Allergenicity and structural properties of new Cor a 1 isoallergens from hazel identified in different plant tissues.

The hazel allergen Cor a 1 is a PR-10 protein, closely related to the major birch pollen allergen Bet v 1. Hazel allergies are caused by cross-reactive IgE antibodies originally directed against Bet v 1. Despite the importance of PR-10 proteins in allergy development, their function and localization in the plant remain largely elusive. Therefore, the presence of Cor a 1 mRNA and proteins was investigated in different tissues, i.e., the female flower, immature and mature nuts, catkins, and pollen. Four yet unknown Cor a 1 isoallergens, i.e., Cor a 1.0501-1.0801, and one new Cor a 1.03 variant were discovered and characterized. Depending on the isoallergen, the occurrence and level of mRNA expression varied in different tissues, suggesting different functions. Interestingly, Cor a 1.04 previously thought to be only present in nuts, was also detected in catkins and pollen. The corresponding Cor a 1 genes were expressed in Escherichia coli. The purified proteins were analysed by CD and NMR spectroscopy. Immunoblots and ELISAs to determine their allergenic potential showed that the new proteins reacted positively with sera from patients allergic to birch, hazel and elder pollen and were recognized as novel isoallergens/variants by the WHO/IUIS Allergen Nomenclature Sub-Committee.

Molecular Mechanism of Sirtuin 1 Inhibition by Human Immunodeficiency Virus 1 Tat Protein.

Sirtuins are NAD+-dependent protein lysine deacylases implicated in metabolic regulation and aging-related dysfunctions. The nuclear isoform Sirt1 deacetylates histones and transcription factors and contributes, e.g., to brain and immune cell functions. Upon infection by human immunodeficiency virus 1 (HIV1), Sirt1 deacetylates the viral transactivator of transcription (Tat) protein to promote the expression of the viral genome. Tat, in turn, inhibits Sirt1, leading to the T cell hyperactivation associated with HIV infection. Here, we describe the molecular mechanism of Tat-dependent sirtuin inhibition. Using Tat-derived peptides and recombinant Tat protein, we mapped the inhibitory activity to Tat residues 34-59, comprising Tat core and basic regions and including the Sirt1 deacetylation site Lys50. Tat binds to the sirtuin catalytic core and inhibits Sirt1, Sirt2, and Sirt3 with comparable potencies. Biochemical data and crystal structures of sirtuin complexes with Tat peptides reveal that Tat exploits its intrinsically extended basic region for binding to the sirtuin substrate binding cleft through substrate-like ß-strand interactions, supported by charge complementarity. Tat Lys50 is positioned in the sirtuin substrate lysine pocket, although binding and inhibition do not require prior acetylation and rely on subtle differences to the binding of regular substrates. Our results provide mechanistic insights into sirtuin regulation by Tat, improving our understanding of physiological sirtuin regulation and the role of this interaction during HIV1 infection.

Allergenicity and IgE Recognition of New Dau c 1 Allergens from Carrot.

SCOPE: Carrot (Daucus carota) allergy is caused by the major carrot allergen Dau c 1, which is a mixture of several isoallergens and variants with sequence identities of >67% or >90%, respectively. However, little is known about the qualitative and quantitative composition of natural Dau c 1. METHODS AND RESULTS: Mass spectrometry of isolated natural Dau c 1 reveals the existence of several yet unknown Dau c 1-like proteins. The study expresses four Dau c 1-like proteins in Escherichia coli. Two of the purified proteins, designated Dau c 1.0501 and 1.0601, exhibit sequence identities to Dau c 1.0101 and 1.0401 between 54% and 87%. They possess allergenic potential and are accepted as new isoallergens. One protein, designated as Dau c 1-like is >50% identical with the new isoallergens but exhibits no allergenicity. Sequence and structural comparisons of this protein with the known Dau c 1 isoallergens offer relevant clues about putative structural IgE epitopes. CONCLUSION: Identification of new isoallergens and the identification of IgE epitopes may contribute to a more refined component resolved diagnosis and may lay ground for further epitope mapping and personalized targeted treatment approaches of carrot allergy in preclinical and clinical studies.

Rapid Detection of Quinolone Resistance Mutations in gyrA of Helicobacter pylori by Real-Time PCR.

The treatment of infections by the gastric pathogen Helicobacter pylori (H. pylori) has become more difficult due to increased rates of resistances against various antibiotics. Typically, atriple therapy, employing a combination of at least two antibiotics and a proton pump inhibitor, is used to cure H. pylori infections. In case of first-line therapy failure, quinolones are commonly applied in a second-line therapy. To prevent second-line treatment failures, we developed an improved method to detect the most common quinolone-resistance mutations located in the quinolone-resistance-determining region (QRDR) of the bacterial gyrA gene. Biopsy material from the gastric mucosa of infected patients was used to identify quinolone-resistant strains before the onset of drug administration. Two different wild-type and six mutant QRDR sequences were included. Melting curve analyses were performed with corresponding gyrA plasmid DNAs using a real-time polymerase chain reaction (RT-PCR) assay. By applying a combination of only two different fluorescent probes, this assay allows wild-type sequences to be unambiguously distinguished from all known mutant QRDR sequences of H. pylori. Next, the Tm values of patient DNAs were established, and the genotypes were confirmed by sequencing. Thus, quinolone-resistant H. pylori strains can be easily and quickly diagnosed before treatment, which will help to avoid the administration of ineffective drug regimes.

Optimizing the Expression of Human Dopamine Receptors in Escherichia coli.

The human dopamine receptors D2S and D3 belong to the group of G protein-coupled receptors (GPCRs) and are important drug targets. Structural analyses and development of new receptor subtype specific drugs have been impeded by low expression yields or receptor instability. Fusing the T4 lysozyme into the intracellular loop 3 improves crystallization but complicates conformational studies. To circumvent these problems, we expressed the human D2S and D3 receptors in Escherichia coli using different N- and C-terminal fusion proteins and thermostabilizing mutations. We optimized expression times and used radioligand binding assays with whole cells and membrane homogenates to evaluate KD-values and the number of receptors in the cell membrane. We show that the presence but not the type of a C-terminal fusion protein is important. Bacteria expressing receptors capable of ligand binding can be selected using FACS analysis and a fluorescently labeled ligand. Improved receptor variants can thus be generated using error-prone PCR. Subsequent analysis of clones showed the distribution of mutations over the whole gene. Repeated cycles of PCR and FACS can be applied for selecting highly expressing receptor variants with high affinity ligand binding, which in the future can be used for analytical studies.

Structures of Substrate Complexes of Foamy Viral Protease-Reverse Transcriptase.

Reverse transcriptases (RTs) use their DNA polymerase and RNase H activities to catalyze the conversion of single-stranded RNA to double-stranded DNA (dsDNA), a crucial process for the replication of retroviruses. Foamy viruses (FVs) possess a unique RT, which is a fusion with the protease (PR) domain. The mechanism of substrate binding by this enzyme has been unknown. Here, we report a crystal structure of monomeric full-length marmoset FV (MFV) PR-RT in complex with an RNA/DNA hybrid substrate. We also describe a structure of MFV PR-RT with an RNase H deletion in complex with a dsDNA substrate in which the enzyme forms an asymmetric homodimer. Cryo-electron microscopy reconstruction of the full-length MFV PR-RT-dsDNA complex confirmed the dimeric architecture. These findings represent the first structural description of nucleic acid binding by a foamy viral RT and demonstrate its ability to change its oligomeric state depending on the type of bound nucleic acid. IMPORTANCE Reverse transcriptases (RTs) are intriguing enzymes converting single-stranded RNA to dsDNA. Their activity is essential for retroviruses, which are divided into two subfamilies differing significantly in their life cycles: Orthoretrovirinae and Spumaretrovirinae. The latter family is much more ancient and comprises five genera. A unique feature of foamy viral RTs is that they contain N-terminal protease (PR) domains, which are not present in orthoretroviral enzymes. So far, no structural information for full-length foamy viral PR-RT interacting with nucleic substrates has been reported. Here, we present crystal and cryo-electron microscopy structures of marmoset foamy virus (MFV) PR-RT. These structures revealed the mode of binding of RNA/DNA and dsDNA substrates. Moreover, unexpectedly, the structures and biochemical data showed that foamy viral PR-RT can adopt both a monomeric configuration, which is observed in our structures in the presence of an RNA/DNA hybrid, and an asymmetric dimer arrangement, which we observed in the presence of dsDNA.

Allergens and their associated small molecule ligands-their dual role in sensitization.

Many allergens feature hydrophobic cavities that allow the binding of primarily hydrophobic small-molecule ligands. Ligand-binding specificities can be strict or promiscuous. Serum albumins from mammals and birds can assume multiple conformations that facilitate the binding of a broad spectrum of compounds. Pollen and plant food allergens of the family 10 of pathogenesis-related proteins bind a variety of small molecules such as glycosylated flavonoid derivatives, flavonoids, cytokinins, and steroids in vitro. However, their natural ligand binding was reported to be highly specific. Insect and mammalian lipocalins transport odorants, pheromones, catecholamines, and fatty acids with a similar level of specificity, while the food allergen ß-lactoglobulin from cow's milk is notably more promiscuous. Non-specific lipid transfer proteins from pollen and plant foods bind a wide variety of lipids, from phospholipids to fatty acids, as well as sterols and prostaglandin B2, aided by the high plasticity and flexibility displayed by their lipid-binding cavities. Ligands increase the stability of allergens to thermal and/or proteolytic degradation. They can also act as immunomodulatory agents that favor a Th2 polarization. In summary, ligand-binding allergens expose the immune system to a variety of biologically active compounds whose impact on the sensitization process has not been well studied thus far.

A Novel Isoallergen Dau c 1.0401 in Carrot: Stability, Allergenicity, and Comparison with Other Isoallergens.

SCOPE: Around 25% of food allergic persons in Central Europe suffer from carrot allergy caused by the major carrot allergen Dau c 1. Three different isoallergens, Dau c 1.01, Dau c 1.02 and Dau c 1.03 are identified. However, information about the qualitative and quantitative composition of natural (n)Dau c 1 is scarce. METHODS AND RESULTS: The new carrot allergen Dau c 1.0401 is identified on the mRNA and protein level by RT-PCR and mass spectrometry. It displays only around 60% sequence identity to the other known Dau c 1 isoallergens. NMR and CD-spectra are typical for a well-folded protein containing both α-helices and ß-strands. It showed a poor refolding capacity after incubation at 95 °C. IgE-binding is impaired in immunoblots, whereas in inhibition assays IgE binding to soluble Dau c 1.0401 is detected and it clearly provoked a response in mediator release assays. CONCLUSION: Dau c 1.0401 is a new isoallergen which contributes to the allergenicity of carrots. The absence of immunoreactivity in immobilized assays indicates that IgE binding is impaired when the protein is blotted on a solid phase. Altogether, the results point out that its allergenicity can be reduced upon carrot processing.

Noncanonical binding of Lck to CD3ε promotes TCR signaling and CAR function.

Initiation of T cell antigen receptor (TCR) signaling involves phosphorylation of CD3 cytoplasmic tails by the tyrosine kinase Lck. How Lck is recruited to the TCR to initiate signaling is not well known. We report a previously unknown binding motif in the CD3ε cytoplasmic tail that interacts in a noncanonical mode with the Lck SH3 domain: the receptor kinase (RK) motif. The RK motif is accessible only upon TCR ligation, demonstrating how ligand binding leads to Lck recruitment. Binding of the Lck SH3 domain to the exposed RK motif resulted in local augmentation of Lck activity, CD3 phosphorylation, T cell activation and thymocyte development. Introducing the RK motif into a well-characterized 41BB-based chimeric antigen receptor enhanced its antitumor function in vitro and in vivo. Our findings underscore how a better understanding of the functioning of the TCR might promote rational improvement of chimeric antigen receptor design for the treatment of cancer.

Food Processing Does Not Abolish the Allergenicity of the Carrot Allergen Dau c 1: Influence of pH, Temperature, and the Food Matrix.

SCOPE: The major carrot allergen Dau c 1 belongs to the group of pathogenesis related class 10 (PR-10) proteins and is homologous to the birch pollen allergen Bet v 1. In contrast to most other PR-10 allergens, Dau c 1 can elicit Bet v 1 independent sensitization. Although Dau c 1 is considered heat labile, allergic reactions against cooked carrots are possible. METHODS AND RESULTS: The pH and temperature stability as well as the allergenic potential before and after treatment of purified natural (n) Dau c 1 and different recombinant (r) isoallergens is investigated: rDau c 1.0104, rDau c 1.0105, rDau c 1.0201, rDau c 1.0301. All proteins except rDau c 1.0201 are able to refold at physiological pH. pH conditions around the pI (4.4-5.5) or the presence of the carrot matrix reduce the refolding capacity. Below the pI, most isoallergens are heat resistant and still able to cause mediator release, indicating allergenicity. Moreover, cooked carrot extract is still able to provoke mediator release due to remaining soluble Dau c 1. CONCLUSION: Patients allergic to carrots should avoid processed carrot containing foodstuff because heating or pH treatment do not completely abolish the allergenicity of Dau c 1.

Structural and Functional Aspects of Foamy Virus Protease-Reverse Transcriptase.

Reverse transcription describes the process of the transformation of single-stranded RNA into double-stranded DNA via an RNA/DNA duplex intermediate, and is catalyzed by the viral enzyme reverse transcriptase (RT). This event is a pivotal step in the life cycle of all retroviruses. In contrast to orthoretroviruses, the domain structure of the mature RT of foamy viruses is different, i.e., it harbors the protease (PR) domain at its N-terminus, thus being a PR-RT. This structural feature has consequences on PR activation, since the enzyme is monomeric in solution and retroviral PRs are only active as dimers. This review focuses on the structural and functional aspects of simian and prototype foamy virus reverse transcription and reverse transcriptase, as well as special features of reverse transcription that deviate from orthoretroviral processes, e.g., PR activation.

Identification of a natural ligand of the hazel allergen Cor a 1.

Hazelnut is one of the most frequent causes of food allergy. The major hazel allergen in Northern Europe is Cor a 1, which is homologous to the major birch pollen allergen Bet v 1. Both allergens belong to the pathogenesis related class PR-10. We determined the solution structure of Cor a 1.0401 from hazelnut and identified a natural ligand of the protein. The structure reveals the protein fold characteristic for PR-10 family members, which consists of a seven-stranded antiparallel ß-sheet, two short α-helices arranged in V-shape and a long C-terminal α-helix encompassing a hydrophobic pocket. However, despite the structural similarities between Cor a 1 and Bet v 1, they bind different ligands. We have shown previously that Bet v 1 binds to quercetin-3-O-sophoroside. Here, we isolated Cor a 1 from hazel pollen and identified the bound ligand, quercetin-3-O-(2"-O-ß-D-glucopyranosyl)-ß-D-galactopyranoside, by mass spectrometry and nuclear magnetic resonance spectroscopy (NMR). NMR experiments were performed to confirm binding. Remarkably, although it has been shown that PR-10 allergens show promiscuous binding behaviour in vitro, we can demonstrate that Cor a 1.0401 and Bet v 1.0101 exhibit highly selective binding for their specific ligand but not for the respective ligand of the other allergen.

Structure and nucleic acid binding properties of KOW domains 4 and 6-7 of human transcription elongation factor DSIF.

The human transcription elongation factor DSIF is highly conserved throughout all kingdoms of life and plays multiple roles during transcription. DSIF is a heterodimer, consisting of Spt4 and Spt5 that interacts with RNA polymerase II (RNAP II). DSIF binds to the elongation complex and induces promoter-proximal pausing of RNAP II. Human Spt5 consists of a NusG N-terminal (NGN) domain motif, which is followed by several KOW domains. We determined the solution structures of the human Spt5 KOW4 and the C-terminal domain by nuclear magnetic resonance spectroscopy. In addition to the typical KOW fold, the solution structure of KOW4 revealed an N-terminal four-stranded ß-sheet, previously designated as the KOW3-KOW4 linker. In solution, the C-terminus of Spt5 consists of two ß-barrel folds typical for KOW domains, designated KOW6 and KOW7. We also analysed the nucleic acid and RNAP II binding properties of the KOW domains. KOW4 variants interacted with nucleic acids, preferentially single stranded RNA, whereas no nucleic acid binding could be detected for KOW6-7. Weak binding of KOW4 to the RNAP II stalk, which is comprised of Rpb4/7, was also detected, consistent with transient interactions between Spt5 and these RNAP II subunits.

Thermotoga maritima NusG: domain interaction mediates autoinhibition and thermostability.

NusG, the only universally conserved transcription factor, comprises an N- and a C-terminal domain (NTD, CTD) that are flexibly connected and move independently in Escherichia coli and other organisms. In NusG from the hyperthermophilic bacterium Thermotoga maritima (tmNusG), however, NTD and CTD interact tightly. This closed state stabilizes the CTD, but masks the binding sites for the interaction partners Rho, NusE and RNA polymerase (RNAP), suggesting that tmNusG is autoinhibited. Furthermore, tmNusG and some other bacterial NusGs have an additional domain, DII, of unknown function. Here we demonstrate that tmNusG is indeed autoinhibited and that binding to RNAP may stabilize the open conformation. We identified two interdomain salt bridges as well as Phe336 as major determinants of the domain interaction. By successive weakening of this interaction we show that after domain dissociation tmNusG-CTD can bind to Rho and NusE, similar to the Escherichia coli NusG-CTD, indicating that these interactions are conserved in bacteria. Furthermore, we show that tmNusG-DII interacts with RNAP as well as nucleic acids with a clear preference for double stranded DNA. We suggest that tmNusG-DII supports tmNusG recruitment to the transcription elongation complex and stabilizes the tmNusG:RNAP complex, a necessary adaptation to high temperatures.

Eleventh International Foamy Virus Conference-Meeting Report.

The Eleventh International Foamy Virus Conference took place on 9-10 June 2016 at the Institut Pasteur, Paris, France. The meeting reviewed progress on foamy virus (FV) research, as well as related current topics in retrovirology. FVs are complex retroviruses that are widespread in several animal species. Several research topics on these viruses are relevant to human health: cross-species transmission and viral emergence, vectors for gene therapy, development of antiretroviral drugs, retroviral evolution and its influence on the human genome. In this article, we review the conference presentations on these viruses and highlight the major questions to be answered.

Biochemical characterization of a multi-drug resistant HIV-1 subtype AG reverse transcriptase: antagonism of AZT discrimination and excision pathways and sensitivity to RNase H inhibitors.

We analyzed a multi-drug resistant (MR) HIV-1 reverse transcriptase (RT), subcloned from a patient-derived subtype CRF02_AG, harboring 45 amino acid exchanges, amongst them four thymidine analog mutations (TAMs) relevant for high-level AZT (azidothymidine) resistance by AZTMP excision (M41L, D67N, T215Y, K219E) as well as four substitutions of the AZTTP discrimination pathway (A62V, V75I, F116Y and Q151M). In addition, K65R, known to antagonize AZTMP excision in HIV-1 subtype B was present. Although MR-RT harbored the most significant amino acid exchanges T215Y and Q151M of each pathway, it exclusively used AZTTP discrimination, indicating that the two mechanisms are mutually exclusive and that the Q151M pathway is obviously preferred since it confers resistance to most nucleoside inhibitors. A derivative was created, additionally harboring the TAM K70R and the reversions M151Q as well as R65K since K65R antagonizes excision. MR-R65K-K70R-M151Q was competent of AZTMP excision, whereas other combinations thereof with only one or two exchanges still promoted discrimination. To tackle the multi-drug resistance problem, we tested if the MR-RTs could still be inhibited by RNase H inhibitors. All MR-RTs exhibited similar sensitivity toward RNase H inhibitors belonging to different inhibitor classes, indicating the importance of developing RNase H inhibitors further as anti-HIV drugs.

The two domains of Mycobacterium tuberculosis NusG protein are dynamically independent.

Transcription elongation factor NusG from Escherichia coli couples transcription and translation. It is the only conserved transcription factor in all three kingdoms of life, playing a variety of roles in gene expression. E. coli NusG consists of two non-interacting domains. While the N-terminal domain interacts with RNA polymerase, the C-terminal domain contacts NusE (S10), or the Rho transcription termination factor. The two corresponding domains of Thermotoga maritima NusG are mutually interacting. Therefore, NusG here forms an autoinhibited state, where the binding sites to RNAP, NusE, and the Rho factor are masked. Recent functional studies showed differences between NusG from E. coli and Mycobacterium tuberculosis. In contrast to E. coli NusG, M. tuberculosis NusG is able to stimulate intrinsic termination, but is not able to bind the Rho factor. To analyze whether this has structural reasons, we determined the solution structure of the carboxyterminal domain of M. tuberculosis NusG by nuclear magnetic resonance spectroscopy. Furthermore, we modeled the wild-type full-length protein, and present evidence that the two domains of this protein do not interact in solution by NMR dynamics measurements.

Exploring RNA polymerase regulation by NMR spectroscopy.

RNA synthesis is a central process in all organisms, with RNA polymerase (RNAP) as the key enzyme. Multisubunit RNAPs are evolutionary related and are tightly regulated by a multitude of transcription factors. Although Escherichia coli RNAP has been studied extensively, only little information is available about its dynamics and transient interactions. This information, however, are crucial for the complete understanding of transcription regulation in atomic detail. To study RNAP by NMR spectroscopy we developed a highly efficient procedure for the assembly of active RNAP from separately expressed subunits that allows specific labeling of the individual constituents. We recorded [(1)H,(13)C] correlation spectra of isoleucine, leucine, and valine methyl groups of complete RNAP and the separately labeled ß' subunit within reconstituted RNAP. We further produced all RNAP subunits individually, established experiments to determine which RNAP subunit a certain regulator binds to, and identified the ß subunit to bind NusE.

AZT resistance alters enzymatic properties and creates an ATP-binding site in SFVmac reverse transcriptase.

BACKGROUND: The replication of simian foamy virus from macaques can be inhibited by the nucleoside reverse transcriptase inhibitor azidothymidine (AZT, zidovudine). Four substitutions in the protease-reverse transcriptase (PR-RT) protein (K211I, I224T, S345T, E350K) are necessary to obtain highly AZT resistant and fully replication competent virus. AZT resistance is based on the excision of the incorporated AZTMP in the presence of ATP. I224T is a polymorphism which is not essential for AZT resistance per se, but is important for regaining efficient replication of the resistant virus. RESULTS: We constructed PR-RT enzymes harboring one to four amino acid substitutions to analyze them biochemically and to determine their ability to remove the incorporated AZTMP. S345T is the only single substitution variant exhibiting significant AZTMP excision activity. Although K211I alone showed no AZTMP excision activity, excision efficiency doubled when K211I was present in combination with S345T and E350K. K211I also decreased nucleotide binding affinity and increased fidelity. NMR titration experiments revealed that a truncated version of the highly AZT resistant mt4 variant, comprising only the fingers-palm subdomains was able to bind ATP with a KD-value of ca. 7.6 mM, whereas no ATP binding could be detected in the corresponding wild type protein. We could show by NMR spectroscopy that S345T is responsible for ATP binding, probably by making a tryptophan residue accessible. CONCLUSION: Although AZT resistance in SFVmac is based on excision of the incorporated AZTMP like in HIV-1, the functions of the resistance substitutions in SFVmac PR-RT appear to be different. No mutation resulting in an aromatic residue like F/Y215 in HIV, which is responsible for π-π-stacking interactions with ATP, is present in SFVmac. Instead, S345T is responsible for creating an ATP binding site, probably by making an already existing tryptophan more accessible, which in turn can interact with ATP. This is in contrast to HIV-1 RT, in which an ATP binding site is present in the WT RT but differs from that of the AZT resistant enzyme.