A high-throughput immobilized bead screen for stable proteins and multi-protein complexes

We describe an in vitro colony screen to identify Escherichia coli expressing soluble proteins and stable, assembled multiprotein complexes. Proteins with an N-terminal 6His tag and C-terminal green fluorescent protein (GFP) S11 tag are fluorescently labeled in cells by complementation with a coexpressed GFP 1–10 fragment. After partial colony lysis, the fluorescent soluble proteins or complexes diffuse through a supporting filtration membrane and are captured on Talon® resin metal affinity beads immobilized in agarose. Images of the fluorescent colonies convey total expression and the level of fluorescence bound to the beads indicates how much protein is soluble. Both pieces of information can be used together when selecting clones. After the assay, colonies can be picked and propagated, eliminating the need to make replica plates. We used the method to screen a DNA fragment library of the human protein p85 and preferentially obtained clones expressing the full-length ‘breakpoint cluster region-homology' and NSH2 domains. The assay also distinguished clones expressing stable multi-protein complexes from those that are unstable due to missing subunits. Clones expressing stable, intact heterotrimeric E.coli YheNML complexes were readily identified in libraries dominated by complexes of YheML missing the N subunit

Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis

Alkaline exonuclease and single-strand DNA (ssDNA) annealing proteins (SSAPs) are key components of DNA recombination and repair systems within many prokaryotes, bacteriophages and virus-like genetic elements. The recently sequenced β-proteobacterium Laribacter hongkongensis (strain HLHK9) encodes putative homologs of alkaline exonuclease (LHK-Exo) and SSAP (LHK-Bet) proteins on its 3.17 Mb genome. Here, we report the biophysical, biochemical and structural characterization of recombinant LHK-Exo protein. LHK-Exo digests linear double-stranded DNA molecules from their 5′-termini in a highly processive manner. Exonuclease activities are optimum at pH 8.2 and essentially require Mg2+ or Mn2+ ions. 5′-phosphorylated DNA substrates are preferred over dephosphorylated ones. The crystal structure of LHK-Exo was resolved to 1.9 Å, revealing a ‘doughnut-shaped’ toroidal trimeric arrangement with a central tapered channel, analogous to that of λ-exonuclease (Exo) from bacteriophage-λ. Active sites containing two bound Mg2+ ions on each of the three monomers were located in clefts exposed to this central channel. Crystal structures of LHK-Exo in complex with dAMP and ssDNA were determined to elucidate the structural basis for substrate recognition and binding. Through structure-guided mutational analysis, we discuss the roles played by various active site residues. A conserved two metal ion catalytic mechanism is proposed for this class of alkaline exonucleases

Coordinated Destruction of Cellular Messages in Translation Complexes by the Gammaherpesvirus Host Shutoff Factor and the Mammalian Exonuclease Xrn1

Several viruses encode factors that promote host mRNA degradation to silence gene expression. It is unclear, however, whether cellular mRNA turnover pathways are engaged to assist in this process. In Kaposi's sarcoma-associated herpesvirus this phenotype is enacted by the host shutoff factor SOX. Here we show that SOX-induced mRNA turnover is a two-step process, in which mRNAs are first cleaved internally by SOX itself then degraded by the cellular exonuclease Xrn1. SOX therefore bypasses the regulatory steps of deadenylation and decapping normally required for Xrn1 activation. SOX is likely recruited to translating mRNAs, as it cosediments with translation initiation complexes and depletes polysomes. Cleaved mRNA intermediates accumulate in the 40S fraction, indicating that recognition occurs at an early stage of translation. This is the first example of a viral protein commandeering cellular mRNA turnover pathways to destroy host mRNAs, and suggests that Xrn1 is poised to deplete messages undergoing translation in mammalian cells

HF-EPR, Raman, UV/VIS Light Spectroscopic, and DFT Studies of the Ribonucleotide Reductase R2 Tyrosyl Radical from Epstein-Barr Virus

Epstein-Barr virus (EBV) belongs to the gamma subfamily of herpes viruses, among the most common pathogenic viruses in humans worldwide. The viral ribonucleotide reductase small subunit (RNR R2) is involved in the biosynthesis of nucleotides, the DNA precursors necessary for viral replication, and is an important drug target for EBV. RNR R2 generates a stable tyrosyl radical required for enzymatic turnover. Here, the electronic and magnetic properties of the tyrosyl radical in EBV R2 have been determined by X-band and high-field/high-frequency electron paramagnetic resonance (EPR) spectroscopy recorded at cryogenic temperatures. The radical exhibits an unusually low g1-tensor component at 2.0080, indicative of a positive charge in the vicinity of the radical. Consistent with these EPR results a relatively high C-O stretching frequency associated with the phenoxyl radical (at 1508 cm−1) is observed with resonance Raman spectroscopy. In contrast to mouse R2, EBV R2 does not show a deuterium shift in the resonance Raman spectra. Thus, the presence of a water molecule as a hydrogen bond donor moiety could not be identified unequivocally. Theoretical simulations showed that a water molecule placed at a distance of 2.6 Å from the tyrosyl-oxygen does not result in a detectable deuterium shift in the calculated Raman spectra. UV/VIS light spectroscopic studies with metal chelators and tyrosyl radical scavengers are consistent with a more accessible dimetal binding/radical site and a lower affinity for Fe2+ in EBV R2 than in Escherichia coli R2. Comparison with previous studies of RNR R2s from mouse, bacteria, and herpes viruses, demonstrates that finely tuned electronic properties of the radical exist within the same RNR R2 Ia class

Global mRNA Degradation during Lytic Gammaherpesvirus Infection Contributes to Establishment of Viral Latency

During a lytic gammaherpesvirus infection, host gene expression is severely restricted by the global degradation and altered 3′ end processing of mRNA. This host shutoff phenotype is orchestrated by the viral SOX protein, yet its functional significance to the viral lifecycle has not been elucidated, in part due to the multifunctional nature of SOX. Using an unbiased mutagenesis screen of the murine gammaherpesvirus 68 (MHV68) SOX homolog, we isolated a single amino acid point mutant that is selectively defective in host shutoff activity. Incorporation of this mutation into MHV68 yielded a virus with significantly reduced capacity for mRNA turnover. Unexpectedly, the MHV68 mutant showed little defect during the acute replication phase in the mouse lung. Instead, the virus exhibited attenuation at later stages of in vivo infections suggestive of defects in both trafficking and latency establishment. Specifically, mice intranasally infected with the host shutoff mutant accumulated to lower levels at 10 days post infection in the lymph nodes, failed to develop splenomegaly, and exhibited reduced viral DNA levels and a lower frequency of latently infected splenocytes. Decreased latency establishment was also observed upon infection via the intraperitoneal route. These results highlight for the first time the importance of global mRNA degradation during a gammaherpesvirus infection and link an exclusively lytic phenomenon with downstream latency establishment

Heterologous Expression of Membrane Proteins: Choosing the Appropriate Host

International audienceBACKGROUND: Membrane proteins are the targets of 50% of drugs, although they only represent 1% of total cellular proteins. The first major bottleneck on the route to their functional and structural characterisation is their overexpression; and simply choosing the right system can involve many months of trial and error. This work is intended as a guide to where to start when faced with heterologous expression of a membrane protein. METHODOLOGY/PRINCIPAL FINDINGS: The expression of 20 membrane proteins, both peripheral and integral, in three prokaryotic (E. coli, L. lactis, R. sphaeroides) and three eukaryotic (A. thaliana, N. benthamiana, Sf9 insect cells) hosts was tested. The proteins tested were of various origins (bacteria, plants and mammals), functions (transporters, receptors, enzymes) and topologies (between 0 and 13 transmembrane segments). The Gateway system was used to clone all 20 genes into appropriate vectors for the hosts to be tested. Culture conditions were optimised for each host, and specific strategies were tested, such as the use of Mistic fusions in E. coli. 17 of the 20 proteins were produced at adequate yields for functional and, in some cases, structural studies. We have formulated general recommendations to assist with choosing an appropriate system based on our observations of protein behaviour in the different hosts. CONCLUSIONS/SIGNIFICANCE: Most of the methods presented here can be quite easily implemented in other laboratories. The results highlight certain factors that should be considered when selecting an expression host. The decision aide provided should help both newcomers and old-hands to select the best system for their favourite membrane protein

Finansinspektionens roll i kontrollen av den svenska finansmarknaden - med fokus på det finansiella företaget

De senaste åren av ekonomisk instabilitet har påverkat finansmarknaden och samhället både nationellt och globalt. Uppsatsen behandlar Finansinspektionens roll i att kontrollera den finansiella marknaden med syftet att begränsa eller förhindra ekonomisk instabilitet och finanskriser. Finansinspektionen ansvarar för tillsynen över svenska finansiella företag, utländska finansiella företag med verksamhet i Sverige samt informationsgivningen i börsbolag och kontrollerar att den finansiella regleringen följs. Genom sina befogenheter och sanktionsmöjligheter kan Finansinspektionen styra och begränsa verksamheten för de som missköter sig. Finansinspektionen ansvarar även för tillståndsprövningen för de finansiella företagen. Tillståndet är en förutsättning för verksamheten i det finansiella företaget samtidigt är det effektivt kontrollverktyg för Finansinspektionen då det skapar en första kontroll vid bildandet av företaget samt ett effektivt påtryckningsmedel då en återkallelse av tillståndet utgör det mest ingripande sanktionsmedlet. Finansinspektionen ansvarar också för normgivningen dels genom utfärdandet av föreskrifter och allmänna råd dels genom att bistå regeringen med utredningar och yttranden i samband med lagstiftningsarbetet. Finansinspektionen skall även för att samråda med andra myndigheter och utländska tillsynsmyndigheter i sitt arbete och ingår i internationella kommittéer. Ramarna för Finansinspektionens befogenheter är långtgående gentemot finansiella företag, som t.ex. kreditinstitut och värdepappersinstitut, genom de särskiljande rörelsereglerna. Det är vida rambestämmelser som samtidigt utgör grund för ingripanden och sanktioner för Finansinspektionen. Det är vidare upp till inspektionen att avgöra vilken åtgärd som är lämplig i förhållande till det finansiella företagets regelöverträdelse. Genom Finansinspektionen beslut skapas praxis som är vägledande för tolkningen av den finansiella regleringen. Finansinspektionens kontroll kan för det finansiella företaget innebära en kvalitetsstämpel för företaget, vara ett incitament för regelefterlevnad, skapa vägledning för styrningen av verksamheten och ge möjlighet att allokera brister och risker i organisationen. Nackdelen för företaget torde vara kostnaden för kontrollen, det merarbete kontrollen skapar och att företagets möjlighet till självbestämmande begränsas. För samhället å andra sidan skapas systemstabilitet och konsumentskydd samt ekonomiska fördelar. En nackdel kan vara den begränsade konkurrensen som därmed skapar färre valmöjligheter för såväl andra företag och privatpersoner. Finansinspektionens huvudsakliga roll i kontrollen av finansmarknaden är att övervaka och styra de finansiella företagen och aktörerna i enlighet med den finansiella regleringen.The latest years of economic instability have influenced the financial market and the society both nationally and globally. The thesis concerns the role of the Swedish Financial Supervisory Authority, Finansinspektionen, in controlling the financial market for the purpose of limiting or preventing economic instability or financial crisis. Finansinspektionen supervises and monitors all the financial companies and other participants operating on the Swedish financial market and ensures the observance of the financial regulations. Through its legal competences and its authority to use sanctions Finansinspektionen can govern and limit the activities of the financial companies which neglect the regulations. Finansinspektionen is also responsible for the authorization of the financial companies. The permit is a prerequisite for any financial company’s business operations. Concurrently it is a withdrawal of the permit a severe intervention which consequently becomes an efficient tool of control for Finansinspektionen. The Authority is also responsible for regulating and publishing complementary rules in the form of regulations that are binding and general guidelines, as well as assisting the Government in the inquiries and issuance of opinions regarding legislation process. Finansinspektionen hold consultations with other national or European authorities within the frames of its work and is part of international committees. The so-called differential business rules are providing wide range of competences towards companies as e.g. credit institutions and securities institutions. These are general rules for the business activity, but concurrently rules for Finansinspektionen’s interventions and sanctions. The Authority is to determine what kind of intervention or sanction measures are adequate to the infringement committed by the financial company. The decisions of Finansinspektionen create guidance practice regarding the interpretations of the financial regulations. For the financial company, the control of Finansinspektionen can result in a mark of quality, an incitement of legal compliance, a guidance of governance and an allocation of risks and deficiencies in the organization of the company. The disadvantages could involve the costs of the control, both in the terms of money and time, and, most importantly, the possible circumscription of the self-governance of the company. For the society, on the other hand, the control creates stability of the financial system and consumer protection and economic advantages. The disadvantage could be the limited competition resulting in fewer options for both other companies and individuals. The main role of Finansinspektionen in controlling the financial market is to monitor and govern the financial companies and the participants of the financial market in accordance with the financial regulatio

Expression and structure-function characterisation of herpesviral proteins

In order to determine and study a protein structure, large amounts of it is needed. The easiest way to obtain a protein is to recombinantly overexpress it in the well-studied bacterium Escherichia coli. However, this expression host has one major disadvantage, overexpressed proteins might not be folded or be insoluble. Within the field of structural genomics, protein production has become one of the most challenging problems and the recombinant overexpression of viral proteins has in particular proven to be difficult. The first part of the thesis concerns the recombinant overexpression of troublesome proteins in E. coli. A method has been developed to screen for soluble overexpression in E. coli at the colony level, making it suitable for screening large gene collections. This method was used to successfully screen deletion libraries of difficult mammalian proteins as well as ORFeomes from five herpesviruses. As a result soluble expression of previously insoluble mammalian proteins was obtained as well as crystals of three proteins from two oncogenic human herpesviruses, all linked to DNA synthesis of the viral genome. The second part of the work presented concerns the structural studies of three herpesviral proteins. SOX from Kaposi’s sarcoma associated herpesvirus is involved in processing and maturation of the viral genome. Recently SOX has also been implicated in host shutoff at the mRNA level. With this structure, we propose a substrate binding site and a likely exonucleolytic mechanism. The holoenzyme ribonucleotide reductase is solely responsible for the production of deoxyribonucleotides and regulates the nucleotide pool of the cell. The small subunit, R2, has been solved from both Epstein Barr virus and KSHV. Both structures show disordered secondary structure elements in their apo-and mono metal forms, located close to the iron binding sites in similarity to the p53 induced R2 indicating that these two R2 proteins might play a similar and important role