ADP-Ribosylation Factor 1 Transiently Activates High-Affinity Adaptor Protein Complex AP-1 Binding Sites On Golgi Membranes

Association of the Golgi-specific adaptor protein complex 1 (AP-1) with the membrane is a prerequisite for clathrin coat assembly on the trans-Golgi network (TGN). The AP-1 adaptor is efficiently recruited from cytosol onto the TGN by myristoylated ADP-ribosylation factor 1 (ARF1) in the presence of the poorly hydrolyzable GTP analog guanosine 5′-O-(3-thiotriphosphate) (GTPγS). Substituting GTP for GTPγS, however, results in only poor AP-1 binding. Here we show that both AP-1 and clathrin can be recruited efficiently onto the TGN in the presence of GTP when cytosol is supplemented with ARF1. Optimal recruitment occurs at 4 μM ARF1 and with 1 mM GTP. The AP-1 recruited by ARF1·GTP is released from the Golgi membrane by treatment with 1 M Tris-HCl (pH 7) or upon reincubation at 37°C, whereas AP-1 recruited with GTPγS or by a constitutively active point mutant, ARF1(Q71L), remains membrane bound after either treatment. An incubation performed with added ARF1, GTP, and AlFn, used to block ARF GTPase-activating protein activity, results in membrane-associated AP-1, which is largely insensitive to Tris extraction. Thus, ARF1·GTP hydrolysis results in lower-affinity binding of AP-1 to the TGN. Using two-stage assays in which ARF1·GTP first primes the Golgi membrane at 37°C, followed by AP-1 binding on ice, we find that the high-affinity nucleating sites generated in the priming stage are rapidly lost. In addition, the AP-1 bound to primed Golgi membranes during a second-stage incubation on ice is fully sensitive to Tris extraction, indicating that the priming stage has passed the ARF1·GTP hydrolysis point. Thus, hydrolysis of ARF1·GTP at the priming sites can occur even before AP-1 binding. Our finding that purified clathrin-coated vesicles contain little ARF1 supports the concept that ARF1 functions in the coat assembly process rather than during the vesicle-uncoating step. We conclude that ARF1 is a limiting factor in the GTP-stimulated recruitment of AP-1 in vitro and that it appears to function in a stoichiometric manner to generate high-affinity AP-1 binding sites that have a relatively short half-life.

Evidence for a Cytoskeleton-Associated Binding Site Involved in Prolamine mRNA Localization to the Protein Bodies in Rice Endosperm Tissue1

Previous studies have demonstrated that the mRNAs encoding the prolamine and glutelin storage proteins are localized to morphologically distinct membranes of the endoplasmic reticulum (ER) complex in developing rice (Oryza sativa L.) endosperm cells. To gain insight about this mRNA localization process, we investigated the association of prolamine polysomes on the ER that delimit the prolamine protein bodies (PBs). The bulk of the prolamine polysomes were resistant to extraction by 1% Triton X-100 either alone or together with puromycin, which suggests that these translation complexes are anchored to the PB surface through a second binding site in addition to the well-characterized ribosome-binding site of the ER-localized protein translocation complex. Suppression of translation initiation shows that these polysomes are bound through the mRNA, as shown by the simultaneous increase in the amounts of ribosome-free prolamine mRNAs and decrease in prolamine polysome content associated with the membrane-stripped PB fraction. The prolamine polysome-binding activity is likely to be associated with the cytoskeleton, based on the association of actin and tubulin with the prolamine polysomes and PBs after sucrose-density centrifugation.

Ice as a matrix for IR-matrix-assisted laser desorption/ionization: mass spectra from a protein single crystal.

Lasers emitting in the ultraviolet wavelength range of 260-360 nm are almost exclusively used for matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) of macromolecules. Reports about the use of lasers emitting in the infrared first appeared in 1990/1991. In contrast to MALDI in the ultraviolet, a very limited number of reports on IR-MALDI have since been published. Several matrices have been identified for infrared MALDI yielding spectra of a quality comparable to those obtained in the ultraviolet. Water (ice) was recognized early as a potential matrix because of its strong O-H stretching mode near 3 microm. Interest in water as matrix derives primarily from the fact that it is the major constituent of most biological tissues. If functional as matrix, it might allow the in situ analysis of macromolecular constituents in frozen cell sections without extraction or exchanging the water. We present results that show that IR-MALDI of lyophilized proteins, air dried protein solutions, or protein crystals up to a molecular mass of 30 kDa is possible without the addition of any separate matrix. Samples must be frozen to retain a sufficient fraction of the water of hydration in the vacuum. The limited current sensitivity, requiring at least 10 pmol of protein for a successful analysis needs to be further improved.

Molecular mechanism of protein-retinal coupling in bacteriorhodopsin.

Bacteriorhodopsin is a membrane protein that functions as a light-driven proton pump. Each cycle of proton transport is initiated by the light-induced isomerization of retinal from the all-trans to 13-cis configuration and is completed by the protein-driven reisomerization of retinal to the all-trans configuration. Previous studies have shown that replacement of Leu-93, a residue in close proximity to the 13-methyl group of retinal, by alanine, resulted in a 250-fold increase in the time required to complete each photocycle. Here, we show that the kinetic defect in the photocycle of the Leu-93-->Ala mutant occurs at a stage after the completion of proton transport and can be overcome in the presence of strong background illumination. Time-resolved retinal-extraction experiments demonstrate the continued presence of a 13-cis intermediate in the photocycle of the Leu-93-->Ala mutant well after the completion of proton release and uptake. These results indicate that retinal reisomerization is kinetically the rate-limiting step in the photocycle of this mutant and that the slow thermal reisomerization can be bypassed by the absorption of a second photon. The effects observed for the Leu-93-->Ala mutant are not observed upon replacement of any other residue in van der Waals contact with retinal or upon replacement of Leu-93 by valine. We conclude that the contact between Leu-93 and the 13-methyl group of retinal plays a key role in controlling the rate of protein conformational changes associated with retinal reisomerization and return of the protein to the initial state.

Different routes to pure food allergens: extraction, recombinant techniques and total chemical synthesis for obtaining the plant-food pan-allergen LTP

La richiesta di allergeni puri è in continuo aumento per scopi diagnostici, come standard per metodi di rilevamento e di quantificazione, per l'immunoterapia e per lo studio a livello molecolare dei meccanismi delle reazioni allergiche, al fine di facilitare lo sviluppo di possibili cure. In questa tesi di dottorato sono descritte diverse strategie per l’ottenimento di forme pure di non-specific Lipid Transfer Proteins (nsLTPs), le quali sono state riconosciute essere rilevanti allergeni alimentari in molti frutti e verdure comunemente consumati e sono state definite come modello di veri allergeni alimentari. Una LTP potenzialmente allergenica, non nota in precedenza, è stata isolata dalle mandorle, mentre una LTP dall’allergenicità nota contenuta nelle noci è stata prodotta mediante tecniche di DNA ricombinante. Oltre a questi approcci classici, metodi per la sintesi chimica totale di proteine sono stati applicati per la prima volta alla produzione di un allergene, utilizzando Pru p 3, la LTP prototipica e principale allergene della pesca nell'area mediterranea, come modello. La sintesi chimica totale di proteinepermette di controllarne completamente la sequenza e di studiare la loro funzione a livello atomico. La sua applicazione alla produzione di allergeni costituisce perciò un importante passo avanti nel campo della ricerca sulle allergie alimentari. La proteina Pru p 3 è stata prodotta nella sua intera lunghezza e sono necessari solo due passaggi finali di deprotezione per ottenere il target nella sua forma nativa. Le condizioni sperimentali per tali deprotezioni sono state messe a punto durante la produzione dei peptidi sPru p 3 (1-37) e sPru p 3 (38-91), componenti insieme l'intera proteina. Tecniche avanzate di spettrometria di massa sono state usate per caratterizzare tutti i composti ottenuti, mentre la loro allergenicità è stata studiata attraverso test immunologici o approcci in silico.

Biomedical events extraction using the hidden vector state model

Objective: Biomedical events extraction concerns about events describing changes on the state of bio-molecules from literature. Comparing to the protein-protein interactions (PPIs) extraction task which often only involves the extraction of binary relations between two proteins, biomedical events extraction is much harder since it needs to deal with complex events consisting of embedded or hierarchical relations among proteins, events, and their textual triggers. In this paper, we propose an information extraction system based on the hidden vector state (HVS) model, called HVS-BioEvent, for biomedical events extraction, and investigate its capability in extracting complex events. Methods and material: HVS has been previously employed for extracting PPIs. In HVS-BioEvent, we propose an automated way to generate abstract annotations for HVS training and further propose novel machine learning approaches for event trigger words identification, and for biomedical events extraction from the HVS parse results. Results: Our proposed system achieves an F-score of 49.57% on the corpus used in the BioNLP'09 shared task, which is only 2.38% lower than the best performing system by UTurku in the BioNLP'09 shared task. Nevertheless, HVS-BioEvent outperforms UTurku's system on complex events extraction with 36.57% vs. 30.52% being achieved for extracting regulation events, and 40.61% vs. 38.99% for negative regulation events. Conclusions: The results suggest that the HVS model with the hierarchical hidden state structure is indeed more suitable for complex event extraction since it could naturally model embedded structural context in sentences.

Extracting protein-protein interactions from MEDLINE using the hidden vector state model

A major challenge in text mining for biomedicine is automatically extracting protein-protein interactions from the vast amount of biomedical literature. We have constructed an information extraction system based on the Hidden Vector State (HVS) model for protein-protein interactions. The HVS model can be trained using only lightly annotated data whilst simultaneously retaining sufficient ability to capture the hierarchical structure. When applied in extracting protein-protein interactions, we found that it performed better than other established statistical methods and achieved 61.5% in F-score with balanced recall and precision values. Moreover, the statistical nature of the pure data-driven HVS model makes it intrinsically robust and it can be easily adapted to other domains.

From biomedical literature to knowledge:mining protein-protein interactions

To date, more than 16 million citations of published articles in biomedical domain are available in the MEDLINE database. These articles describe the new discoveries which accompany a tremendous development in biomedicine during the last decade. It is crucial for biomedical researchers to retrieve and mine some specific knowledge from the huge quantity of published articles with high efficiency. Researchers have been engaged in the development of text mining tools to find knowledge such as protein-protein interactions, which are most relevant and useful for specific analysis tasks. This chapter provides a road map to the various information extraction methods in biomedical domain, such as protein name recognition and discovery of protein-protein interactions. Disciplines involved in analyzing and processing unstructured-text are summarized. Current work in biomedical information extracting is categorized. Challenges in the field are also presented and possible solutions are discussed.

Novel strategies to improve recombinant membrane protein production in yeast

Approximately 60% of pharmaceuticals target membrane proteins; 30% of the human genome codes for membrane proteins yet they represent less than 1% of known unique crystal structures deposited in the Protein Data Bank (PDB), with 50% of structures derived from recombinant membrane proteins having been synthesized in yeasts. G protein-coupled receptors (GPCRs) are an important class of membrane proteins that are not naturally abundant in their native membranes. Unfortunately their recombinant synthesis often suffers from low yields; moreover, function may be lost during extraction and purification from cell membranes, impeding research aimed at structural and functional determination. We therefore devised two novel strategies to improve functional yields of recombinant membrane proteins in the yeast Saccharomyces cerevisiae. We used human adenosine A2A receptor (hA2AR) as a model GPRC since it is functionally and structurally well characterised.In the first strategy, we investigated whether it is possible to provide yeast cells with a selective advantage (SA) in producing the fusion protein hA2AR-Ura3p when grown in medium lacking uracil; Ura3p is a decarboxylase that catalyzes the sixth enzymatic step in the de novo biosynthesis of pyrimidines, generating uridine monophosphate. The first transformant (H1) selected using the SA strategy gave high total yields of hA2AR-Ura3p, but low functional yields as determined by radio-ligand binding, leading to the discovery that the majority of the hA2AR-Ura3p had been internalized to the vacuole. The yeast deletion strain spt3Δ is thought to have slower translation rates and improved folding capabilities compared to wild-type cells and was therefore utilised for the SA strategy to generate a second transformant, SU1, which gave higher functional yields than H1. Subsequently hA2AR-Ura3p from H1 was solubilised with n-dodecyl-β-D-maltoside and cholesteryl hemisuccinate, which yielded functional hA2AR-Ura3p at the highest yield of all approaches used. The second strategy involved using knowledge of translational processes to improve recombinant protein synthesis to increase functional yield. Modification of existing expression vectors with an internal ribosome entry site (IRES) inserted into the 5ˊ untranslated region (UTR) of the gene encoding hA2AR was employed to circumvent regulatory controls on recombinant synthesis in the yeast host cell. The mechanisms involved were investigated through the use of yeast deletion strains and drugs that cause translation inhibition, which is known to improve protein folding and yield. The data highlight the potential to use deletion strains to increase IRES-mediated expression of recombinant hA2AR. Overall, the data presented in this thesis provide mechanistic insights into two novel strategies that can increase functional membrane protein yields in the eukaryotic microbe, S. cerevisiae.

Editorial overview:new protein production tools for structural biology

Full text: The idea of producing proteins from recombinant DNA hatched almost half a century ago. In his PhD thesis, Peter Lobban foresaw the prospect of inserting foreign DNA (from any source, including mammalian cells) into the genome of a λ phage in order to detect and recover protein products from Escherichia coli [ 1 and 2]. Only a few years later, in 1977, Herbert Boyer and his colleagues succeeded in the first ever expression of a peptide-coding gene in E. coli — they produced recombinant somatostatin [ 3] followed shortly after by human insulin. The field has advanced enormously since those early days and today recombinant proteins have become indispensable in advancing research and development in all fields of the life sciences. Structural biology, in particular, has benefitted tremendously from recombinant protein biotechnology, and an overwhelming proportion of the entries in the Protein Data Bank (PDB) are based on heterologously expressed proteins. Nonetheless, synthesizing, purifying and stabilizing recombinant proteins can still be thoroughly challenging. For example, the soluble proteome is organized to a large part into multicomponent complexes (in humans often comprising ten or more subunits), posing critical challenges for recombinant production. A third of all proteins in cells are located in the membrane, and pose special challenges that require a more bespoke approach. Recent advances may now mean that even these most recalcitrant of proteins could become tenable structural biology targets on a more routine basis. In this special issue, we examine progress in key areas that suggests this is indeed the case. Our first contribution examines the importance of understanding quality control in the host cell during recombinant protein production, and pays particular attention to the synthesis of recombinant membrane proteins. A major challenge faced by any host cell factory is the balance it must strike between its own requirements for growth and the fact that its cellular machinery has essentially been hijacked by an expression construct. In this context, Bill and von der Haar examine emerging insights into the role of the dependent pathways of translation and protein folding in defining high-yielding recombinant membrane protein production experiments for the common prokaryotic and eukaryotic expression hosts. Rather than acting as isolated entities, many membrane proteins form complexes to carry out their functions. To understand their biological mechanisms, it is essential to study the molecular structure of the intact membrane protein assemblies. Recombinant production of membrane protein complexes is still a formidable, at times insurmountable, challenge. In these cases, extraction from natural sources is the only option to prepare samples for structural and functional studies. Zorman and co-workers, in our second contribution, provide an overview of recent advances in the production of multi-subunit membrane protein complexes and highlight recent achievements in membrane protein structural research brought about by state-of-the-art near-atomic resolution cryo-electron microscopy techniques. E. coli has been the dominant host cell for recombinant protein production. Nonetheless, eukaryotic expression systems, including yeasts, insect cells and mammalian cells, are increasingly gaining prominence in the field. The yeast species Pichia pastoris, is a well-established recombinant expression system for a number of applications, including the production of a range of different membrane proteins. Byrne reviews high-resolution structures that have been determined using this methylotroph as an expression host. Although it is not yet clear why P. pastoris is suited to producing such a wide range of membrane proteins, its ease of use and the availability of diverse tools that can be readily implemented in standard bioscience laboratories mean that it is likely to become an increasingly popular option in structural biology pipelines. The contribution by Columbus concludes the membrane protein section of this volume. In her overview of post-expression strategies, Columbus surveys the four most common biochemical approaches for the structural investigation of membrane proteins. Limited proteolysis has successfully aided structure determination of membrane proteins in many cases. Deglycosylation of membrane proteins following production and purification analysis has also facilitated membrane protein structure analysis. Moreover, chemical modifications, such as lysine methylation and cysteine alkylation, have proven their worth to facilitate crystallization of membrane proteins, as well as NMR investigations of membrane protein conformational sampling. Together these approaches have greatly facilitated the structure determination of more than 40 membrane proteins to date. It may be an advantage to produce a target protein in mammalian cells, especially if authentic post-translational modifications such as glycosylation are required for proper activity. Chinese Hamster Ovary (CHO) cells and Human Embryonic Kidney (HEK) 293 cell lines have emerged as excellent hosts for heterologous production. The generation of stable cell-lines is often an aspiration for synthesizing proteins expressed in mammalian cells, in particular if high volumetric yields are to be achieved. In his report, Buessow surveys recent structures of proteins produced using stable mammalian cells and summarizes both well-established and novel approaches to facilitate stable cell-line generation for structural biology applications. The ambition of many biologists is to observe a protein's structure in the native environment of the cell itself. Until recently, this seemed to be more of a dream than a reality. Advances in nuclear magnetic resonance (NMR) spectroscopy techniques, however, have now made possible the observation of mechanistic events at the molecular level of protein structure. Smith and colleagues, in an exciting contribution, review emerging ‘in-cell NMR’ techniques that demonstrate the potential to monitor biological activities by NMR in real time in native physiological environments. A current drawback of NMR as a structure determination tool derives from size limitations of the molecule under investigation and the structures of large proteins and their complexes are therefore typically intractable by NMR. A solution to this challenge is the use of selective isotope labeling of the target protein, which results in a marked reduction of the complexity of NMR spectra and allows dynamic processes even in very large proteins and even ribosomes to be investigated. Kerfah and co-workers introduce methyl-specific isotopic labeling as a molecular tool-box, and review its applications to the solution NMR analysis of large proteins. Tyagi and Lemke next examine single-molecule FRET and crosslinking following the co-translational incorporation of non-canonical amino acids (ncAAs); the goal here is to move beyond static snap-shots of proteins and their complexes and to observe them as dynamic entities. The encoding of ncAAs through codon-suppression technology allows biomolecules to be investigated with diverse structural biology methods. In their article, Tyagi and Lemke discuss these approaches and speculate on the design of improved host organisms for ‘integrative structural biology research’. Our volume concludes with two contributions that resolve particular bottlenecks in the protein structure determination pipeline. The contribution by Crepin and co-workers introduces the concept of polyproteins in contemporary structural biology. Polyproteins are widespread in nature. They represent long polypeptide chains in which individual smaller proteins with different biological function are covalently linked together. Highly specific proteases then tailor the polyprotein into its constituent proteins. Many viruses use polyproteins as a means of organizing their proteome. The concept of polyproteins has now been exploited successfully to produce hitherto inaccessible recombinant protein complexes. For instance, by means of a self-processing synthetic polyprotein, the influenza polymerase, a high-value drug target that had remained elusive for decades, has been produced, and its high-resolution structure determined. In the contribution by Desmyter and co-workers, a further, often imposing, bottleneck in high-resolution protein structure determination is addressed: The requirement to form stable three-dimensional crystal lattices that diffract incident X-ray radiation to high resolution. Nanobodies have proven to be uniquely useful as crystallization chaperones, to coax challenging targets into suitable crystal lattices. Desmyter and co-workers review the generation of nanobodies by immunization, and highlight the application of this powerful technology to the crystallography of important protein specimens including G protein-coupled receptors (GPCRs). Recombinant protein production has come a long way since Peter Lobban's hypothesis in the late 1960s, with recombinant proteins now a dominant force in structural biology. The contributions in this volume showcase an impressive array of inventive approaches that are being developed and implemented, ever increasing the scope of recombinant technology to facilitate the determination of elusive protein structures. Powerful new methods from synthetic biology are further accelerating progress. Structure determination is now reaching into the living cell with the ultimate goal of observing functional molecular architectures in action in their native physiological environment. We anticipate that even the most challenging protein assemblies will be tackled by recombinant technology in the near future.

Biomedical relation extraction:from binary to complex

Biomedical relation extraction aims to uncover high-quality relations from life science literature with high accuracy and efficiency. Early biomedical relation extraction tasks focused on capturing binary relations, such as protein-protein interactions, which are crucial for virtually every process in a living cell. Information about these interactions provides the foundations for new therapeutic approaches. In recent years, more interests have been shifted to the extraction of complex relations such as biomolecular events. While complex relations go beyond binary relations and involve more than two arguments, they might also take another relation as an argument. In the paper, we conduct a thorough survey on the research in biomedical relation extraction. We first present a general framework for biomedical relation extraction and then discuss the approaches proposed for binary and complex relation extraction with focus on the latter since it is a much more difficult task compared to binary relation extraction. Finally, we discuss challenges that we are facing with complex relation extraction and outline possible solutions and future directions.

Detergent-free membrane protein purification

Membrane proteins are localised within a lipid bilayer; in order to purify them for functional and structural studies the first step must involve solubilising or extracting the protein from these lipids. To date this has been achieved using detergents which disrupt the bilayer and bind to the protein in the transmembrane region. However finding conditions for optimal extraction, without destabilising protein structure is time consuming and expensive. Here we present a recently-developed method using a styrene maleic acid (SMA) co-polymer instead of detergents. The SMA co-polymer extracts membrane proteins in a small disc of lipid bilayer which can be used for affinity chromatography purification, thus enabling the purification of membrane proteins while maintaining their native lipid bilayer environment.