used to study intact biomolecules and biomolecular complexes in their native state, and we review which other factors determine mass resolving power in native MS analyses. Recent examples from the literature are given to illustrate the current status and limitations.
Mitochondrial Carnitine Palmitoyltransferase 1a (CPT1a) Is Part of an Outer Membrane Fatty Acid Transfer Complex*

PubMed Central

Lee, Kwangwon; Kerner, Janos; Hoppel, Charles L.

2011-01-01

CPT1a (carnitine palmitoyltransferase 1a) in the liver mitochondrial outer membrane (MOM) catalyzes the primary regulated step in overall mitochondrial fatty acid oxidation. It has been suggested that the fundamental unit of CPT1a exists as a trimer, which, under native conditions, could form a dimer of the trimers, creating a hexamer channel for acylcarnitine translocation. To examine the state of CPT1a in the MOM, we employed a combined approach of sizing by mass and isolation using an immunological method. Blue native electrophoresis followed by detection with immunoblotting and mass spectrometry identified large molecular mass complexes that contained not only CPT1a but also long chain acyl-CoA synthetase (ACSL) and the voltage-dependent anion channel (VDAC). Immunoprecipitation with antisera against the proteins revealed a strong interaction between the three proteins. Immobilized CPT1a-specific antibodies immunocaptured not only CPT1a but also ACSL and VDAC, further strengthening findings with blue native electrophoresis and immunoprecipitation. This study shows strong protein-protein interaction between CPT1a, ACSL, and VDAC. We propose that this complex transfers activated fatty acids through the MOM. PMID:21622568
Enhancement of sensitivity using hybrid stimulus for the diagnosis of prostate cancer based on polydiacetylene (PDA) supramolecules.

PubMed

Kwon, Il Kyoung; Kim, Jun Pyo; Sim, Sang Jun

2010-12-15

In this study, hybrid stimulus was initially introduced to improve the sensitivity of PDA vesicle chip for detection of prostate-specific antigen-α1-antichymotrypsin (PSA-ACT) complex. The strategy of hybrid stimulus on PDA vesicle chip offers the amplification method of fluorescent signal which combines a primary response by the immune reaction of antigen-antibody and a secondary response by the mechanical pressure of pAb-conjugated magnetic beads. As the primary response result on PDA vesicle chip, the PSA-ACT complex in PBS buffer was detected at 10 ng/mL. However, this detection sensitivity was insufficient for diagnosis of prostate cancer because the normal human PSA concentration is less than 4.0 ng/mL. To solve this problem, polyclonal PSA antibody-conjugated magnetic beads were used as an amplifying agent after primary immunoresponse. As a result, the PSA-ACT complex concentrations (as low as 0.1 ng/mL) could be detected in the PBS buffer sample. Therefore, this result can be applied to various fields, such as the detection of cells, proteins, and DNA for sensitive and specific biosensing based on PDA supramolecules. Copyright © 2010 Elsevier B.V. All rights reserved.
Radioimmunoassay of the marjor group specific protein of endogenous baboon type C viruses: relation to the RD-114/CCC group and detection of antigen in normal baboon tissues

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sherr, C.J.; Todaro, G.J.

1974-09-01

The major group specific (gs) protein of the endogenous baboon type C virus M7 was purified to homogeneity by gel filtration and isoelectric focusing. The protein has a molecular weight of approximately 33,000, as determined by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate, and an isoelectric point (pl) of 7.1, different from the pls of similarly purified gs proteins from six other mammalian type C viruses. Detergent disrupted M7 virus, whether grown in canine thymus or human rhabdomyosarcoma cells, fully displaced radiolabeled M7 gs protein from antigen-antibody complexes in a competitive radioimmunoassay. No antigenic differences were detected among themore » gs proteins of five independent isolates of baboon type C viruses grown in various cultured cell lines. The gs proteins of six independently isolated feline viruses of the RD-114/CCC group were antigenically related to, but could be distinguished from, the gs proteins of baboon type C viruses. No significant cross-reactions were observed in the radioimmunoassay for M7 gs protein using several other type C viruses, including two previously isolated from a woolly monkey and a gibbon ape. Group specific antigen was found in normal baboon testicular and splenic tissues using the M7 radioimmunoassay; no gs antigen could be detected in these same tissues using a radioimmunoassay for the gs protein of the woolly monkey type C virus. No gs antigen was found in baboon liver or in the tissues of several other primates. (auth)« less
Bimolecular fluorescence complementation: lighting up seven transmembrane domain receptor signalling networks

PubMed Central

Rose, Rachel H; Briddon, Stephen J; Holliday, Nicholas D

2010-01-01

There is increasing complexity in the organization of seven transmembrane domain (7TM) receptor signalling pathways, and in the ability of their ligands to modulate and direct this signalling. Underlying these events is a network of protein interactions between the 7TM receptors themselves and associated effectors, such as G proteins and β-arrestins. Bimolecular fluorescence complementation, or BiFC, is a technique capable of detecting these protein–protein events essential for 7TM receptor function. Fluorescent proteins, such as those from Aequorea victoria, are split into two non-fluorescent halves, which then tag the proteins under study. On association, these fragments refold and regenerate a mature fluorescent protein, producing a BiFC signal indicative of complex formation. Here, we review the experimental criteria for successful application of BiFC, considered in the context of 7TM receptor signalling events such as receptor dimerization, G protein and β-arrestin signalling. The advantages and limitations of BiFC imaging are compared with alternative resonance energy transfer techniques. We show that the essential simplicity of the fluorescent BiFC measurement allows high-content and advanced imaging applications, and that it can probe more complex multi-protein interactions alone or in combination with resonance energy transfer. These capabilities suggest that BiFC techniques will become ever more useful in the analysis of ligand and 7TM receptor pharmacology at the molecular level of protein–protein interactions. This article is part of a themed section on Imaging in Pharmacology. To view the editorial for this themed section visit http://dx.doi.org/10.1111/j.1476-5381.2010.00685.x PMID:20015298
Crystallization of the Large Membrane Protein Complex Photosystem I in a Microfluidic Channel

PubMed Central

Abdallah, Bahige G.; Kupitz, Christopher; Fromme, Petra; Ros, Alexandra

2014-01-01

Traditional macroscale protein crystallization is accomplished non-trivially by exploring a range of protein concentrations and buffers in solution until a suitable combination is attained. This methodology is time consuming and resource intensive, hindering protein structure determination. Even more difficulties arise when crystallizing large membrane protein complexes such as photosystem I (PSI) due to their large unit cells dominated by solvent and complex characteristics that call for even stricter buffer requirements. Structure determination techniques tailored for these ‘difficult to crystallize’ proteins such as femtosecond nanocrystallography are being developed, yet still need specific crystal characteristics. Here, we demonstrate a simple and robust method to screen protein crystallization conditions at low ionic strength in a microfluidic device. This is realized in one microfluidic experiment using low sample amounts, unlike traditional methods where each solution condition is set up separately. Second harmonic generation microscopy via Second Order Nonlinear Imaging of Chiral Crystals (SONICC) was applied for the detection of nanometer and micrometer sized PSI crystals within microchannels. To develop a crystallization phase diagram, crystals imaged with SONICC at specific channel locations were correlated to protein and salt concentrations determined by numerical simulations of the time-dependent diffusion process along the channel. Our method demonstrated that a portion of the PSI crystallization phase diagram could be reconstructed in excellent agreement with crystallization conditions determined by traditional methods. We postulate that this approach could be utilized to efficiently study and optimize crystallization conditions for a wide range of proteins that are poorly understood to date. PMID:24191698
Quantitative analysis of residual protein contamination of podiatry instruments reprocessed through local and central decontamination units

PubMed Central

2011-01-01

Background The cleaning stage of the instrument decontamination process has come under increased scrutiny due to the increasing complexity of surgical instruments and the adverse affects of residual protein contamination on surgical instruments. Instruments used in the podiatry field have a complex surface topography and are exposed to a wide range of biological contamination. Currently, podiatry instruments are reprocessed locally within surgeries while national strategies are favouring a move toward reprocessing in central facilities. The aim of this study was to determine the efficacy of local and central reprocessing on podiatry instruments by measuring residual protein contamination of instruments reprocessed by both methods. Methods The residual protein of 189 instruments reprocessed centrally and 189 instruments reprocessed locally was determined using a fluorescent assay based on the reaction of proteins with o-phthaldialdehyde/sodium 2-mercaptoethanesulfonate. Results Residual protein was detected on 72% (n = 136) of instruments reprocessed centrally and 90% (n = 170) of instruments reprocessed locally. Significantly less protein (p < 0.001) was recovered from instruments reprocessed centrally (median 20.62 μg, range 0 - 5705 μg) than local reprocessing (median 111.9 μg, range 0 - 6344 μg). Conclusions Overall, the results show the superiority of central reprocessing for complex podiatry instruments when protein contamination is considered, though no significant difference was found in residual protein between local decontamination unit and central decontamination unit processes for Blacks files. Further research is needed to undertake qualitative identification of protein contamination to identify any cross contamination risks and a standard for acceptable residual protein contamination applicable to different instruments and specialities should be considered as a matter of urgency. PMID:21219613
Quantitative analysis of residual protein contamination of podiatry instruments reprocessed through local and central decontamination units.

PubMed

Smith, Gordon Wg; Goldie, Frank; Long, Steven; Lappin, David F; Ramage, Gordon; Smith, Andrew J

2011-01-10

The cleaning stage of the instrument decontamination process has come under increased scrutiny due to the increasing complexity of surgical instruments and the adverse affects of residual protein contamination on surgical instruments. Instruments used in the podiatry field have a complex surface topography and are exposed to a wide range of biological contamination. Currently, podiatry instruments are reprocessed locally within surgeries while national strategies are favouring a move toward reprocessing in central facilities. The aim of this study was to determine the efficacy of local and central reprocessing on podiatry instruments by measuring residual protein contamination of instruments reprocessed by both methods. The residual protein of 189 instruments reprocessed centrally and 189 instruments reprocessed locally was determined using a fluorescent assay based on the reaction of proteins with o-phthaldialdehyde/sodium 2-mercaptoethanesulfonate. Residual protein was detected on 72% (n = 136) of instruments reprocessed centrally and 90% (n = 170) of instruments reprocessed locally. Significantly less protein (p < 0.001) was recovered from instruments reprocessed centrally (median 20.62 μg, range 0 - 5705 μg) than local reprocessing (median 111.9 μg, range 0 - 6344 μg). Overall, the results show the superiority of central reprocessing for complex podiatry instruments when protein contamination is considered, though no significant difference was found in residual protein between local decontamination unit and central decontamination unit processes for Blacks files. Further research is needed to undertake qualitative identification of protein contamination to identify any cross contamination risks and a standard for acceptable residual protein contamination applicable to different instruments and specialities should be considered as a matter of urgency.

Effective Identification of Akt Interacting Proteins by Two-Step Chemical Crosslinking, Co-Immunoprecipitation and Mass Spectrometry

PubMed Central

Huang, Bill X.; Kim, Hee-Yong

2013-01-01

Akt is a critical protein for cell survival and known to interact with various proteins. However, Akt binding partners that modulate or regulate Akt activation have not been fully elucidated. Identification of Akt-interacting proteins has been customarily achieved by co-immunoprecipitation combined with western blot and/or MS analysis. An intrinsic problem of the method is loss of interacting proteins during procedures to remove non-specific proteins. Moreover, antibody contamination often interferes with the detection of less abundant proteins. Here, we developed a novel two-step chemical crosslinking strategy to overcome these problems which resulted in a dramatic improvement in identifying Akt interacting partners. Akt antibody was first immobilized on protein A/G beads using disuccinimidyl suberate and allowed to bind to cellular Akt along with its interacting proteins. Subsequently, dithiobis[succinimidylpropionate], a cleavable crosslinker, was introduced to produce stable complexes between Akt and binding partners prior to the SDS-PAGE and nanoLC-MS/MS analysis. This approach enabled identification of ten Akt partners from cell lysates containing as low as 1.5 mg proteins, including two new potential Akt interacting partners. None of these but one protein was detectable without crosslinking procedures. The present method provides a sensitive and effective tool to probe Akt-interacting proteins. This strategy should also prove useful for other protein interactions, particularly those involving less abundant or weakly associating partners. PMID:23613850
A new multi-scale method to reveal hierarchical modular structures in biological networks.

PubMed

Jiao, Qing-Ju; Huang, Yan; Shen, Hong-Bin

2016-11-15

Biological networks are effective tools for studying molecular interactions. Modular structure, in which genes or proteins may tend to be associated with functional modules or protein complexes, is a remarkable feature of biological networks. Mining modular structure from biological networks enables us to focus on a set of potentially important nodes, which provides a reliable guide to future biological experiments. The first fundamental challenge in mining modular structure from biological networks is that the quality of the observed network data is usually low owing to noise and incompleteness in the obtained networks. The second problem that poses a challenge to existing approaches to the mining of modular structure is that the organization of both functional modules and protein complexes in networks is far more complicated than was ever thought. For instance, the sizes of different modules vary considerably from each other and they often form multi-scale hierarchical structures. To solve these problems, we propose a new multi-scale protocol for mining modular structure (named ISIMB) driven by a node similarity metric, which works in an iteratively converged space to reduce the effects of the low data quality of the observed network data. The multi-scale node similarity metric couples both the local and the global topology of the network with a resolution regulator. By varying this resolution regulator to give different weightings to the local and global terms in the metric, the ISIMB method is able to fit the shape of modules and to detect them on different scales. Experiments on protein-protein interaction and genetic interaction networks show that our method can not only mine functional modules and protein complexes successfully, but can also predict functional modules from specific to general and reveal the hierarchical organization of protein complexes.
The relative vertex clustering value - a new criterion for the fast discovery of functional modules in protein interaction networks

PubMed Central

2015-01-01

Background Cellular processes are known to be modular and are realized by groups of proteins implicated in common biological functions. Such groups of proteins are called functional modules, and many community detection methods have been devised for their discovery from protein interaction networks (PINs) data. In current agglomerative clustering approaches, vertices with just a very few neighbors are often classified as separate clusters, which does not make sense biologically. Also, a major limitation of agglomerative techniques is that their computational efficiency do not scale well to large PINs. Finally, PIN data obtained from large scale experiments generally contain many false positives, and this makes it hard for agglomerative clustering methods to find the correct clusters, since they are known to be sensitive to noisy data. Results We propose a local similarity premetric, the relative vertex clustering value, as a new criterion allowing to decide when a node can be added to a given node's cluster and which addresses the above three issues. Based on this criterion, we introduce a novel and very fast agglomerative clustering technique, FAC-PIN, for discovering functional modules and protein complexes from a PIN data. Conclusions Our proposed FAC-PIN algorithm is applied to nine PIN data from eight different species including the yeast PIN, and the identified functional modules are validated using Gene Ontology (GO) annotations from DAVID Bioinformatics Resources. Identified protein complexes are also validated using experimentally verified complexes. Computational results show that FAC-PIN can discover functional modules or protein complexes from PINs more accurately and more efficiently than HC-PIN and CNM, the current state-of-the-art approaches for clustering PINs in an agglomerative manner. PMID:25734691
Evolutionary gradient of predicted nuclear localization signals (NLS)-bearing proteins in genomes of family Planctomycetaceae.

PubMed

Guo, Min; Yang, Ruifu; Huang, Chen; Liao, Qiwen; Fan, Guangyi; Sun, Chenghang; Lee, Simon Ming-Yuen

2017-04-04

The nuclear envelope is considered a key classification marker that distinguishes prokaryotes from eukaryotes. However, this marker does not apply to the family Planctomycetaceae, which has intracellular spaces divided by lipidic intracytoplasmic membranes (ICMs). Nuclear localization signal (NLS), a short stretch of amino acid sequence, destines to transport proteins from cytoplasm into nucleus, and is also associated with the development of nuclear envelope. We attempted to investigate the NLS motifs in Planctomycetaceae genomes to demonstrate the potential molecular transition in the development of intracellular membrane system. In this study, we identified NLS-like motifs that have the same amino acid compositions as experimentally identified NLSs in genomes of 11 representative species of family Planctomycetaceae. A total of 15 NLS types and 170 NLS-bearing proteins were detected in the 11 strains. To determine the molecular transformation, we compared NLS-bearing protein abundances in the 11 representative Planctomycetaceae genomes with them in genomes of 16 taxonomically varied microorganisms: nine bacteria, two archaea and five fungi. In the 27 strains, 29 NLS types and 1101 NLS-bearing proteins were identified, principal component analysis showed a significant transitional gradient from bacteria to Planctomycetaceae to fungi on their NLS-bearing protein abundance profiles. Then, we clustered the 993 non-redundant NLS-bearing proteins into 181 families and annotated their involved metabolic pathways. Afterwards, we aligned the ten types of NLS motifs from the 13 families containing NLS-bearing proteins among bacteria, Planctomycetaceae or fungi, considering their diversity, length and origin. A transition towards increased complexity from non-planctomycete bacteria to Planctomycetaceae to archaea and fungi was detected based on the complexity of the 10 types of NLS-like motifs in the 13 NLS-bearing proteins families. The results of this study reveal that Planctomycetaceae separates slightly from the members of non-planctomycete bacteria but still has substantial differences from fungi, based on the NLS-like motifs and NLS-bearing protein analysis.
Nanoporous Anodic Alumina Surface Modification by Electrostatic, Covalent, and Immune Complexation Binding Investigated by Capillary Filling.

PubMed

Eckstein, Chris; Acosta, Laura K; Pol, Laura; Xifré-Pérez, Elisabet; Pallares, Josep; Ferré-Borrull, Josep; Marsal, Lluis F

2018-03-28

The fluid imbibition-coupled laser interferometry (FICLI) technique has been applied to detect and quantify surface changes and pore dimension variations in nanoporous anodic alumina (NAA) structures. FICLI is a noninvasive optical technique that permits the determination of the NAA average pore radius with high accuracy. In this work, the technique is applied after each step of different surface modification paths of the NAA pores: (i) electrostatic immobilization of bovine serum albumin (BSA), (ii) covalent attachment of streptavidin via (3-aminipropyl)-triethoxysilane and glutaraldehyde grafting, and (iii) immune complexation. Results show that BSA attachment can be detected as a reduction in estimated radius from FICLI with high accuracy and reproducibility. In the case of the covalent attachment of streptavidin, FICLI is able to recognize a multilayer formation of the silane and the protein. For immune complexation, the technique is able to detect different antibody-antigen bindings and distinguish different dynamics among different immune species.
Species and tissues specific differentiation of processed animal proteins in aquafeeds using proteomics tools.

PubMed

Rasinger, J D; Marbaix, H; Dieu, M; Fumière, O; Mauro, S; Palmblad, M; Raes, M; Berntssen, M H G

2016-09-16

The rapidly growing aquaculture industry drives the search for sustainable protein sources in fish feed. In the European Union (EU) since 2013 non-ruminant processed animal proteins (PAP) are again permitted to be used in aquafeeds. To ensure that commercial fish feeds do not contain PAP from prohibited species, EU reference methods were established. However, due to the heterogeneous and complex nature of PAP complementary methods are required to guarantee the safe use of this fish feed ingredient. In addition, there is a need for tissue specific PAP detection to identify the sources (i.e. bovine carcass, blood, or meat) of illegal PAP use. In the present study, we investigated and compared different protein extraction, solubilisation and digestion protocols on different proteomics platforms for the detection and differentiation of prohibited PAP. In addition, we assessed if tissue specific PAP detection was feasible using proteomics tools. All work was performed independently in two different laboratories. We found that irrespective of sample preparation gel-based proteomics tools were inappropriate when working with PAP. Gel-free shotgun proteomics approaches in combination with direct spectral comparison were able to provide quality species and tissue specific data to complement and refine current methods of PAP detection and identification. To guarantee the safe use of processed animal protein (PAP) in aquafeeds efficient PAP detection and monitoring tools are required. The present study investigated and compared various proteomics workflows and shows that the application of shotgun proteomics in combination with direct comparison of spectral libraries provides for the desired species and tissue specific classification of this heat sterilized and pressure treated (≥133°C, at 3bar for 20min) protein feed ingredient. Copyright © 2016 Elsevier B.V. All rights reserved.
Single gene deletions of mrpA to mrpG and mrpE point mutations affect activity of the Mrp Na+/H+ antiporter of alkaliphilic Bacillus and formation of hetero-oligomeric Mrp complexes.

PubMed

Morino, Masato; Natsui, Shinsuke; Swartz, Talia H; Krulwich, Terry A; Ito, Masahiro

2008-06-01

Mrp antiporters catalyze secondary Na(+)(Li(+))/H(+) antiport and/or K(+)/H(+) antiport that is physiologically important in diverse bacteria. An additional capacity for anion flux has been observed for a few systems. Mrp is unique among antiporters in that it requires all six or seven hydrophobic gene products (MrpA to MrpG) of the mrp operon for full antiporter activity, but MrpE has been reported to be dispensable. Here, the membrane complexes formed by Mrp proteins were examined using a cloned mrp operon from alkaliphilic Bacillus pseudofirmus OF4. The operon was engineered so that the seven Mrp proteins could be detected in single samples. Membrane extracts of an antiporter-deficient Escherichia coli strain expressing this construct were analyzed by blue native-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Mrp complexes of two sizes were identified containing all seven Mrp proteins. Studies of the single nonpolar mrp gene deletions in the construct showed that a subcomplex of MrpA, MrpB, MrpC, and MrpD was formed in the absence of MrpE, MrpF, or MrpG. By contrast, MrpE, MrpF, and MrpG were not observed in membranes lacking MrpA, MrpB, MrpC, or MrpD. Although MrpA and MrpD have been hypothesized to be the antiporter proteins, the MrpA-to-D complex was inactive. Every Mrp protein was required for an activity level near that of the wild-type Na(+)/H(+) antiporter, but a very low activity level was observed in the absence of MrpE. The introduction of an MrpE(P114G) mutation into the full Mrp complex led to antiport activity with a greatly increased apparent K(m) value for Na(+). The results suggested that interactions among the proteins of heterooligomeric Mrp complexes strongly impact antiporter properties.
Identification of a basic protein of Mr 75,000 as an accessory desmosomal plaque protein in stratified and complex epithelia.

PubMed

Kapprell, H P; Owaribe, K; Franke, W W

1988-05-01

Desmosomes are intercellular adhering junctions characterized by a special structure and certain obligatory constituent proteins such as the cytoplasmic protein, desmoglein. Desmosomal fractions from bovine muzzle epidermis contain, in addition, a major polypeptide of Mr approximately 75,000 ("band 6 protein") which differs from all other desmosomal proteins so far identified by its positive charge (isoelectric at pH approximately 8.5 in the denatured state) and its avidity to bind certain type I cytokeratins under stringent conditions. We purified this protein from bovine muzzle epidermis and raised antibodies to it. Using affinity-purified antibodies, we identified a protein of identical SDS-PAGE mobility and isoelectric pH in all epithelia of higher complexity, including representatives of stratified, complex (pseudostratified) and transitional epithelia as well as benign and malignant human tumors derived from such epithelia. Immunolocalization studies revealed the location of this protein along cell boundaries in stratified and complex epithelia, often resolved into punctate arrays. In some epithelia it seemed to be restricted to certain cell types and layers; in rat cornea, for example, it was only detected in upper strata. Electron microscopic immunolocalization showed that this protein is a component of the desmosomal plaque. However, it was not found in the desmosomes of all simple epithelia examined, in the tumors and cultured cells derived thereof, in myocardiac and Purkinje fiber cells, in arachnoideal cells and meningiomas, and in dendritic reticulum cells of lymphoid tissue, i.e., all cells containing typical desmosomes. The protein was also absent in all nondesmosomal adhering junctions. From these results we conclude that this basic protein is not an obligatory desmosomal plaque constituent but an accessory component specific to the desmosomes of certain kinds of epithelial cells with stratified tissue architecture. This suggests that the Mr 75,000 basic protein does not serve general desmosomal functions but rather cell type-specific ones and that the composition of the desmosomal plaque can be different in different cell types. The possible diagnostic value of this protein as a marker in cell typing is discussed.
Endoplasmic reticulum chaperone glucose regulated protein 170-Pokemon complexes elicit a robust antitumor immune response in vivo.

PubMed

Yuan, Bangqing; Xian, Ronghua; Wu, Xianqu; Jing, Junjie; Chen, Kangning; Liu, Guojun; Zhou, Zhenhua

2012-07-01

Previous evidence suggested that the stress protein grp170 can function as a highly efficient molecular chaperone, binding to large protein substrates and acting as a potent vaccine against specific tumors when purified from the same tumor. In addition, Pokemon can be found in almost all malignant tumor cells and is regarded to be a promising candidate for the treatment of tumors. However, the potential of the grp170-Pokemon chaperone complex has not been well described. In the present study, the natural chaperone complex between grp170 and the Pokemon was formed by heat shock, and its immunogenicity was detected by ELISPOT and (51)Cr-release assays in vitro and by tumor bearing models in vivo. Our results demonstrated that the grp170-Pokemon chaperone complex could elicit T cell responses as determined by ELISPOT and (51)Cr-release assays. In addition, immunized C57BL/6 mice were challenged with subcutaneous (s.c.) injection of Lewis cancer cells to induce primary tumors. Treatment of mice with the grp170-Pokemon chaperone complex also significantly inhibited tumor growth and prolonged the life span of tumor-bearing mice. Our results indicated that the grp170-Pokemon chaperone complex might represent a powerful approach to tumor immunotherapy and have significant potential for clinical application. Copyright © 2012 Elsevier GmbH. All rights reserved.
Analysis of Multiplexed Nanosensor Arrays Based on Near-Infrared Fluorescent Single-Walled Carbon Nanotubes.

PubMed

Dong, Juyao; Salem, Daniel P; Sun, Jessica H; Strano, Michael S

2018-04-24

The high-throughput, label-free detection of biomolecules remains an important challenge in analytical chemistry with the potential of nanosensors to significantly increase the ability to multiplex such assays. In this work, we develop an optical sensor array, printable from a single-walled carbon nanotube/chitosan ink and functionalized to enable a divalent ion-based proximity quenching mechanism for transducing binding between a capture protein or an antibody with the target analyte. Arrays of 5 × 6, 200 μm near-infrared (nIR) spots at a density of ≈300 spots/cm 2 are conjugated with immunoglobulin-binding proteins (proteins A, G, and L) for the detection of human IgG, mouse IgM, rat IgG2a, and human IgD. Binding kinetics are measured in a parallel, multiplexed fashion from each sensor spot using a custom laser scanning imaging configuration with an nIR photomultiplier tube detector. These arrays are used to examine cross-reactivity, competitive and nonspecific binding of analyte mixtures. We find that protein G and protein L functionalized sensors report selective responses to mouse IgM on the latter, as anticipated. Optically addressable platforms such as the one examined in this work have potential to significantly advance the real-time, multiplexed biomolecular detection of complex mixtures.
Carbohydrate-based electrochemical biosensor for detection of a cancer biomarker in human plasma.

PubMed

Devillers, Marion; Ahmad, Lama; Korri-Youssoufi, Hafsa; Salmon, Laurent

2017-10-15

Autocrine motility factor (AMF) is a tumor-secreted cytokine that stimulates tumor cell motility in vitro and metastasis in vivo. AMF could be detected in serum or urine of cancer patients with worse prognosis. Reported as a cancer biomarker, AMF secretion into body fluids might be closely related to metastases formation. In this study, a sensitive and specific carbohydrate-based electrochemical biosensor was designed for the detection and quantification of a protein model of AMF, namely phosphoglucose isomerase from rabbit muscle (RmPGI). Indeed, RmPGI displays high homology with AMF and has been shown to have AMF activity. The biosensor was constructed by covalent binding of the enzyme substrate d-fructose 6-phosphate (F6P). Immobilization was achieved on a gold surface electrode following a bottom-up approach through an aminated surface obtained by electrochemical patterning of ethylene diamine and terminal amine polyethylene glycol chain to prevent non-specific interactions. Carbohydrate-protein interactions were quantified in a range of 10 fM to 100nM. Complex formation was analyzed through monitoring of the redox couple Fe 2+ /Fe 3+ by electrochemical impedance spectroscopy and square wave voltammetry. The F6P-biosensor demonstrates a detection limit of 6.6 fM and high selectivity when compared to other non-specific glycolytic proteins such as d-glucose-6-phosphate dehydrogenase. Detection of protein in spiked plasma was demonstrated and accuracy of 95% is obtained compared to result obtained in PBS (phosphate buffered saline). F6P-biosensor is a very promising proof of concept required for the design of a carbohydrate-based electrochemical biosensor using the enzyme substrate as bioreceptor. Such biosensor could be generalized to detect other protein biomarkers of interest. Copyright © 2017 Elsevier B.V. All rights reserved.
Gene expression patterns combined with network analysis identify hub genes associated with bladder cancer.

PubMed

Bi, Dongbin; Ning, Hao; Liu, Shuai; Que, Xinxiang; Ding, Kejia

2015-06-01

To explore molecular mechanisms of bladder cancer (BC), network strategy was used to find biomarkers for early detection and diagnosis. The differentially expressed genes (DEGs) between bladder carcinoma patients and normal subjects were screened using empirical Bayes method of the linear models for microarray data package. Co-expression networks were constructed by differentially co-expressed genes and links. Regulatory impact factors (RIF) metric was used to identify critical transcription factors (TFs). The protein-protein interaction (PPI) networks were constructed by the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) and clusters were obtained through molecular complex detection (MCODE) algorithm. Centralities analyses for complex networks were performed based on degree, stress and betweenness. Enrichment analyses were performed based on Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Co-expression networks and TFs (based on expression data of global DEGs and DEGs in different stages and grades) were identified. Hub genes of complex networks, such as UBE2C, ACTA2, FABP4, CKS2, FN1 and TOP2A, were also obtained according to analysis of degree. In gene enrichment analyses of global DEGs, cell adhesion, proteinaceous extracellular matrix and extracellular matrix structural constituent were top three GO terms. ECM-receptor interaction, focal adhesion, and cell cycle were significant pathways. Our results provide some potential underlying biomarkers of BC. However, further validation is required and deep studies are needed to elucidate the pathogenesis of BC. Copyright © 2015 Elsevier Ltd. All rights reserved.
Fluorescence Polarization and Fluctuation Analysis Monitors Subunit Proximity, Stoichiometry, and Protein Complex Hydrodynamics

PubMed Central

Nguyen, Tuan A.; Sarkar, Pabak; Veetil, Jithesh V.; Koushik, Srinagesh V.; Vogel, Steven S.

2012-01-01

Förster resonance energy transfer (FRET) microscopy is frequently used to study protein interactions and conformational changes in living cells. The utility of FRET is limited by false positive and negative signals. To overcome these limitations we have developed Fluorescence Polarization and Fluctuation Analysis (FPFA), a hybrid single-molecule based method combining time-resolved fluorescence anisotropy (homo-FRET) and fluorescence correlation spectroscopy. Using FPFA, homo-FRET (a 1–10 nm proximity gauge), brightness (a measure of the number of fluorescent subunits in a complex), and correlation time (an attribute sensitive to the mass and shape of a protein complex) can be simultaneously measured. These measurements together rigorously constrain the interpretation of FRET signals. Venus based control-constructs were used to validate FPFA. The utility of FPFA was demonstrated by measuring in living cells the number of subunits in the α-isoform of Venus-tagged calcium-calmodulin dependent protein kinase-II (CaMKIIα) holoenzyme. Brightness analysis revealed that the holoenzyme has, on average, 11.9±1.2 subunit, but values ranged from 10–14 in individual cells. Homo-FRET analysis simultaneously detected that catalytic domains were arranged as dimers in the dodecameric holoenzyme, and this paired organization was confirmed by quantitative hetero-FRET analysis. In freshly prepared cell homogenates FPFA detected only 10.2±1.3 subunits in the holoenzyme with values ranging from 9–12. Despite the reduction in subunit number, catalytic domains were still arranged as pairs in homogenates. Thus, FPFA suggests that while the absolute number of subunits in an auto-inhibited holoenzyme might vary from cell to cell, the organization of catalytic domains into pairs is preserved. PMID:22666486
Short communication: Labeling Listeria with anaerobic fluorescent protein for food safety studies.

PubMed

Landete, José M; Peirotén, Ángela; Medina, Margarita; Arqués, Juan L

2017-01-01

Many food safety-related studies require the tracking of inoculated food-borne pathogens to monitor their fate in food complex environments. In the current study, we demonstrate the potential of plasmids containing the fluorescence protein gene evoglow-Pp1 (Evocatal, Dusseldorf, Germany) as a real-time reporter system for Listeria strains. This anaerobic fluorescent protein provides an easily detectable phenotype of microorganisms for food safety studies. This work is the first to report a reliable method to identify fluorescently labeled Listeria strains in food ecosystems. Copyright © 2017 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.
Interactions between soy protein from water-soluble soy extract and polysaccharides in solutions with polydextrose.

PubMed

Spada, Jordana C; Marczak, Ligia D F; Tessaro, Isabel C; Cardozo, Nilo S M

2015-12-10

This study focuses on the investigation of the interactions between polysaccharides (carrageenan and carboxymethylcellulose--CMC) and soy proteins from the water-soluble soy extract. The influence of pH (2-7) and protein-polysaccharide ratio (5:1-40:1) on the interaction between these polyelectrolytes was investigated in aqueous solutions with 10% of polydextrose and without polydextrose. The studied systems were analyzed in terms of pH-solubility profile of protein, ζ-potential, methylene blue-polysaccharide interactions, differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FTIR), and confocal laser scanning microscopy. Although the mixtures of soy extract with both carrageenan and CMC showed dependency on the pH and protein-polysaccharide ratio, they did not present the same behavior. Both polysaccharides modified the pH-solubility profile of the soy protein, shifting the pH range in which the coacervate is formed to a lower pH region with the decrease of the soy extract-polysaccharide ratio. The samples also presented detectable differences regarding to ζ-potential, DSC, FTIR and microscopy analyses. The complex formation was also detected even in a pH range where both biopolymers were net-negatively charged. The changes promoted by the presence of polydextrose were mainly detected by blue-polysaccharide interactions measures and confocal microscopy. Copyright © 2015 Elsevier Ltd. All rights reserved.
A Routine 'Top-Down' Approach to Analysis of the Human Serum Proteome.

PubMed

D'Silva, Arlene M; Hyett, Jon A; Coorssen, Jens R

2017-06-06

Serum provides a rich source of potential biomarker proteoforms. One of the major obstacles in analysing serum proteomes is detecting lower abundance proteins owing to the presence of hyper-abundant species (e.g., serum albumin and immunoglobulins). Although depletion methods have been used to address this, these can lead to the concomitant removal of non-targeted protein species, and thus raise issues of specificity, reproducibility, and the capacity for meaningful quantitative analyses. Altering the native stoichiometry of the proteome components may thus yield a more complex series of issues than dealing directly with the inherent complexity of the sample. Hence, here we targeted method refinements so as to ensure optimum resolution of serum proteomes via a top down two-dimensional gel electrophoresis (2DE) approach that enables the routine assessment of proteoforms and is fully compatible with subsequent mass spectrometric analyses. Testing included various fractionation and non-fractionation approaches. The data show that resolving 500 µg protein on 17 cm 3-10 non-linear immobilised pH gradient strips in the first dimension followed by second dimension resolution on 7-20% gradient gels with a combination of lithium dodecyl sulfate (LDS) and sodium dodecyl sulfate (SDS) detergents markedly improves the resolution and detection of proteoforms in serum. In addition, well established third dimension electrophoretic separations in combination with deep imaging further contributed to the best available resolution, detection, and thus quantitative top-down analysis of serum proteomes.
Proteomic Interaction Patterns between Human Cyclins, the Cyclin-Dependent Kinase Ortholog pUL97 and Additional Cytomegalovirus Proteins

PubMed Central

Steingruber, Mirjam; Kraut, Alexandra; Socher, Eileen; Sticht, Heinrich; Reichel, Anna; Stamminger, Thomas; Amin, Bushra; Couté, Yohann; Hutterer, Corina; Marschall, Manfred

2016-01-01

The human cytomegalovirus (HCMV)-encoded cyclin-dependent kinase (CDK) ortholog pUL97 associates with human cyclin B1 and other types of cyclins. Here, the question was addressed whether cyclin interaction of pUL97 and additional viral proteins is detectable by mass spectrometry-based approaches. Proteomic data were validated by coimmunoprecipitation (CoIP), Western blot, in vitro kinase and bioinformatic analyses. Our findings suggest that: (i) pUL97 shows differential affinities to human cyclins; (ii) pUL97 inhibitor maribavir (MBV) disrupts the interaction with cyclin B1, but not with other cyclin types; (iii) cyclin H is identified as a new high-affinity interactor of pUL97 in HCMV-infected cells; (iv) even more viral phosphoproteins, including all known substrates of pUL97, are detectable in the cyclin-associated complexes; and (v) a first functional validation of pUL97-cyclin B1 interaction, analyzed by in vitro kinase assay, points to a cyclin-mediated modulation of pUL97 substrate preference. In addition, our bioinformatic analyses suggest individual, cyclin-specific binding interfaces for pUL97-cyclin interaction, which could explain the different strengths of interactions and the selective inhibitory effect of MBV on pUL97-cyclin B1 interaction. Combined, the detection of cyclin-associated proteins in HCMV-infected cells suggests a complex pattern of substrate phosphorylation and a role of cyclins in the fine-modulation of pUL97 activities. PMID:27548200
Capillary zone electrophoresis for analysis of complex proteomes using an electrokinetically pumped sheath flow nanospray interface

PubMed Central

Sun, Liangliang; Zhu, Guijie; Yan, Xiaojing; Champion, Mathew M.

2014-01-01

The vast majority of proteomic studies employ reversed-phase high-performance liquid chromatography coupled with tandem mass spectrometry for analysis of the tryptic digest of a cellular lysate. This technology is quite mature, and typically provides identification of hundreds to thousands of peptides, which is used to infer the identity of hundreds to thousands of proteins. These approaches usually require milligrams to micrograms of starting material. Capillary zone electrophoresis provides an interesting alternative separation method based on a different separation mechanism than HPLC. Capillary electrophoresis received some attention for protein analysis beginning 25 years ago. Those efforts stalled because of the limited performance of the electrospray interfaces and the limited speed and sensitivity of mass spectrometers of that era. This review considers a new electrospray interface design coupled with Orbitrap Velos and linear Q-trap mass spectrometers. Capillary zone electrophoresis coupled with this interface and these detectors provides single shot detection of >1,250 peptides from an E. coli digest in less than one hour, identification of nearly 5,000 peptides from analysis of seven fractions produced by solid-phase extraction of the E. coli digest in a six hour total analysis time, low attomole detection limits for peptides generated from standard proteins, and high zeptomole detection limits for selected ion monitoring of peptides. Incorporation of an integrated on-line immobilized trypsin microreactor allows digestion and analysis of picogram amounts of a complex eukaryotic proteome. PMID:24277677
Cross-linking by protein oxidation in the rapidly setting gel-based glues of slugs.

PubMed

Bradshaw, Andrew; Salt, Michael; Bell, Ashley; Zeitler, Matt; Litra, Noelle; Smith, Andrew M

2011-05-15

The terrestrial slug Arion subfuscus secretes a glue that is a dilute gel with remarkable adhesive and cohesive strength. The function of this glue depends on metals, raising the possibility that metal-catalyzed oxidation plays a role. The extent and time course of protein oxidation was measured by immunoblotting to detect the resulting carbonyl groups. Several proteins, particularly one with a relative molecular mass (M(r)) of 165 x 10³, were heavily oxidized. Of the proteins known to distinguish the glue from non-adhesive mucus, only specific size variants were oxidized. The oxidation appears to occur within the first few seconds of secretion. Although carbonyls were detected by 2,4-dinitrophenylhydrazine (DNPH) in denatured proteins, they were not easily detected in the native state. The presence of reversible cross-links derived from carbonyls was tested for by treatment with sodium borohydride, which would reduce uncross-linked carbonyls to alcohols, but stabilize imine bonds formed by carbonyls and thus lead to less soluble complexes. Consistent with imine bond formation, sodium borohydride led to a 20-35% decrease in the amount of soluble protein with a M(r) of 40-165 (x 10³) without changing the carbonyl content per protein. In contrast, the nucleophile hydroxylamine, which would competitively disrupt imine bonds, increased protein solubility in the glue. Finally, the primary amine groups on a protein with a M(r) of 15 x 10³ were not accessible to acid anhydrides. The results suggest that cross-links between aldehydes and primary amines contribute to the cohesive strength of the glue.
Cross-linking by protein oxidation in the rapidly setting gel-based glues of slugs

PubMed Central

Bradshaw, Andrew; Salt, Michael; Bell, Ashley; Zeitler, Matt; Litra, Noelle; Smith, Andrew M.

2011-01-01

SUMMARY The terrestrial slug Arion subfuscus secretes a glue that is a dilute gel with remarkable adhesive and cohesive strength. The function of this glue depends on metals, raising the possibility that metal-catalyzed oxidation plays a role. The extent and time course of protein oxidation was measured by immunoblotting to detect the resulting carbonyl groups. Several proteins, particularly one with a relative molecular mass (Mr) of 165×103, were heavily oxidized. Of the proteins known to distinguish the glue from non-adhesive mucus, only specific size variants were oxidized. The oxidation appears to occur within the first few seconds of secretion. Although carbonyls were detected by 2,4-dinitrophenylhydrazine (DNPH) in denatured proteins, they were not easily detected in the native state. The presence of reversible cross-links derived from carbonyls was tested for by treatment with sodium borohydride, which would reduce uncross-linked carbonyls to alcohols, but stabilize imine bonds formed by carbonyls and thus lead to less soluble complexes. Consistent with imine bond formation, sodium borohydride led to a 20–35% decrease in the amount of soluble protein with a Mr of 40–165 (×103) without changing the carbonyl content per protein. In contrast, the nucleophile hydroxylamine, which would competitively disrupt imine bonds, increased protein solubility in the glue. Finally, the primary amine groups on a protein with a Mr of 15×103 were not accessible to acid anhydrides. The results suggest that cross-links between aldehydes and primary amines contribute to the cohesive strength of the glue. PMID:21525316

PROPER: global protein interaction network alignment through percolation matching.

PubMed

Kazemi, Ehsan; Hassani, Hamed; Grossglauser, Matthias; Pezeshgi Modarres, Hassan

2016-12-12

The alignment of protein-protein interaction (PPI) networks enables us to uncover the relationships between different species, which leads to a deeper understanding of biological systems. Network alignment can be used to transfer biological knowledge between species. Although different PPI-network alignment algorithms were introduced during the last decade, developing an accurate and scalable algorithm that can find alignments with high biological and structural similarities among PPI networks is still challenging. In this paper, we introduce a new global network alignment algorithm for PPI networks called PROPER. Compared to other global network alignment methods, our algorithm shows higher accuracy and speed over real PPI datasets and synthetic networks. We show that the PROPER algorithm can detect large portions of conserved biological pathways between species. Also, using a simple parsimonious evolutionary model, we explain why PROPER performs well based on several different comparison criteria. We highlight that PROPER has high potential in further applications such as detecting biological pathways, finding protein complexes and PPI prediction. The PROPER algorithm is available at http://proper.epfl.ch .
Clinical proteomics: Applications for prostate cancer biomarker discovery and detection.

PubMed

Petricoin, Emanuel F; Ornstein, David K; Liotta, Lance A

2004-01-01

The science of proteomics comprises much more than simply generating lists of proteins that change in expression as a cause of or consequence of pathophysiology. The goal of proteomics should be to characterize the information flow through the intercellular protein circuitry that communicates with the extracellular microenvironment and then ultimately to the serum/plasma macroenvironment. Serum proteomic pattern diagnostics is a new type of proteomic concept in which patterns of ion signatures generated from high dimensional mass spectrometry data are used as diagnostic classifiers. This recent approach has exciting potential for clinical utility of diagnostic patterns because low molecular weight metabolites, peptides, and protein fragments may have higher accuracy than traditional biomarkers of cancer detection. Intriguingly, we now have discovered that this diagnostic information exists in a bound state, complexed with circulating highly abundant carrier proteins. These diagnostic fragments may one day be harvested by circulating nanoparticles, designed to absorb, enrich, and amplify the repertoire of diagnostic biomarkers generated-even at the critical, initial stages of carcinogenesis. Copyright 2004 Elsevier Inc.
Compositions and methods for cancer treatment using targeted carbon nanotubes

DOEpatents

Harrison, Jr., Roger G; Resasco, Daniel E; Neves, Luis Filipe Ferreira

2013-08-27

The present invention is a method for detecting and destroying cancer tumors. The method is based on the concept of associating a linking protein or linking peptide such as, but not limited to, annexin V or other annexins to carbon nanotubes such as single-walled carbon nanotubes (SWNTs) to form a protein-CNT complex. Said linking protein or peptide can selectively bind to cancerous cells, especially tumor vasculature endothelial cells, rather than to healthy ones by binding to cancer-specific external receptors such as anionic phospholipids including phosphatidylserine expressed on the outer surfaces of cancer cells only. Irradiation of bound CNTs with one or more specific electromagnetic wavelengths is then used to detect and destroy those cells to which the CNTs are bound via the linking protein or peptide thereby destroying the tumor or cancer cells and preferably an immunostimulant is provided to the patient to enhance the immune response against antigens released from the tumor or cancer cells.
Interlaboratory Study Characterizing a Yeast Performance Standard for Benchmarking LC-MS Platform Performance*

PubMed Central

Paulovich, Amanda G.; Billheimer, Dean; Ham, Amy-Joan L.; Vega-Montoto, Lorenzo; Rudnick, Paul A.; Tabb, David L.; Wang, Pei; Blackman, Ronald K.; Bunk, David M.; Cardasis, Helene L.; Clauser, Karl R.; Kinsinger, Christopher R.; Schilling, Birgit; Tegeler, Tony J.; Variyath, Asokan Mulayath; Wang, Mu; Whiteaker, Jeffrey R.; Zimmerman, Lisa J.; Fenyo, David; Carr, Steven A.; Fisher, Susan J.; Gibson, Bradford W.; Mesri, Mehdi; Neubert, Thomas A.; Regnier, Fred E.; Rodriguez, Henry; Spiegelman, Cliff; Stein, Stephen E.; Tempst, Paul; Liebler, Daniel C.

2010-01-01

Optimal performance of LC-MS/MS platforms is critical to generating high quality proteomics data. Although individual laboratories have developed quality control samples, there is no widely available performance standard of biological complexity (and associated reference data sets) for benchmarking of platform performance for analysis of complex biological proteomes across different laboratories in the community. Individual preparations of the yeast Saccharomyces cerevisiae proteome have been used extensively by laboratories in the proteomics community to characterize LC-MS platform performance. The yeast proteome is uniquely attractive as a performance standard because it is the most extensively characterized complex biological proteome and the only one associated with several large scale studies estimating the abundance of all detectable proteins. In this study, we describe a standard operating protocol for large scale production of the yeast performance standard and offer aliquots to the community through the National Institute of Standards and Technology where the yeast proteome is under development as a certified reference material to meet the long term needs of the community. Using a series of metrics that characterize LC-MS performance, we provide a reference data set demonstrating typical performance of commonly used ion trap instrument platforms in expert laboratories; the results provide a basis for laboratories to benchmark their own performance, to improve upon current methods, and to evaluate new technologies. Additionally, we demonstrate how the yeast reference, spiked with human proteins, can be used to benchmark the power of proteomics platforms for detection of differentially expressed proteins at different levels of concentration in a complex matrix, thereby providing a metric to evaluate and minimize preanalytical and analytical variation in comparative proteomics experiments. PMID:19858499
The Arabidopsis THO/TREX component TEX1 functionally interacts with MOS11 and modulates mRNA export and alternative splicing events.

PubMed

Sørensen, Brian B; Ehrnsberger, Hans F; Esposito, Silvia; Pfab, Alexander; Bruckmann, Astrid; Hauptmann, Judith; Meister, Gunter; Merkl, Rainer; Schubert, Thomas; Längst, Gernot; Melzer, Michael; Grasser, Marion; Grasser, Klaus D

2017-02-01

We identify proteins that associate with the THO core complex, and show that the TEX1 and MOS11 components functionally interact, affecting mRNA export and splicing as well as plant development. TREX (TRanscription-EXport) is a multiprotein complex that plays a central role in the coordination of synthesis, processing and nuclear export of mRNAs. Using targeted proteomics, we identified proteins that associate with the THO core complex of Arabidopsis TREX. In addition to the RNA helicase UAP56 and the mRNA export factors ALY2-4 and MOS11 we detected interactions with the mRNA export complex TREX-2 and multiple spliceosomal components. Plants defective in the THO component TEX1 or in the mRNA export factor MOS11 (orthologue of human CIP29) are mildly affected. However, tex1 mos11 double-mutant plants show marked defects in vegetative and reproductive development. In tex1 plants, the levels of tasiRNAs are reduced, while miR173 levels are decreased in mos11 mutants. In nuclei of mos11 cells increased mRNA accumulation was observed, while no mRNA export defect was detected with tex1 cells. Nevertheless, in tex1 mos11 double-mutants, the mRNA export defect was clearly enhanced relative to mos11. The subnuclear distribution of TEX1 substantially overlaps with that of splicing-related SR proteins and in tex1 plants the ratio of certain alternative splicing events is altered. Our results demonstrate that Arabidopsis TEX1 and MOS11 are involved in distinct steps of the biogenesis of mRNAs and small RNAs, and that they interact regarding some aspects, but act independently in others.
Prostate Secretory Protein of 94 Amino Acids (PSP94) Binds to Prostatic Acid Phosphatase (PAP) in Human Seminal Plasma

PubMed Central

Anklesaria, Jenifer H.; Jagtap, Dhanashree D.; Pathak, Bhakti R.; Kadam, Kaushiki M.; Joseph, Shaini; Mahale, Smita D.

2013-01-01

Prostate Secretory Protein of 94 amino acids (PSP94) is one of the major proteins present in the human seminal plasma. Though several functions have been predicted for this protein, its exact role either in sperm function or in prostate pathophysiology has not been clearly defined. Attempts to understand the mechanism of action of PSP94 has led to the search for its probable binding partners. This has resulted in the identification of PSP94 binding proteins in plasma and seminal plasma from human. During the chromatographic separation step of proteins from human seminal plasma by reversed phase HPLC, we had observed that in addition to the main fraction of PSP94, other fractions containing higher molecular weight proteins also showed the presence of detectable amounts of PSP94. This prompted us to hypothesize that PSP94 could be present in the seminal plasma complexed with other protein/s of higher molecular weight. One such fraction containing a major protein of ∼47 kDa, on characterization by mass spectrometric analysis, was identified to be Prostatic Acid Phosphatase (PAP). The ability of PAP present in this fraction to bind to PSP94 was demonstrated by affinity chromatography. Co-immunoprecipitation experiments confirmed the presence of PSP94-PAP complex both in the fraction studied and in the fresh seminal plasma. In silico molecular modeling of the PSP94-PAP complex suggests that β-strands 1 and 6 of PSP94 appear to interact with domain 2 of PAP, while β-strands 7 and 10 with domain 1 of PAP. This is the first report which suggests that PSP94 can bind to PAP and the PAP-bound PSP94 is present in human seminal plasma. PMID:23469287
Evaluation of a Bead-Free Coimmunoprecipitation Technique for Identification of Virus-Host Protein Interactions Using High-Resolution Mass Spectrometry.

PubMed

DeBlasio, Stacy L; Bereman, Michael S; Mahoney, Jaclyn; Thannhauser, Theodore W; Gray, Stewart M; MacCoss, Michael J; Cilia Heck, Michelle

2017-09-01

Protein interactions between virus and host are essential for viral propagation and movement, as viruses lack most of the proteins required to thrive on their own. Precision methods aimed at disrupting virus-host interactions represent new approaches to disease management but require in-depth knowledge of the identity and binding specificity of host proteins within these interaction networks. Protein coimmunoprecipitation (co-IP) coupled with mass spectrometry (MS) provides a high-throughput way to characterize virus-host interactomes in a single experiment. Common co-IP methods use antibodies immobilized on agarose or magnetic beads to isolate virus-host complexes in solutions of host tissue homogenate. Although these workflows are well established, they can be fairly laborious and expensive. Therefore, we evaluated the feasibility of using antibody-coated microtiter plates coupled with MS analysis as an easy, less expensive way to identify host proteins that interact with Potato leafroll virus (PLRV), an insect-borne RNA virus that infects potatoes. With the use of the bead-free platform, we were able to detect 36 plant and 1 nonstructural viral protein significantly coimmunoprecipitating with PLRV. Two of these proteins, a 14-3-3 signal transduction protein and malate dehydrogenase 2 (mMDH2), were detected as having a weakened or lost association with a structural mutant of the virus, demonstrating that the bead-free method is sensitive enough to detect quantitative differences that can be used to pin-point domains of interaction. Collectively, our analysis shows that the bead-free platform is a low-cost alternative that can be used by core facilities and other investigators to identify plant and viral proteins interacting with virions and/or the viral structural proteins.
Single-Molecule Spectroscopy and Imaging Studies of Protein Dynamics

NASA Astrophysics Data System (ADS)

Lu, H. Peter

2012-04-01

Enzymatic reactions and protein-protein interactions are traditionally studied at the ensemble level, despite significant static and dynamic inhomogeneities. Subtle conformational changes play a crucial role in protein functions, and these protein conformations are highly dynamic rather than being static. We applied AFM-enhanced single-molecule spectroscopy to study the mechanisms and dynamics of enzymatic reactions involved with kinase and lysozyme proteins. Enzymatic reaction turnovers and the associated structure changes of individual protein molecules were observed simultaneously in real-time by single-molecule FRET detections. Our single-molecule spectroscopy measurements of T4 lysozyme and HPPK enzymatic conformational dynamics have revealed time bunching effect and intermittent coherence in conformational state change dynamics involving in enzymatic reaction cycles. The coherent conformational state dynamics suggests that the enzymatic catalysis involves a multi-step conformational motion along the coordinates of substrate-enzyme complex formation and product releasing, presenting as an extreme dynamic behavior intrinsically related to the time bunching effect that we have reported previously. Our results of HPPK interaction with substrate support a multiple-conformational state model, being consistent with a complementary conformation selection and induced-fit enzymatic loop-gated conformational change mechanism in substrate-enzyme active complex formation. Our new approach is applicable to a wide range of single-molecule FRET measurements for protein conformational changes under enzymatic reactions.
Quantitative identification of proteins that influence miRNA biogenesis by RNA pull-down-SILAC mass spectrometry (RP-SMS).

PubMed

Choudhury, Nila Roy; Michlewski, Gracjan

2018-06-08

RNA-binding proteins mediate and control gene expression. As some examples, they regulate pre-mRNA synthesis and processing; mRNA localisation, translation and decay; and microRNA (miRNA) biogenesis and function. Here, we present a detailed protocol for RNA pull-down coupled to stable isotope labelling by amino acids in cell culture (SILAC) mass spectrometry (RP-SMS) that enables quantitative, fast and specific detection of RNA-binding proteins that regulate miRNA biogenesis. In general, this method allows for the identification of RNA-protein complexes formed using in vitro or chemically synthesized RNAs and protein extracts derived from cultured cells. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.
Deconstructing the core dynamics from a complex time-lagged regulatory biological circuit.

PubMed

Eriksson, O; Brinne, B; Zhou, Y; Björkegren, J; Tegnér, J

2009-03-01

Complex regulatory dynamics is ubiquitous in molecular networks composed of genes and proteins. Recent progress in computational biology and its application to molecular data generate a growing number of complex networks. Yet, it has been difficult to understand the governing principles of these networks beyond graphical analysis or extensive numerical simulations. Here the authors exploit several simplifying biological circumstances which thereby enable to directly detect the underlying dynamical regularities driving periodic oscillations in a dynamical nonlinear computational model of a protein-protein network. System analysis is performed using the cell cycle, a mathematically well-described complex regulatory circuit driven by external signals. By introducing an explicit time delay and using a 'tearing-and-zooming' approach the authors reduce the system to a piecewise linear system with two variables that capture the dynamics of this complex network. A key step in the analysis is the identification of functional subsystems by identifying the relations between state-variables within the model. These functional subsystems are referred to as dynamical modules operating as sensitive switches in the original complex model. By using reduced mathematical representations of the subsystems the authors derive explicit conditions on how the cell cycle dynamics depends on system parameters, and can, for the first time, analyse and prove global conditions for system stability. The approach which includes utilising biological simplifying conditions, identification of dynamical modules and mathematical reduction of the model complexity may be applicable to other well-characterised biological regulatory circuits. [Includes supplementary material].
Time-course, negative-stain electron microscopy-based analysis for investigating protein-protein interactions at the single-molecule level.

PubMed

Nogal, Bartek; Bowman, Charles A; Ward, Andrew B

2017-11-24

Several biophysical approaches are available to study protein-protein interactions. Most approaches are conducted in bulk solution, and are therefore limited to an average measurement of the ensemble of molecular interactions. Here, we show how single-particle EM can enrich our understanding of protein-protein interactions at the single-molecule level and potentially capture states that are unobservable with ensemble methods because they are below the limit of detection or not conducted on an appropriate time scale. Using the HIV-1 envelope glycoprotein (Env) and its interaction with receptor CD4-binding site neutralizing antibodies as a model system, we both corroborate ensemble kinetics-derived parameters and demonstrate how time-course EM can further dissect stoichiometric states of complexes that are not readily observable with other methods. Visualization of the kinetics and stoichiometry of Env-antibody complexes demonstrated the applicability of our approach to qualitatively and semi-quantitatively differentiate two highly similar neutralizing antibodies. Furthermore, implementation of machine-learning techniques for sorting class averages of these complexes into discrete subclasses of particles helped reduce human bias. Our data provide proof of concept that single-particle EM can be used to generate a "visual" kinetic profile that should be amenable to studying many other protein-protein interactions, is relatively simple and complementary to well-established biophysical approaches. Moreover, our method provides critical insights into broadly neutralizing antibody recognition of Env, which may inform vaccine immunogen design and immunotherapeutic development. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Sensitive ionization of non-volatile analytes using protein solutions as spray liquid in desorption electrospray ionization mass spectrometry.

PubMed

Zhu, Zhiqiang; Han, Jing; Zhang, Yan; Zhou, Yafei; Xu, Ning; Zhang, Bo; Gu, Haiwei; Chen, Huanwen

2012-12-15

Desorption electrospray ionization (DESI) is the most popular ambient ionization technique for direct analysis of complex samples without sample pretreatment. However, for many applications, especially for trace analysis, it is of interest to improve the sensitivity of DESI-mass spectrometry (MS). In traditional DESI-MS, a mixture of methanol/water/acetic acid is usually used to generate the primary ions. In this article, dilute protein solutions were electrosprayed in the DESI method to create multiply charged primary ions for the desorption ionization of trace analytes on various surfaces (e.g., filter paper, glass, Al-foil) without any sample pretreatment. The analyte ions were then detected and structurally characterized using a LTQ XL mass spectrometer. Compared with the methanol/water/acetic acid (49:49:2, v/v/v) solution, protein solutions significantly increased the signal levels of non-volatile compounds such as benzoic acid, TNT, o-toluidine, peptide and insulin in either positive or negative ion detection mode. For all the analytes tested, the limits of detection (LODs) were reduced to about half of the original values which were obtained using traditional DESI. The results showed that the signal enhancement is highly correlated with the molecular weight of the proteins and the selected solid surfaces. The proposed DESI method is a universal strategy for rapid and sensitive detection of trace amounts of strongly bound and/or non-volatile analytes, including explosives, peptides, and proteins. The results indicate that the sensitivity of DESI can be further improved by selecting larger proteins and appropriate solid surfaces. Copyright © 2012 John Wiley & Sons, Ltd.
Nanoporous-Gold-Based Electrode Morphology Libraries for Investigating Structure-Property Relationships in Nucleic Acid Based Electrochemical Biosensors.

PubMed

Matharu, Zimple; Daggumati, Pallavi; Wang, Ling; Dorofeeva, Tatiana S; Li, Zidong; Seker, Erkin

2017-04-19

Nanoporous gold (np-Au) electrode coatings significantly enhance the performance of electrochemical nucleic acid biosensors because of their three-dimensional nanoscale network, high electrical conductivity, facile surface functionalization, and biocompatibility. Contrary to planar electrodes, the np-Au electrodes also exhibit sensitive detection in the presence of common biofouling media due to their porous structure. However, the pore size of the nanomatrix plays a critical role in dictating the extent of biomolecular capture and transport. Small pores perform better in the case of target detection in complex samples by filtering out the large nonspecific proteins. On the other hand, larger pores increase the accessibility of target nucleic acids in the nanoporous structure, enhancing the detection limits of the sensor at the expense of more interference from biofouling molecules. Here, we report a microfabricated np-Au multiple electrode array that displays a range of electrode morphologies on the same chip for identifying feature sizes that reduce the nonspecific adsorption of proteins but facilitate the permeation of target DNA molecules into the pores. We demonstrate the utility of the electrode morphology library in studying DNA functionalization and target detection in complex biological media with a special emphasis on revealing ranges of electrode morphologies that mutually enhance the limit of detection and biofouling resilience. We expect this technique to assist in the development of high-performance biosensors for point-of-care diagnostics and facilitate studies on the electrode structure-property relationships in potential applications ranging from neural electrodes to catalysts.
Simple horizontal magnetic tweezers for micromanipulation of single DNA molecules and DNA–protein complexes

PubMed Central

McAndrew, Christopher P.; Tyson, Christopher; Zischkau, Joseph; Mehl, Patrick; Tuma, Pamela L.; Pegg, Ian L.; Sarkar, Abhijit

2016-01-01

We report the development of a simple-to-implement magnetic force transducer that can apply a wide range of piconewton (pN) scale forces on single DNA molecules and DNA–protein complexes in the horizontal plane. The resulting low-noise force-extension data enable very high-resolution detection of changes in the DNA tether’s extension: ~0.05 pN in force and <10 nm change in extension. We have also verified that we can manipulate DNA in near equilibrium conditions through the wide range of forces by ramping the force from low to high and back again, and observing minimal hysteresis in the molecule’s force response. Using a calibration technique based on Stokes’ drag law, we have confirmed our force measurements from DNA force-extension experiments obtained using the fluctuation-dissipation theorem applied to transverse fluctuations of the magnetic microsphere. We present data on the force-distance characteristics of a DNA molecule complexed with histones. The results illustrate how the tweezers can be used to study DNA binding proteins at the single molecule level. PMID:26757808
Quantification of proteins in urine samples using targeted mass spectrometry methods.

PubMed

Khristenko, Nina; Domon, Bruno

2015-01-01

Numerous clinical proteomics studies are focused on the development of biomarkers to improve either diagnostics for early disease detection or the monitoring of the response to the treatment. Although, a wealth of biomarker candidates are available, their evaluation and validation in a true clinical setup remains challenging. In biomarkers evaluation studies, a panel of proteins of interest are systematically analyzed in a large cohort of samples. However, in spite of the latest progresses in mass spectrometry, the consistent detection of pertinent proteins in high complex biological samples is still a challenging task. Thus, targeted LC-MS/MS methods are better suited for the systematic analysis of biomarkers rather than shotgun approaches. This chapter describes the workflow used to perform targeted quantitative analyses of proteins in urinary samples. The peptides, as surrogates of the protein of interest, are commonly measured using a triple quadrupole mass spectrometers operated in selected reaction monitoring (SRM) mode. More recently, the advances in targeted LC-MS/MS analysis based on parallel reaction monitoring (PRM) performed on a quadrupole-orbitrap instrument have allowed to increase the specificity and selectivity of the measurements.
Combining atomic force and fluorescence microscopy for analysis of quantum-dot labeled protein–DNA complexes

PubMed Central

Ebenstein, Yuval; Gassman, Natalie; Kim, Soohong; Weiss, Shimon

2011-01-01

Atomic force microscopy (AFM) and fluorescence microscopy are widely used for the study of protein-DNA interactions. While AFM excels in its ability to elucidate structural detail and spatial arrangement, it lacks the ability to distinguish between similarly sized objects in a complex system. This information is readily accessible to optical imaging techniques via site-specific fluorescent labels, which enable the direct detection and identification of multiple components simultaneously. Here, we show how the utilization of semiconductor quantum dots (QDs), serving as contrast agents for both AFM topography and fluorescence imaging, facilitates the combination of both imaging techniques, and with the addition of a flow based DNA extension method for sample deposition, results in a powerful tool for the study of protein-DNA complexes. We demonstrate the inherent advantages of this novel combination of techniques by imaging individual RNA polymerases (RNAP) on T7 genomic DNA. PMID:19452448
Highly sensitive protein detection using a plasmonic field effect transistor (Conference Presentation)

NASA Astrophysics Data System (ADS)

Shokri-Kojori, Hossein; Ji, Yiwen; Han, Xu; Paik, Younghun; Braunschweig, Adam; Kim, Sung Jin

2016-03-01

Localized surface Plasmon Resonance (LSPR) is a nanoscale phenomenon which presents strong resonance associated with noble metal nanostructures. This plasmon resonance based technology enables highly sensitive detection for chemical and biological applications. Recently, we have developed a plasmon field effect transistor (FET) that enables direct plasmonic-to-electric signal conversion with signal amplification. The plasmon FET consists of back-gated field effect transistor incorporated with gold nanoparticles on top of the FET channel. The gold nanostructures are physically separated from transistor electrodes and can be functionalized for a specific biological application. In this presentation, we report a successful demonstration of a model system to detect Con A proteins using Carbohydrate linkers as a capture molecule. The plasmon FET detected a very low concentration of Con A (0.006 mg/L) while it offers a wide dynamic range of 0.006-50 mg/L. In this demonstration, we used two-color light sources instead of a bulky spectrometer to achieve high sensitivity and wide dynamic range. The details of two-color based differential measurement method will be discussed. This novel protein-based sensor has several advantages such as extremely small size for point-of-care system, multiplexing capability, no need of complex optical geometry.
Simultaneous and sensitive detection of six serotypes of botulinum neurotoxin using enzyme-linked immunosorbent assay-based protein antibody microarrays

PubMed Central

Zhang, Yanfeng; Lou, Jianlong; Jenko, Kathy L.; Marks, James D.; Varnum, Susan M.

2012-01-01

Botulinum neurotoxins (BoNTs), produced by Clostridium botulinum, are a group of seven (A–G) immunologically distinct proteins and cause the paralytic disease botulism. These toxins are the most poisonous substances known to humans and are potential bioweapon agents. Therefore, it is necessary to develop highly sensitive assays for the detection of BoNTs in both clinical and environmental samples. In the current study, we have developed an enzyme-linked immunosorbent assay (ELISA)-based protein antibody microarray for the sensitive and simultaneous detection of BoNT serotypes A, B, C, D, E, and F. With engineered high-affinity antibodies, the BoNT assays have sensitivities in buffer ranging from 1.3 fM (0.2 pg/ml) to 14.7 fM (2.2 pg/ml). Using clinical and food matrices (serum and milk), the microarray is capable of detecting BoNT serotypes A to F to similar levels as in standard buffer. Cross-reactivity between assays for individual serotype was also analyzed. These simultaneous, rapid, and sensitive assays have the potential to measure botulinum toxins in a high-throughput manner in complex clinical, food, and environmental samples. PMID:22935296
A Method for WD40 Repeat Detection and Secondary Structure Prediction

PubMed Central

Wang, Yang; Jiang, Fan; Zhuo, Zhu; Wu, Xian-Hui; Wu, Yun-Dong

2013-01-01

WD40-repeat proteins (WD40s), as one of the largest protein families in eukaryotes, play vital roles in assembling protein-protein/DNA/RNA complexes. WD40s fold into similar β-propeller structures despite diversified sequences. A program WDSP (WD40 repeat protein Structure Predictor) has been developed to accurately identify WD40 repeats and predict their secondary structures. The method is designed specifically for WD40 proteins by incorporating both local residue information and non-local family-specific structural features. It overcomes the problem of highly diversified protein sequences and variable loops. In addition, WDSP achieves a better prediction in identifying multiple WD40-domain proteins by taking the global combination of repeats into consideration. In secondary structure prediction, the average Q3 accuracy of WDSP in jack-knife test reaches 93.7%. A disease related protein LRRK2 was used as a representive example to demonstrate the structure prediction. PMID:23776530
Detection of dysregulated protein-association networks by high-throughput proteomics predicts cancer vulnerabilities.

PubMed

Lapek, John D; Greninger, Patricia; Morris, Robert; Amzallag, Arnaud; Pruteanu-Malinici, Iulian; Benes, Cyril H; Haas, Wilhelm

2017-10-01

The formation of protein complexes and the co-regulation of the cellular concentrations of proteins are essential mechanisms for cellular signaling and for maintaining homeostasis. Here we use isobaric-labeling multiplexed proteomics to analyze protein co-regulation and show that this allows the identification of protein-protein associations with high accuracy. We apply this 'interactome mapping by high-throughput quantitative proteome analysis' (IMAHP) method to a panel of 41 breast cancer cell lines and show that deviations of the observed protein co-regulations in specific cell lines from the consensus network affects cellular fitness. Furthermore, these aberrant interactions serve as biomarkers that predict the drug sensitivity of cell lines in screens across 195 drugs. We expect that IMAHP can be broadly used to gain insight into how changing landscapes of protein-protein associations affect the phenotype of biological systems.

A microarray immunoassay for simultaneous detection of proteins and bacteria

NASA Technical Reports Server (NTRS)

Delehanty, James B.; Ligler, Frances S.

2002-01-01

We report the development and characterization of an antibody microarray biosensor for the rapid detection of both protein and bacterial analytes under flow conditions. Using a noncontact microarray printer, biotinylated capture antibodies were immobilized at discrete locations on the surface of an avidin-coated glass microscope slide. Preservation of capture antibody function during the deposition process was accomplished with the use of a low-salt buffer containing sucrose and bovine serum albumin. The slide was fitted with a six-channel flow module that conducted analyte-containing solutions over the array of capture antibody microspots. Detection of bound analyte was subsequently achieved using fluorescent tracer antibodies. The pattern of fluorescent complexes was interrogated using a scanning confocal microscope equipped with a 635-nm laser. This microarray system was employed to detect protein and bacterial analytes both individually and in samples containing mixtures of analytes. Assays were completed in 15 min, and detection of cholera toxin, staphylococcal enterotoxin B, ricin, and Bacillus globigii was demonstrated at levels as low as 8 ng/mL, 4 ng/mL, 10 ng/mL, and 6.2 x 10(4) cfu/mL, respectively. The assays presented here are very fast, as compared to previously published methods for measuring antibody-antigen interactions using microarrays (minutes versus hours).
Molecular Approaches for High Throughput Detection and Quantification of Genetically Modified Crops: A Review

PubMed Central

Salisu, Ibrahim B.; Shahid, Ahmad A.; Yaqoob, Amina; Ali, Qurban; Bajwa, Kamran S.; Rao, Abdul Q.; Husnain, Tayyab

2017-01-01

As long as the genetically modified crops are gaining attention globally, their proper approval and commercialization need accurate and reliable diagnostic methods for the transgenic content. These diagnostic techniques are mainly divided into two major groups, i.e., identification of transgenic (1) DNA and (2) proteins from GMOs and their products. Conventional methods such as PCR (polymerase chain reaction) and enzyme-linked immunosorbent assay (ELISA) were routinely employed for DNA and protein based quantification respectively. Although, these Techniques (PCR and ELISA) are considered as significantly convenient and productive, but there is need for more advance technologies that allow for high throughput detection and the quantification of GM event as the production of more complex GMO is increasing day by day. Therefore, recent approaches like microarray, capillary gel electrophoresis, digital PCR and next generation sequencing are more promising due to their accuracy and precise detection of transgenic contents. The present article is a brief comparative study of all such detection techniques on the basis of their advent, feasibility, accuracy, and cost effectiveness. However, these emerging technologies have a lot to do with detection of a specific event, contamination of different events and determination of fusion as well as stacked gene protein are the critical issues to be addressed in future. PMID:29085378
Transcriptional regulation of the Borrelia burgdorferi antigenically variable VlsE surface protein.

PubMed

Bykowski, Tomasz; Babb, Kelly; von Lackum, Kate; Riley, Sean P; Norris, Steven J; Stevenson, Brian

2006-07-01

The Lyme disease agent Borrelia burgdorferi can persistently infect humans and other animals despite host active immune responses. This is facilitated, in part, by the vls locus, a complex system consisting of the vlsE expression site and an adjacent set of 11 to 15 silent vls cassettes. Segments of nonexpressed cassettes recombine with the vlsE region during infection of mammalian hosts, resulting in combinatorial antigenic variation of the VlsE outer surface protein. We now demonstrate that synthesis of VlsE is regulated during the natural mammal-tick infectious cycle, being activated in mammals but repressed during tick colonization. Examination of cultured B. burgdorferi cells indicated that the spirochete controls vlsE transcription levels in response to environmental cues. Analysis of PvlsE::gfp fusions in B. burgdorferi indicated that VlsE production is controlled at the level of transcriptional initiation, and regions of 5' DNA involved in the regulation were identified. Electrophoretic mobility shift assays detected qualitative and quantitative changes in patterns of protein-DNA complexes formed between the vlsE promoter and cytoplasmic proteins, suggesting the involvement of DNA-binding proteins in the regulation of vlsE, with at least one protein acting as a transcriptional activator.
Translating Mendelian and complex inheritance of Alzheimer's disease genes for predicting unique personal genome variants

PubMed Central

Regan, Kelly; Wang, Kanix; Doughty, Emily; Li, Haiquan; Li, Jianrong; Lee, Younghee; Kann, Maricel G

2012-01-01

Objective Although trait-associated genes identified as complex versus single-gene inheritance differ substantially in odds ratio, the authors nonetheless posit that their mechanistic concordance can reveal fundamental properties of the genetic architecture, allowing the automated interpretation of unique polymorphisms within a personal genome. Materials and methods An analytical method, SPADE-gen, spanning three biological scales was developed to demonstrate the mechanistic concordance between Mendelian and complex inheritance of Alzheimer's disease (AD) genes: biological functions (BP), protein interaction modeling, and protein domain implicated in the disease-associated polymorphism. Results Among Gene Ontology (GO) biological processes (BP) enriched at a false detection rate <5% in 15 AD genes of Mendelian inheritance (Online Mendelian Inheritance in Man) and independently in those of complex inheritance (25 host genes of intragenic AD single-nucleotide polymorphisms confirmed in genome-wide association studies), 16 overlapped (empirical p=0.007) and 45 were similar (empirical p<0.009; information theory). SPAN network modeling extended the canonical pathway of AD (KEGG) with 26 new protein interactions (empirical p<0.0001). Discussion The study prioritized new AD-associated biological mechanisms and focused the analysis on previously unreported interactions associated with the biological processes of polymorphisms that affect specific protein domains within characterized AD genes and their direct interactors using (1) concordant GO-BP and (2) domain interactions within STRING protein–protein interactions corresponding to the genomic location of the AD polymorphism (eg, EPHA1, APOE, and CD2AP). Conclusion These results are in line with unique-event polymorphism theory, indicating how disease-associated polymorphisms of Mendelian or complex inheritance relate genetically to those observed as ‘unique personal variants’. They also provide insight for identifying novel targets, for repositioning drugs, and for personal therapeutics. PMID:22319180
Overexpression of MutSα Complex Proteins Predicts Poor Prognosis in Oral Squamous Cell Carcinoma.

PubMed

Wagner, Vivian Petersen; Webber, Liana Preto; Salvadori, Gabriela; Meurer, Luise; Fonseca, Felipe Paiva; Castilho, Rogério Moraes; Squarize, Cristiane Helena; Vargas, Pablo Agustin; Martins, Manoela Domingues

2016-05-01

The DNA mismatch repair (MMR) system is responsible for the detection and correction of errors created during DNA replication, thereby avoiding the incorporation of mutations in dividing cells. The prognostic value of alterations in MMR system has not previously been analyzed in oral squamous cell carcinoma (OSCC).The study comprised 115 cases of OSCC diagnosed between 1996 and 2010. The specimens collected were constructed into tissue microarray blocks. Immunohistochemical staining for MutSα complex proteins hMSH2 and hMSH6 was performed. The slides were subsequently scanned into high-resolution images, and nuclear staining of hMSH2 and hMSH6 was analyzed using the Nuclear V9 algorithm. Univariable and multivariable Cox proportional hazard regression models were performed to evaluate the prognostic value of hMSH2 and hMSH6 in OSCC.All cases in the present cohort were positive for hMSH2 and hMSH6 and a direct correlation was found between the expression of the proteins (P < 0.05). The mean number of positive cells for hMSH2 and hMSH6 was 64.44 ± 15.21 and 31.46 ± 22.38, respectively. These values were used as cutoff points to determine high protein expression. Cases with high expression of both proteins simultaneously were classified as having high MutSα complex expression. In the multivariable analysis, high expression of the MutSα complex was an independent prognostic factor for poor overall survival (hazard ratio: 2.75, P = 0.02).This study provides a first insight of the prognostic value of alterations in MMR system in OSCC. We found that MutSα complex may constitute a molecular marker for the poor prognosis of OSCC.
Isolation of a complementary DNA clone for the human complement protein C2 and its use in the identification of a restriction fragment length polymorphism.

PubMed Central

Woods, D E; Edge, M D; Colten, H R

1984-01-01

Complementary DNA (cDNA) clones corresponding to the major histocompatibility (MHC) class III antigen, complement protein C2, have been isolated from human liver cDNA libraries with the use of a complex mixture of synthetic oligonucleotides (17 mer) that contains 576 different oligonucleotide sequences. The C2 cDNA were used to identify a DNA restriction enzyme fragment length polymorphism that provides a genetic marker within the MHC that was not detectable at the protein level. An extensive search for genomic polymorphisms using a cDNA clone for another MHC class III gene, factor B, failed to reveal any DNA variants. The genomic variants detected with the C2 cDNA probe provide an additional genetic marker for analysis of MHC-linked diseases. Images PMID:6086718
Detection of ASC Speck Formation by Flow Cytometry and Chemical Cross-linking.

PubMed

Hoss, Florian; Rolfes, Verena; Davanso, Mariana R; Braga, Tarcio T; Franklin, Bernardo S

2018-01-01

Assembly of a relatively large protein aggregate or "speck" formed by the adaptor protein ASC is a common downstream step in the activation of most inflammasomes. This unique feature of ASC allows its visualization by several imaging techniques and constitutes a reliable and feasible readout for inflammasome activation in cells and tissues. We have previously described step-by-step protocols to generate immortalized cell lines stably expressing ASC fused to a fluorescent protein for measuring inflammasome activation by confocal microscopy, and immunofluorescence of endogenous ASC in primary cells. Here, we present two more methods to detect ASC speck formation: (1) Assessment of ASC speck formation by flow cytometry; and (2) Chemical cross-linking of ASC followed by immunoblotting. These methods allow for the discrimination of inflammasome-activated versus non-activated cells, the identification of lineage-specific inflammasome activation in complex cell mixtures, and sorting of inflammasome-activated cells for further analysis.
Demonstration of the salmonid humoral response to Renibacterium salmoninarum using a monoclonal antibody against salmonid immunoglobulin

USGS Publications Warehouse

Bartholomew, J.L.; Arkoosh , M.R.; Rohovec, J.S.

1991-01-01

The specificity of the antibody response of salmonids to Renibacterium salmoninarum antigens was demonstrated by western blotting techniques that utilized a monoclonal antibody against salmonid immunoglobulin. In this study, the specificity of the response in immunized chinook salmon Oncorhynchus tshawytschawas compared with the response in naturally infected chinook salmon and coho salmon O. kisutch, and immunized rabbits. The antibody response in immunized salmon and rabbits and the naturally infected fish was primarily against the 57–58kilodalton protein complex. In addition to recognizing these proteins in the extracellular fraction and whole-cell preparations, antibody from the immunized salmon and rabbits detected four proteins with lower molecular masses. Western blotting techniques allow identification of the specific antigens recognized and are a useful tool for comparing the immunogenicity of different R. salmoninarumpreparations. Immunofluorescent techniques with whole bacteria were less sensitive than western blotting in detecting salmonid anti-R. salmoninarumantibody.
Serial interactome capture of the human cell nucleus.

PubMed

Conrad, Thomas; Albrecht, Anne-Susann; de Melo Costa, Veronica Rodrigues; Sauer, Sascha; Meierhofer, David; Ørom, Ulf Andersson

2016-04-04

Novel RNA-guided cellular functions are paralleled by an increasing number of RNA-binding proteins (RBPs). Here we present 'serial RNA interactome capture' (serIC), a multiple purification procedure of ultraviolet-crosslinked poly(A)-RNA-protein complexes that enables global RBP detection with high specificity. We apply serIC to the nuclei of proliferating K562 cells to obtain the first human nuclear RNA interactome. The domain composition of the 382 identified nuclear RBPs markedly differs from previous IC experiments, including few factors without known RNA-binding domains that are in good agreement with computationally predicted RNA binding. serIC extends the number of DNA-RNA-binding proteins (DRBPs), and reveals a network of RBPs involved in p53 signalling and double-strand break repair. serIC is an effective tool to couple global RBP capture with additional selection or labelling steps for specific detection of highly purified RBPs.
Production of antibodies against glycolipids from the Mycobacterium tuberculosis cell wall in aerosol murine models of tuberculosis.

PubMed

Cardona, P J; Julián, E; Vallès, X; Gordillo, S; Muñoz, M; Luquin, M; Ausina, V

2002-06-01

Evolution of antibodies against glycolipids from the Mycobacterium tuberculosis cell wall has been studied for the first time in experimental murine models of tuberculosis induced by aerosol, in which infection, reinfection, reactivation, prophylaxis and treatment with antibiotics have been assayed. Results show a significant humoral response against these antigens, where diacyltrehaloses (DAT) and sulpholipid I (SL-I) elicited higher antibody levels than protein antigens like antigen 85 protein complex (Ag85), culture filtrate proteins (CFP) and purified protein derivative (PPD). Only immunoglobulin M (IgM) antibodies have been detected against DAT and SL-I. Their evolution has a positive correlation with bacillary concentration in tissues.
The Salivary Secretome of the Tsetse Fly Glossina pallidipes (Diptera: Glossinidae) Infected by Salivary Gland Hypertrophy Virus

PubMed Central

Kariithi, Henry M.; Ince, Ikbal A.; Boeren, Sjef; Abd-Alla, Adly M. M.; Parker, Andrew G.; Aksoy, Serap; Vlak, Just M.; van Oers, Monique M.

2011-01-01

Background The competence of the tsetse fly Glossina pallidipes (Diptera; Glossinidae) to acquire salivary gland hypertrophy virus (SGHV), to support virus replication and successfully transmit the virus depends on complex interactions between Glossina and SGHV macromolecules. Critical requisites to SGHV transmission are its replication and secretion of mature virions into the fly's salivary gland (SG) lumen. However, secretion of host proteins is of equal importance for successful transmission and requires cataloging of G. pallidipes secretome proteins from hypertrophied and non-hypertrophied SGs. Methodology/Principal Findings After electrophoretic profiling and in-gel trypsin digestion, saliva proteins were analyzed by nano-LC-MS/MS. MaxQuant/Andromeda search of the MS data against the non-redundant (nr) GenBank database and a G. morsitans morsitans SG EST database, yielded a total of 521 hits, 31 of which were SGHV-encoded. On a false discovery rate limit of 1% and detection threshold of least 2 unique peptides per protein, the analysis resulted in 292 Glossina and 25 SGHV MS-supported proteins. When annotated by the Blast2GO suite, at least one gene ontology (GO) term could be assigned to 89.9% (285/317) of the detected proteins. Five (∼1.8%) Glossina and three (∼12%) SGHV proteins remained without a predicted function after blast searches against the nr database. Sixty-five of the 292 detected Glossina proteins contained an N-terminal signal/secretion peptide sequence. Eight of the SGHV proteins were predicted to be non-structural (NS), and fourteen are known structural (VP) proteins. Conclusions/Significance SGHV alters the protein expression pattern in Glossina. The G. pallidipes SG secretome encompasses a spectrum of proteins that may be required during the SGHV infection cycle. These detected proteins have putative interactions with at least 21 of the 25 SGHV-encoded proteins. Our findings opens venues for developing novel SGHV mitigation strategies to block SGHV infections in tsetse production facilities such as using SGHV-specific antibodies and phage display-selected gut epithelia-binding peptides. PMID:22132244
Probing structurally altered and aggregated states of therapeutically relevant proteins using GroEL coupled to bio-layer interferometry

PubMed Central

Naik, Subhashchandra; Kumru, Ozan S; Cullom, Melissa; Telikepalli, Srivalli N; Lindboe, Elizabeth; Roop, Taylor L; Joshi, Sangeeta B; Amin, Divya; Gao, Phillip; Middaugh, C Russell; Volkin, David B; Fisher, Mark T

2014-01-01

The ability of a GroEL-based bio-layer interferometry (BLI) assay to detect structurally altered and/or aggregated species of pharmaceutically relevant proteins is demonstrated. Assay development included optimizing biotinylated-GroEL immobilization to streptavidin biosensors, combined with biophysical and activity measurements showing native and biotinylated GroEL are both stable and active. First, acidic fibroblast growth factor (FGF-1) was incubated under conditions known to promote (40°C) and inhibit (heparin addition) molten globule formation. Heat exposed (40°C) FGF-1 exhibited binding to GroEL-biosensors, which was significantly diminished in the presence of heparin. Second, a polyclonal human IgG solution containing 6–8% non-native dimer showed an increase in higher molecular weight aggregates upon heating by size exclusion chromatography (SEC). The poly IgG solution displayed binding to GroEL-biosensors initially with progressively increased binding upon heating. Enriched preparations of the IgG dimers or monomers showed significant binding to GroEL-biosensors. Finally, a thermally treated IgG1 monoclonal antibody (mAb) solution also demonstrated increased GroEL-biosensor binding, but with different kinetics. The bound complexes could be partially to fully dissociated after ATP addition (i.e., specific GroEL binding) depending on the protein, environmental stress, and the assay’s experimental conditions. Transmission electron microscopy (TEM) images of GroEL-mAb complexes, released from the biosensor, also confirmed interaction of bound complexes at the GroEL binding site with heat-stressed mAb. Results indicate that the GroEL-biosensor-BLI method can detect conformationally altered and/or early aggregation states of proteins, and may potentially be useful as a rapid, stability-indicating biosensor assay for monitoring the structural integrity and physical stability of therapeutic protein candidates. PMID:25043635
A Highly Expressed High-Molecular-Weight S-Layer Complex of Pelosinus sp. Strain UFO1 Binds Uranium

PubMed Central

Thorgersen, Michael P.; Lancaster, W. Andrew; Rajeev, Lara; Ge, Xiaoxuan; Vaccaro, Brian J.; Poole, Farris L.; Arkin, Adam P.; Mukhopadhyay, Aindrila

2016-01-01

ABSTRACT Cell suspensions of Pelosinus sp. strain UFO1 were previously shown, using spectroscopic analysis, to sequester uranium as U(IV) complexed with carboxyl and phosphoryl group ligands on proteins. The goal of our present study was to characterize the proteins involved in uranium binding. Virtually all of the uranium in UFO1 cells was associated with a heterodimeric protein, which was termed the uranium-binding complex (UBC). The UBC was composed of two S-layer domain proteins encoded by UFO1_4202 and UFO1_4203. Samples of UBC purified from the membrane fraction contained 3.3 U atoms/heterodimer, but significant amounts of phosphate were not detected. The UBC had an estimated molecular mass by gel filtration chromatography of 15 MDa, and it was proposed to contain 150 heterodimers (UFO1_4203 and UFO1_4202) and about 500 uranium atoms. The UBC was also the dominant extracellular protein, but when purified from the growth medium, it contained only 0.3 U atoms/heterodimer. The two genes encoding the UBC were among the most highly expressed genes within the UFO1 genome, and their expressions were unchanged by the presence or absence of uranium. Therefore, the UBC appears to be constitutively expressed and is the first line of defense against uranium, including by secretion into the extracellular medium. Although S-layer proteins were previously shown to bind U(VI), here we showed that U(IV) binds to S-layer proteins, we identified the proteins involved, and we quantitated the amount of uranium bound. IMPORTANCE Widespread uranium contamination from industrial sources poses hazards to human health and to the environment. Herein, we identified a highly abundant uranium-binding complex (UBC) from Pelosinus sp. strain UFO1. The complex makes up the primary protein component of the S-layer of strain UFO1 and binds 3.3 atoms of U(IV) per heterodimer. While other bacteria have been shown to bind U(VI) on their S-layer, we demonstrate here an example of U(IV) bound by an S-layer complex. The UBC provides a potential tool for the microbiological sequestration of uranium for the cleaning of contaminated environments. PMID:27913415
A Highly Expressed High-Molecular-Weight S-Layer Complex of Pelosinus sp. Strain UFO1 Binds Uranium

DOE Office of Scientific and Technical Information (OSTI.GOV)

Thorgersen, Michael P.; Lancaster, W. Andrew; Rajeev, Lara

Cell suspensions of Pelosinus sp. strain UFO1 were previously shown, using spectroscopic analysis, to sequester uranium as U(IV) complexed with carboxyl and phosphoryl group ligands on proteins. The goal of our present study was to characterize the proteins involved in uranium binding. Virtually all of the uranium in UFO1 cells was associated with a heterodimeric protein, which was termed the uranium-binding complex (UBC). The UBC was composed of two S-layer domain proteins encoded by UFO1_4202 and UFO1_4203. Samples of UBC purified from the membrane fraction contained 3.3 U atoms/heterodimer, but significant amounts of phosphate were not detected. The UBC hadmore » an estimated molecular mass by gel filtration chromatography of 15 MDa, and it was proposed to contain 150 heterodimers (UFO1_4203 and UFO1_4202) and about 500 uranium atoms. The UBC was also the dominant extracellular protein, but when purified from the growth medium, it contained only 0.3 U atoms/heterodimer. The two genes encoding the UBC were among the most highly expressed genes within the UFO1 genome, and their expressions were unchanged by the presence or absence of uranium. Therefore, the UBC appears to be constitutively expressed and is the first line of defense against uranium, including by secretion into the extracellular medium. Although S-layer proteins were previously shown to bind U(VI), here we showed that U(IV) binds to S-layer proteins, we identified the proteins involved, and we quantitated the amount of uranium bound. Widespread uranium contamination from industrial sources poses hazards to human health and to the environment. Here in this paper, we identified a highly abundant uranium-binding complex (UBC) from Pelosinus sp. strain UFO1. The complex makes up the primary protein component of the S-layer of strain UFO1 and binds 3.3 atoms of U(IV) per heterodimer. Finally, while other bacteria have been shown to bind U(VI) on their S-layer, we demonstrate here an example of U(IV) bound by an S-layer complex. The UBC provides a potential tool for the microbiological sequestration of uranium for the cleaning of contaminated environments.« less
A Highly Expressed High-Molecular-Weight S-Layer Complex of Pelosinus sp. Strain UFO1 Binds Uranium.

PubMed

Thorgersen, Michael P; Lancaster, W Andrew; Rajeev, Lara; Ge, Xiaoxuan; Vaccaro, Brian J; Poole, Farris L; Arkin, Adam P; Mukhopadhyay, Aindrila; Adams, Michael W W

2017-02-15

Cell suspensions of Pelosinus sp. strain UFO1 were previously shown, using spectroscopic analysis, to sequester uranium as U(IV) complexed with carboxyl and phosphoryl group ligands on proteins. The goal of our present study was to characterize the proteins involved in uranium binding. Virtually all of the uranium in UFO1 cells was associated with a heterodimeric protein, which was termed the uranium-binding complex (UBC). The UBC was composed of two S-layer domain proteins encoded by UFO1_4202 and UFO1_4203. Samples of UBC purified from the membrane fraction contained 3.3 U atoms/heterodimer, but significant amounts of phosphate were not detected. The UBC had an estimated molecular mass by gel filtration chromatography of 15 MDa, and it was proposed to contain 150 heterodimers (UFO1_4203 and UFO1_4202) and about 500 uranium atoms. The UBC was also the dominant extracellular protein, but when purified from the growth medium, it contained only 0.3 U atoms/heterodimer. The two genes encoding the UBC were among the most highly expressed genes within the UFO1 genome, and their expressions were unchanged by the presence or absence of uranium. Therefore, the UBC appears to be constitutively expressed and is the first line of defense against uranium, including by secretion into the extracellular medium. Although S-layer proteins were previously shown to bind U(VI), here we showed that U(IV) binds to S-layer proteins, we identified the proteins involved, and we quantitated the amount of uranium bound. Widespread uranium contamination from industrial sources poses hazards to human health and to the environment. Herein, we identified a highly abundant uranium-binding complex (UBC) from Pelosinus sp. strain UFO1. The complex makes up the primary protein component of the S-layer of strain UFO1 and binds 3.3 atoms of U(IV) per heterodimer. While other bacteria have been shown to bind U(VI) on their S-layer, we demonstrate here an example of U(IV) bound by an S-layer complex. The UBC provides a potential tool for the microbiological sequestration of uranium for the cleaning of contaminated environments. Copyright © 2017 American Society for Microbiology.
Detection of ricin by using gold nanoclusters functionalized with chicken egg white proteins as sensing probes.

PubMed

Selvaprakash, Karuppuchamy; Chen, Yu-Chie

2017-06-15

Ricin produced from the castor oil plant, Ricinus communis, is a well-known toxin. The toxin comprises A and B chains. Ricin A chain can cause toxicity by inhibiting protein synthesis, and ricin B can bind to the galactose ligand on the cell membrane of host cells. Inhalation or ingestion of ricin may even lead to death. Therefore, rapid and convenient sensing methods for detecting ricin in suspicious samples must be developed. In this study, we generated protein encapsulated gold nanoclusters (AuNCs@ew) with bright photoluminescence by using chicken egg white proteins as starting materials to react with aqueous tetrachloroaurate. The generated nanoclusters, which were mainly composed of chicken ovalbumin-encapsulated AuNCs, can recognize ricin B because of the presence of Galβ(1→4)GlcNAc ligands on chicken ovalbumin. The generated conjugates of AuNCs@ew and ricin B were heavy and readily settled down under centrifugation (13,000rpm, 60min). Thus, bright spots resulting from the conjugates at the bottom of the sample vials were easily visualized by the naked eye under ultraviolet light illumination. The limit of detection (LOD) was ~4.6µM. The LOD was reduced to ~400nM when fluorescence spectroscopy was used as the detection tool, while the LOD can be further improved to ~7.8nM when using matrix-assisted laser desorption/ionization mass spectrometry as the detection method. We also demonstrated the feasibility of using the proposed approach to selectively detect ricin B chain in complex samples. Copyright © 2016 Elsevier B.V. All rights reserved.
Isolation and preliminary characterization of circulating immune complexes from rabbits with experimental syphilis.

PubMed Central

Baughn, R E; Musher, D M

1983-01-01

Immune complexes isolated from sera of rabbits with experimental, disseminated syphilis were found to have sedimentation coefficients greater than 19s. By radioimmunoblot assays, materials precipitated with 2.5% polyethylene glycol or chromatographed on DEAE-Affi-Gel Blue were found to contain albumin, C3, immunoglobulin M (IgM), IgG, and treponemal antigen(s), whereas control materials contained only albumin and IgG. When polyethylene glycol precipitation of immune complexes from syphilitic rabbits was followed by immobilization on protein A and acid elution, radioimmunoblots detected only IgG and treponemal antigen(s). Images PMID:6358025
Isolation and characterization of an RNA aptamer for the HPV-16 E7 oncoprotein.

PubMed

Toscano-Garibay, Julia D; Benítez-Hess, María L; Alvarez-Salas, Luis M

2011-02-01

Cervical cancer is a common neoplastic disease affecting women worldwide. Expression of human papillomavirus type 16 (HPV-16) E6/E7 genes is frequently associated with cervical cancer, representing ideal targets for diagnostic and therapeutic strategies. Aptamers are oligonucleotide ligands capable of binding with high affinity and specificity to relevant markers in therapeutics and disease detection. The aim of the study was to isolate an RNA aptamer specific for the HPV-16 E7 protein. Aptamers were selected from a randomized oligonucleotide library using a modified SELEX method and recombinant HPV-16 E7 protein. Isolated aptamers were cloned and sequenced for in silico analysis. Interaction and electromobility shift assays (EMSA) were performed to establish aptamer specificity and affinity for E7. RNase footprinting and serial deletions of the aptamer and the E7 protein were made to characterize the aptamer-protein complex. Sandwich slot-blot assays were used for K(D) determination. After several rounds of SELEX, an aptamer (G5α3N.4) exhibited specificity for E7 using cell-free and protein extracts. G5α3N.4 binding yielded a K(D) comparable to aptamers directed to other small targets. Enzymatic and genetic analysis of G5α3N.4 binding showed a secondary structure with two stem-loop domains joined by single-stranded region contacting E7 in a clamp-like manner. The G5α3N.4 aptamer also produced specific complexes in HPV-positive cervical carcinoma cells. The affinity and specificity of G5α3N.4 binding domains for the HPV-16 E7 protein may be used for the detection of papillomavirus infection and cervical cancer. Copyright © 2011 IMSS. Published by Elsevier Inc. All rights reserved.
Luminescence properties of the dinuclear copper complex in the active site of hemocyanins.

PubMed

Beltramini, M; di Muro, P; Rocco, G P; Salvato, B

1994-09-01

The deoxygenated form of hemocyanin, containing a dinuclear Cu(I) active site, emits luminescence in the red with maximum around 1.54 microns-1 (650 nm). The luminescence of deoxyhemocyanin (deoxy-Hc) from arthropod species is detectable at room temperature, the quantum yield being 2.4-2.7 x 10(-3); in contrast, the emission from molluscan proteins can be detected only at liquid nitrogen temperature. The luminescence emission is an inherent property of the bis[Cu(I)-(histidine)3] complex of the deoxygenated form of the protein to which both Cu(I) ions contribute equally to the overall emission. Luminescence is not observed with the oxygenated and the oxidized forms of hemocyanin, in which the metal is in the Cu(II) state, and in the metal-depleted or apo-Hc form. Based on steady-state and time-resolved measurements and references to Cu(I) model compounds, the luminescence emission is attributed to a triplet excited state of a Cu(I)-to-N (histidine) charge transfer transition 3d-pi*. Acrylamide quenching experiments indicate that the metal active site is very shielded from the solvent. This property of deoxy-Hc enables us to directly follow reactions that modify either the copper oxidation number or the metal-to-protein stoichiometry.
Post-translational quantitation by SRM/MRM: applications in cardiology.

PubMed

Gianazza, Erica; Banfi, Cristina

2018-06-04

Post-translational modifications (PTMs) have an important role in the regulation of protein function, localization and interaction with other molecules. PTMs apply a dynamic control of proteins both in physiological and pathological conditions. The study of disease-specific PTMs allows identifying potential biomarkers and developing effective drugs. Enrichment techniques combined with high-resolution MS/MS analysis provide attractive results on PTMs characterization. Selected reaction monitoring/multiple reaction monitoring (SRM/MRM) is a powerful targeted assay for the quantitation and validation of PTMs in complex biological samples. Areas covered: The most frequent PTMs are described in terms of biological role and analytical methods commonly used to detect them. The applications of SRM/MRM for the absolute quantitation of PTMs are reported and a specific section is focused on PTMs detection in proteins that are involved in cardiovascular system and heart diseases. Expert commentary: PTMs characterization in relation to disease pathology is still in progress, but targeted proteomics by LC-MS/MS has significantly upgraded the knowledge in the last years. Advances in enrichment strategies and software tools will facilitate the interpretation of high PTMs complexity. Promising studies confirm the great potentiality of SRM/MRM to study PTMs in cardiovascular field and PTMomics could be very useful in a clinical perspective.

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