A full-genomic sequence-verified protein-coding gene collection for Francisella tularensis

The rapid development of new technologies for the high throughput (HT) study of proteins has increased the demand for comprehensive plasmid clone resources that support protein expression. These clones must be full-length, sequence-verified and in a flexible format. The generation of these resources requires automated pipelines supported by software management systems. Although the availability of clone resources is growing, current collections are either not complete or not fully sequence-verified. We report an automated pipeline, supported by several software applications that enabled the construction of the first comprehensive sequence-verified plasmid clone resource for more than 96% of protein coding sequences of the genome of F. tularensis, a highly virulent human pathogen and the causative agent of tularemia. This clone resource was applied to a HT protein purification pipeline successfully producing recombinant proteins for 72% of the genes. These methods and resources represent significant technological steps towards exploiting the genomic information of F. tularensis in discovery applications.     

Development of a full‐length human protein production pipeline

There are many proteomic applications that require large collections of purified protein, but parallel production of large numbers of different proteins remains a very challenging task. To help meet the needs of the scientific community, we have developed a human protein production pipeline. Using high‐throughput (HT) methods, we transferred the genes of 31 full‐length proteins into three expression vectors, and expressed the collection as N‐terminal HaloTag fusion proteins in Escherichia coli and two commercial cell‐free (CF) systems, wheat germ extract (WGE) and HeLa cell extract (HCE). Expression was assessed by labeling the fusion proteins specifically and covalently with a fluorescent HaloTag ligand and detecting its fluorescence on a LabChip^® GX microfluidic capillary gel electrophoresis instrument. This automated, HT assay provided both qualitative and quantitative assessment of recombinant protein. E. coli was only capable of expressing 20% of the test collection in the supernatant fraction with ≥20 μg yields, whereas CF systems had ≥83% success rates. We purified expressed proteins using an automated HaloTag purification method. We purified 20, 33, and 42% of the test collection from E. coli, WGE, and HCE, respectively, with yields ≥1 μg and ≥90% purity. Based on these observations, we have developed a triage strategy for producing full‐length human proteins in these three expression systems.     

Protein glycosylation in bacterial mucosal pathogens

In eukaryotes, glycosylated proteins are ubiquitous components of extracellular matrices and cellular surfaces. Their oligosaccharide moieties are implicated in a wide range of cell-cell and cell-matrix recognition events that are required for biological processes ranging from immune recognition to cancer development. Glycosylation was previously considered to be restricted to eukaryotes; however, through advances in analytical methods and genome sequencing, there have been increasing reports of both O-linked and N-linked protein glycosylation pathways in bacteria, particularly amongst mucosal-associated pathogens. Studying glycosylation in relatively less-complicated bacterial systems provides the opportunity to elucidate and exploit glycoprotein biosynthetic pathways. We will review the genetic organization, glycan structures and function of glycosylation systems in mucosal bacterial pathogens, and speculate on how this knowledge may help us to understand glycosylation processes in more complex eukaryotic systems and how it can be used for glycoengineering.     

Recreating ancestral proteins

Tracing the history of molecular changes using phylogenetic methods can provide powerful insights into how and why molecules work the way they do. It is now possible to recreate inferred ancestral proteins in the laboratory and study the function of these molecules. This provides a unique opportunity to study the paths and the mechanisms of functional change during molecular evolution. What insights have already emerged from such phylogenetic studies of protein evolution and function, what are the impediments to progress and what are the prospects for the future?     

Identification of tyrosine-nitrated proteins in HT22 hippocampal cells during glutamate-induced oxidative stress

Objectives: Nitration of tyrosine residues in protein is a post‐translational modification, which occurs under oxidative stress, and is associated with several neurodegenerative diseases. To understand the role of nitrated proteins in oxidative stress‐induced cell death, we identified nitrated proteins and checked correlation of their nitration in glutamate‐induced HT22 cell death. Materials and methods: Nitrated proteins were detected by western blotting using an anti‐nitrotyrosine antibody, extracted from matching reference 2‐dimensional electrophoresis gels, and identified with matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry. Results: Glutamate treatment induced apoptosis in HT22 cells, while reactive oxygen species (ROS) inhibitor or neuronal nitric oxide synthase (nNOS) inhibitor blocked glutamate‐induced HT22 cell death. Nitration levels of 13 proteins were increased after glutamate stimulation; six of them were involved in regulation of energy production and two were related to apoptosis. The other nitrated proteins were associated with calcium signal modulation, ER dysfunction, or were of unknown function. Conclusions: The 13 tyrosine‐nitrated proteins were detected in these glutamate‐treated HT22 cells. Results demonstrated that cell death, ROS accumulation and nNOS expression were related to nitration of protein tyrosine in the glutamate‐stimulated cells.     

Protein folding at single-molecule resolution

The protein folding reaction carries great significance for cellular function and hence continues to be the research focus of a large interdisciplinary protein science community. Single-molecule methods are providing new and powerful tools for dissecting the mechanisms of this complex process by virtue of their ability to provide views of protein structure and dynamics without associated ensemble averaging. This review briefly introduces common FRET and force methods, and then explores several areas of protein folding where single-molecule experiments have yielded insights. These include exciting new information about folding landscapes, dynamics, intermediates, unfolded ensembles, intrinsically disordered proteins, assisted folding and biomechanical unfolding. Emerging and future work is expected to include advances in single-molecule techniques aimed at such investigations, and increasing work on more complex systems from both the physics and biology standpoints, including folding and dynamics of systems of interacting proteins and of proteins in cells and organisms. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches.     

Quantitative methods for the analysis of protein phosphorylation in drug development

Most signal transduction and cell signaling pathways are mediated by protein kinases. Protein kinases have emerged as important cellular regulatory proteins in many aspects of neoplasia. Protein kinase inhibitors offer the opportunity to target diseases such as cancer with chemotherapeutic agents specific for the causative molecular defect. In order to identify possible targets and assess kinase inhibitors, quantitative methods for analyzing protein phosphorylation have been developed. This review examines some of the current formats used for quantifying kinase function for drug development.     

Quantitative methods for the analysis of protein phosphorylation in drug development

Most signal transduction and cell signaling pathways are mediated by protein kinases. Protein kinases have emerged as important cellular regulatory proteins in many aspects of neoplasia. Protein kinase inhibitors offer the opportunity to target diseases such as cancer with chemotherapeutic agents specific for the causative molecular defect. In order to identify possible targets and assess kinase inhibitors, quantitative methods for analyzing protein phosphorylation have been developed. This review examines some of the current formats used for quantifying kinase function for drug development.     

In vivo protein complex topologies: sights through a cross-linking lens

Proteins are a remarkable class of molecules that exhibit wide diversity of shapes or topological features that underpin protein interactions and give rise to biological function. In addition to quantitation of abundance levels of proteins in biological systems under a variety of conditions, the field of proteome research has as a primary mission the assignment of function for proteins and if possible, illumination of factors that enable function. For many years, chemical cross-linking methods have been used to provide structural data on single purified proteins and purified protein complexes. However, these methods also offer the alluring possibility to extend capabilities to complex biological samples such as cell lysates or intact living cells where proteins may exhibit native topological features that do not exist in purified form. Recent efforts are beginning to provide glimpses of protein complexes and topologies in cells that suggest continued development will yield novel capabilities to view functional topological features of many proteins and complexes as they exist in cells, tissues, or other complex samples. This review will describe rationale, challenges, and a few success stories along the path of development of cross-linking technologies for measurement of in vivo protein interaction topologies.     

Preparative parallel protein purification (P4)

In state of the art drug discovery, it is essential to gain structural information of pharmacologically relevant proteins. Increasing the output of novel protein structures requires improved preparative methods for high throughput (HT) protein purification. Currently, most HT platforms are limited to small-scale and available technology for increasing throughput at larger scales is scarce. We have adapted a 10-channel parallel flash chromatography system for protein purification applications. The system enables us to perform 10 different purifications in parallel with individual gradients and UV monitoring. Typical protein purification applications were set up. Methods for ion exchange chromatography were developed for different sample proteins and columns. Affinity chromatography was optimized for His-tagged proteins using metal chelating media and buffer exchange by gel filtration was also tested. The results from the present system were comparable, with respect to resolution and reproducibility, with those from control experiments on an AKTA purifier system. Finally, lysates from 10 E. coli cultures expressing different His-tagged proteins were subjected to a three-step parallel purification procedure, combining the above-mentioned procedures. Nine proteins were successfully purified whereas one failed probably due to lack of expression.     

Protein nanomechanics in biological context

How proteins respond to pulling forces, or protein nanomechanics, is a key contributor to the form and function of biological systems. Indeed, the conventional view that proteins are able to diffuse in solution does not apply to the many polypeptides that are anchored to rigid supramolecular structures. These tethered proteins typically have important mechanical roles that enable cells to generate, sense, and transduce mechanical forces. To fully comprehend the interplay between mechanical forces and biology, we must understand how protein nanomechanics emerge in living matter. This endeavor is definitely challenging and only recently has it started to appear tractable. Here, I introduce the main in vitro single-molecule biophysics methods that have been instrumental to investigate protein nanomechanics over the last 2 decades. Then, I present the contemporary view on how mechanical force shapes the free energy of tethered proteins, as well as the effect of biological factors such as post-translational modifications and mutations. To illustrate the contribution of protein nanomechanics to biological function, I review current knowledge on the mechanobiology of selected muscle and cell adhesion proteins including titin, talin, and bacterial pilins. Finally, I discuss emerging methods to modulate protein nanomechanics in living matter, for instance by inducing specific mechanical loss-of-function (mLOF). By interrogating biological systems in a causative manner, these new tools can contribute to further place protein nanomechanics in a biological context.     

A HT/PEXEL Motif in Toxoplasma Dense Granule Proteins is a Signal for Protein Cleavage but not Export into the Host Cell

Apicomplexan parasites, such as Toxoplasma gondii and Plasmodium, secrete proteins for attachment, invasion and modulation of their host cells. The host targeting (HT), also known as the Plasmodium export element (PEXEL), directs Plasmodium proteins into erythrocytes to remodel the host cell and establish infection. Bioinformatic analysis of Toxoplasma revealed a HT/PEXEL‐like motif at the N‐terminus of several hypothetical unknown and dense granule proteins. Hemagglutinin‐tagged versions of these uncharacterized proteins show co‐localization with dense granule proteins found on the parasitophorous vacuole membrane (PVM). In contrast to Plasmodium, these Toxoplasma HT/PEXEL containing proteins are not exported into the host cell. Site directed mutagenesis of the Toxoplasma HT/PEXEL motif, RxLxD/E, shows that the arginine and leucine residues are permissible for protein cleavage. Mutations within the HT/PEXEL motif that prevent protein cleavage still allow for targeting to the PV but the proteins have a reduced association with the PVM. Addition of a Myc tag before and after the cleavage site shows that processed HT/PEXEL protein has increased PVM association. These findings suggest that while Toxoplasma and Plasmodium share similar HT/PEXEL motifs, Toxoplasma HT/PEXEL containing proteins interact with but do not cross the PVM .     

Proline transport across the intestinal microvillus membrane may be regulated by membrane physical properties.

There is now abundant evidence that integral membrane protein function may be modulated by the physical properties of membrane lipids. The intestinal brush border membrane represents a membrane system highly specialized for nutrient absorption and, thus, provides an opportunity to study the interaction between integral membrane transport proteins and their lipid environment. We have previously demonstrated that alterations in this environment may modulate the function of the sodium-dependent glucose transporter in terms of its affinity for glucose. In this communication we report that membrane lipid-protein interactions are distinctly different for the proline transport proteins. Maximal transport rates for L-proline by either the neutral brush border or imino transport systems are reduced 10-fold when the surrounding membrane environment is made more fluid over the physiological range that exists along the crypt-villus axis. Furthermore, in microvillus membrane vesicles prepared from enterocytes isolated from along the crypt-villus axis a similar gradient exists in the functional activity of these transport systems. This would imply that either the functional activity of these transporters are regulated by membrane physical properties or that the synthesis and insertion of these proteins is coordinated in concert with membrane physical properties as the enterocyte migrates up the crypt-villus axis.     

Systems approach to explore components and interactions in the presynapse

The application of proteomic techniques to neuroscientific research provides an opportunity for a greater understanding of nervous system structure and function. As increasing amounts of neuroproteomic data become available, it is necessary to formulate methods to integrate these data in a meaningful way to obtain a more comprehensive picture of neuronal subcompartments. Furthermore, computational methods can be used to make biologically relevant predictions from large proteomic data sets. Here, we applied an integrated proteomics and systems biology approach to characterize the presynaptic (PRE) nerve terminal. For this, we carried out proteomic analyses of presynaptically enriched fractions, and generated a PRE literature‐based protein–protein interaction network. We combined these with other proteomic analyses to generate a core list of 117 PRE proteins, and used graph theory‐inspired algorithms to predict 92 additional components and a PRE complex containing 17 proteins. Some of these predictions were validated experimentally, indicating that the computational analyses can identify novel proteins and complexes in a subcellular compartment. We conclude that the combination of techniques (proteomics, data integration, and computational analyses) used in this study are useful in obtaining a comprehensive understanding of functional components, especially low‐abundance entities and/or interactions in the PRE nerve terminal.     

A Plasmodium falciparum host-targeting motif functions in export during blood stage infection of the rodent malarial parasite Plasmodium berghei

Plasmodium falciparum (P. falciparum) secretes hundreds of proteins--including major virulence proteins--into the host erythrocyte. In order to reach the host cytoplasm, most P. falciparum proteins contain an N terminal host-targeting (HT) motif composed of 11 amino acids. In silico analyses have suggested that the HT motif is conserved throughout the Plasmodium species but experimental evidence only exists for P. falciparum. Here, we show that in the rodent malaria parasite Plasmodium berghei (P. berghei) a reporter-like green fluorescent protein expressed by the parasite can be exported to the erythrocyte cytoplasm in a HT-specific manner. This provides the first experimental proof that the HT motif can function as a signal for protein delivery to the erythrocyte across Plasmodium species. Further, it suggests that P. berghei may serve as a model for validation of P. falciparum secretome proteins. We also show that tubovesicular membranes extend from the vacuolar parasite into the erythrocyte cytoplasm and speculate that these structures may facilitate protein export to the erythrocyte.     

Multiple mechanisms of serotonergic signal transduction

In this article we review serotonergic signal transduction mechanisms in the central and peripheral nervous systems and in a variety of target organs. The various classes of pharmacologically defined serotonergic receptors are coupled to three major effector systems: (1) adenylate cyclase; (2) phospholipase C mediated phosphoinositide (PI) hydrolysis and (3) ion channels (K+ and Ca++). Long term occupancy of serotonergic receptors also appears to induce alterations in mRNA and protein synthesis. For all three types of signal transduction there is evidence accumulating which suggests the involvement of guanine nucleotide regulatory proteins. Recent findings suggest that the distinct types of pharmacologically defined serotonergic receptors (5HT1A, 5HT1B, 5HT1c, 5HT2) may be coupled to one or more signal transduction systems. Thus, 5HT1 receptors may both activate and inhibit adenylate cyclase and increase K+-ion conductance in the hippocampus. 5HT2 receptors which activate PI hydrolysis in the brain, both open voltage-gated calcium channels and activate PI metabolism in certain smooth muscle preparations. Thus, each class of serotonergic receptor may be linked to one or more distinct biochemical transduction mechanisms. The possibility is raised that selective agonists and antagonists might be developed which have specific effects on a particular receptor-linked effector system.     

The chronic ingestion of diets containing different proteins produces marked variations in brain tryptophan levels and serotonin synthesis in the rat

Serotonin (5HT) synthesis in brain is influenced by precursor (tryptophan (TRP)) concentrations, which are modified by food ingestion. Hence, in rats, a carbohydrate meal raises brain TRP and 5HT; a protein-containing meal does not, but little attention has focused on differences among dietary proteins. Recently, single meals containing different proteins have been shown to produce marked changes in TRP and 5HT. The present studies evaluate if such differences persist when rats ingest such diets chronically. Male rats were studied that ingested diets for 9 days containing zein, wheat gluten, soy protein, casein, or α-lactalbumin (17% dry weight). Brain TRP varied up to eightfold, and 5HT synthesis fivefold among the different protein groups. TYR and LEU concentrations, and catecholamine synthesis rate in brain varied much less. The effects of dietary protein on brain TRP and 5HT previously noted after single meals thus continue undiminished when such diets are consumed chronically.     

Identification of New PNEPs Indicates a Substantial Non-PEXEL Exportome and Underpins Common Features in Plasmodium falciparum Protein Export

Malaria blood stage parasites export a large number of proteins into their host erythrocyte to change it from a container of predominantly hemoglobin optimized for the transport of oxygen into a niche for parasite propagation. To understand this process, it is crucial to know which parasite proteins are exported into the host cell. This has been aided by the PEXEL/HT sequence, a five-residue motif found in many exported proteins, leading to the prediction of the exportome. However, several PEXEL/HT negative exported proteins (PNEPs) indicate that this exportome is incomplete and it remains unknown if and how many further PNEPs exist. Here we report the identification of new PNEPs in the most virulent malaria parasite Plasmodium falciparum. This includes proteins with a domain structure deviating from previously known PNEPs and indicates that PNEPs are not a rare exception. Unexpectedly, this included members of the MSP-7 related protein (MSRP) family, suggesting unanticipated functions of MSRPs. Analyzing regions mediating export of selected new PNEPs, we show that the first 20 amino acids of PNEPs without a classical N-terminal signal peptide are sufficient to promote export of a reporter, confirming the concept that this is a shared property of all PNEPs of this type. Moreover, we took advantage of newly found soluble PNEPs to show that this type of exported protein requires unfolding to move from the parasitophorous vacuole (PV) into the host cell. This indicates that soluble PNEPs, like PEXEL/HT proteins, are exported by translocation across the PV membrane (PVM), highlighting protein translocation in the parasite periphery as a general means in protein export of malaria parasites.     

Separation and detection methods for covalent drug-protein adducts

Covalent binding of reactive metabolites of drugs to proteins has been a predominant hypothesis for the mechanism of toxicity caused by numerous drugs. The development of efficient and sensitive analytical methods for the separation, identification, quantification of drug-protein adducts have important clinical and toxicological implications. In the last few decades, continuous progress in analytical methodology has been achieved with substantial increase in the number of new, more specific and more sensitive methods for drug-protein adducts. The methods used for drug-protein adduct studies include those for separation and for subsequent detection and identification. Various chromatographic (e.g., affinity chromatography, ion-exchange chromatography, and high-performance liquid chromatography) and electrophoretic techniques [e.g., sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional SDS-PAGE, and capillary electrophoresis], used alone or in combination, offer an opportunity to purify proteins adducted by reactive drug metabolites. Conventionally, mass spectrometric (MS), nuclear magnetic resonance, and immunological and radioisotope methods are used to detect and identify protein targets for reactive drug metabolites. However, these methods are labor-intensive, and have provided very limited sequence information on the target proteins adducted, and thus the identities of the protein targets are usually unknown. Moreover, the antibody-based methods are limited by the availability, quality, and specificity of antibodies to protein adducts, which greatly hindered the identification of specific protein targets of drugs and their clinical applications. Recently, the use of powerful MS technologies (e.g., matrix-assisted laser desorption/ionization time-of-flight) together with analytical proteomics have enabled one to separate, identify unknown protein adducts, and establish the sequence context of specific adducts by offering the opportunity to search for adducts in proteomes containing a large number of proteins with protein adducts and unmodified proteins. The present review highlights the separation and detection technologies for drug-protein adducts, with an emphasis on methodology, advantages and limitations to these techniques. Furthermore, a brief discussion of the application of these techniques to individual drugs and their target proteins will be outlined.