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.

NMR-Based Metabolite Profiling and the Application of STOCSY toward the Quality and Authentication Assessment of European EVOOs.

Extra virgin olive oil (EVOO) possesses a high-value rank in the food industry, thus making it a common target for adulteration. Hence, several methods have been essentially made available over the years. However, the issue of authentication remains unresolved with national and food safety organizations globally struggling to regulate and control its market. Over the course of this study, the aim was to determine the origin of EVOOs suggesting a high-throughput, state-of-the-art method that could be easily adopted. A rapid, NMR-based untargeted metabolite profiling method was applied and complemented by multivariate analysis (MVA) and statistical total correlation spectroscopy (STOCSY). STOCSY is a valuable statistical tool contributing to the biomarker identification process and was employed for the first time in EVOO analysis. Market samples from three Mediterranean countries of Spain, Italy, and Greece, blended samples from these countries, as well as monocultivar samples from Greece were analyzed. The NMR spectra were collected, with the help of chemometrics acting as "fingerprints" leading to the discovery of certain chemical classes and single biomarkers that were related to the classification of the samples into groups based on their origin.

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.

Reversible fold-switching controls the functional cycle of the antitermination factor RfaH.

RfaH, member of the NusG/Spt5 family, activates virulence genes in Gram-negative pathogens. RfaH exists in two states, with its C-terminal domain (CTD) folded either as α-helical hairpin or ß-barrel. In free RfaH, the α-helical CTD interacts with, and masks the RNA polymerase binding site on, the N-terminal domain, autoinhibiting RfaH and restricting its recruitment to opsDNA sequences. Upon activation, the domains separate and the CTD refolds into the ß-barrel, which recruits a ribosome, activating translation. Using NMR spectroscopy, we show that only a complete ops-paused transcription elongation complex activates RfaH, probably via a transient encounter complex, allowing the refolded CTD to bind ribosomal protein S10. We also demonstrate that upon release from the elongation complex, the CTD transforms back into the autoinhibitory α-state, resetting the cycle. Transformation-coupled autoinhibition allows RfaH to achieve high specificity and potent activation of gene expression.

Quality and potency profile of eight recombinant isoallergens, largely mimicking total Bet v 1-specific IgE binding of birch pollen.

BACKGROUND: To date, only limited information on structure, expression levels and IgE binding of Bet v 1 variants, which are simultaneously expressed in birch pollen, is available. OBJECTIVE: To analyse and compare structure and serum IgE/IgG binding of rBet v 1 variants to Bet v 1.0101. METHODS: Recombinant Bet v 1 variants were studied with sera of 20 subjects allergic to birch pollen. Folding, aggregation and solubility of the rBet v 1 variants were analysed to attribute diverging IgE binding to either allergen structure or methodological features. IgE/IgG binding was studied with rBet v 1 in solution or adsorbed to solid phases. Allergen-mediated cross-linking of FcεRI receptors was determined by mediator release of sensitized humanized rat basophil leukaemia cells. RESULTS: All variants, except for rBet v 1.0113, were monomeric and had Bet v 1-type conformation. Serum IgE binding to variants adsorbed to solid phase was reduced to 6.6%-36.5% compared with Bet v 1.0101. In contrast, inhibition of IgE binding to Bet v 1.0101 by rBet v 1 variants ranged from 62% to 83%. Similarly, mediator release ranged from 30.7% to 55.2% for all variants and was only clearly reduced for rBet v 1.0301 (10.4%). The IgE-binding potency of rBet v 1 variants representing their native quantities in birch pollen was only slightly lower compared to extract. IgG binding to variants was between 50.9% and 134.5% compared with rBet v 1.0101 (100%). CONCLUSION AND CLINICAL RELEVANCE: Bet v 1 variants previously classified as hypoallergenic can exhibit similar functional IgE binding as Bet v 1.0101. Eight rBet v 1 variants largely reproduce total Bet v 1-specific IgE binding of birch pollen extracts. Assay format-dependent variation in IgE-binding properties needs to be considered in the development of diagnostic or therapeutic products.

Formal comment on "Systematic review of the predictors of statin adherence for the primary prevention of cardiovascular disease".

Statins have been prescribed for primary prevention of cardiovascular disease (CVD) for nearly 3 decades. Throughout this period key opinion leaders in the field have been dismayed by the high rate of non-adherence of patients to follow their statin regimen. Hope et al., [1] have addressed this issue by providing a systematic review of research on predictors of statin adherence for primary prevention of CVD. However, their review does not address the ongoing debate as to whether statin treatment is warranted for primary prevention of CVD, nor does it adequately address concerns regarding adverse effects of statins. We have therefore written a commentary which provides a broader perspective on the benefits versus harms of statin therapy. Our perspective of the literature is that non-adherence to statin treatment for primary prevention of CVD is justified because the meager benefits are more than offset by the extensive harms.

LDL-C does not cause cardiovascular disease: a comprehensive review of the current literature.

INTRODUCTION: For half a century, a high level of total cholesterol (TC) or low-density lipoprotein cholesterol (LDL-C) has been considered to be the major cause of atherosclerosis and cardiovascular disease (CVD), and statin treatment has been widely promoted for cardiovascular prevention. However, there is an increasing understanding that the mechanisms are more complicated and that statin treatment, in particular when used as primary prevention, is of doubtful benefit. Areas covered: The authors of three large reviews recently published by statin advocates have attempted to validate the current dogma. This article delineates the serious errors in these three reviews as well as other obvious falsifications of the cholesterol hypothesis. Expert commentary: Our search for falsifications of the cholesterol hypothesis confirms that it is unable to satisfy any of the Bradford Hill criteria for causality and that the conclusions of the authors of the three reviews are based on misleading statistics, exclusion of unsuccessful trials and by ignoring numerous contradictory observations.

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.

The universally-conserved transcription factor RfaH is recruited to a hairpin structure of the non-template DNA strand.

RfaH, a transcription regulator of the universally conserved NusG/Spt5 family, utilizes a unique mode of recruitment to elongating RNA polymerase to activate virulence genes. RfaH function depends critically on an ops sequence, an exemplar of a consensus pause, in the non-template DNA strand of the transcription bubble. We used structural and functional analyses to elucidate the role of ops in RfaH recruitment. Our results demonstrate that ops induces pausing to facilitate RfaH binding and establishes direct contacts with RfaH. Strikingly, the non-template DNA forms a hairpin in the RfaH:ops complex structure, flipping out a conserved T residue that is specifically recognized by RfaH. Molecular modeling and genetic evidence support the notion that ops hairpin is required for RfaH recruitment. We argue that both the sequence and the structure of the non-template strand are read out by transcription factors, expanding the repertoire of transcriptional regulators in all domains of life.

pH and Heat Resistance of the Major Celery Allergen Api g 1.

SCOPE: The major celery allergen Api g 1 is a member of the pathogenesis-related 10 class protein family. This study aims to investigate the impact of heat and pH on the native protein conformation required for Immunoglobulin E (IgE) recognition. METHODS AND RESULTS: Spectroscopic methods, MS and IgE-binding analyses are used to study the effects of pH and thermal treatment on Api g 1.0101. Heat processing results in a loss of the native protein fold via denaturation, oligomerization, and precipitation along with a subsequent reduction of IgE recognition. The induced effects and timescales are strongly pH dependent. While Api g 1 refolds partially into an IgE-binding conformation at physiological pH, acidic pH treatment leads to the formation of structurally heat-resistant, IgE-reactive oligomers. Thermal processing in the presence of a celery matrix or at pH conditions close to the isoelectric point (pI = 4.63) of Api g 1.0101 results in almost instant precipitation. CONCLUSION: This study demonstrates that Api g 1.0101 is not intrinsically susceptible to heat treatment in vitro. However, the pH and the celery matrix strongly influence the stability of Api g 1.0101 and might be the main reasons for the observed temperature lability of this important food allergen.

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.

Development, synthesis and structure-activity-relationships of inhibitors of the macrophage infectivity potentiator (Mip) proteins of Legionella pneumophila and Burkholderia pseudomallei.

The bacteria Burkholderia pseudomallei and Legionella pneumophila cause severe diseases like melioidosis and Legionnaire's disease with high mortality rates despite antibiotic treatment. Due to increasing antibiotic resistances against these and other Gram-negative bacteria, alternative therapeutical strategies are in urgent demand. As a virulence factor, the macrophage infectivity potentiator (Mip) protein constitutes an attractive target. The Mip proteins of B. pseudomallei and L. pneumophila exhibit peptidyl-prolyl cis/trans isomerase (PPIase) activity and belong to the PPIase superfamily. In previous studies, the pipecolic acid moiety proved to be a valuable scaffold for inhibiting this PPIase activity. Thus, a library of pipecolic acid derivatives was established guided by structural information and computational analyses of the binding site and possible binding modes. Stability and toxicity considerations were taken into account in iterative extensions of the library. Synthesis and evaluation of the compounds in PPIase assays resulted in highly active inhibitors. The activities can be interpreted in terms of a common binding mode obtained by docking calculations.

Lack of an association or an inverse association between low-density-lipoprotein cholesterol and mortality in the elderly: a systematic review.

OBJECTIVE: It is well known that total cholesterol becomes less of a risk factor or not at all for all-cause and cardiovascular (CV) mortality with increasing age, but as little is known as to whether low-density lipoprotein cholesterol (LDL-C), one component of total cholesterol, is associated with mortality in the elderly, we decided to investigate this issue. SETTING, PARTICIPANTS AND OUTCOME MEASURES: We sought PubMed for cohort studies, where LDL-C had been investigated as a risk factor for all-cause and/or CV mortality in individuals ≥60 years from the general population. RESULTS: We identified 19 cohort studies including 30 cohorts with a total of 68 094 elderly people, where all-cause mortality was recorded in 28 cohorts and CV mortality in 9 cohorts. Inverse association between all-cause mortality and LDL-C was seen in 16 cohorts (in 14 with statistical significance) representing 92% of the number of participants, where this association was recorded. In the rest, no association was found. In two cohorts, CV mortality was highest in the lowest LDL-C quartile and with statistical significance; in seven cohorts, no association was found. CONCLUSIONS: High LDL-C is inversely associated with mortality in most people over 60 years. This finding is inconsistent with the cholesterol hypothesis (ie, that cholesterol, particularly LDL-C, is inherently atherogenic). Since elderly people with high LDL-C live as long or longer than those with low LDL-C, our analysis provides reason to question the validity of the cholesterol hypothesis. Moreover, our study provides the rationale for a re-evaluation of guidelines recommending pharmacological reduction of LDL-C in the elderly as a component of cardiovascular disease prevention strategies.

Transcription is regulated by NusA:NusG interaction.

NusA and NusG are major regulators of bacterial transcription elongation, which act either in concert or antagonistically. Both bind to RNA polymerase (RNAP), regulating pausing as well as intrinsic and Rho-dependent termination. Here, we demonstrate by nuclear magnetic resonance spectroscopy that the Escherichia coli NusG amino-terminal domain forms a complex with the acidic repeat domain 2 (AR2) of NusA. The interaction surface of either transcription factor overlaps with the respective binding site for RNAP. We show that NusA-AR2 is able to remove NusG from RNAP. Our in vivo and in vitro results suggest that interaction between NusA and NusG could play various regulatory roles during transcription, including recruitment of NusG to RNAP, resynchronization of transcription:translation coupling, and modulation of termination efficiency.

Resonance assignment of an engineered amino-terminal domain of a major ampullate spider silk with neutralized charge cluster.

Spider dragline fibers are predominantly made out of the major ampullate spidroins (MaSp) 1 and 2. The assembly of dissolved spidroin into a stable fiber is highly controlled for example by dimerization of its amino-terminal domain (NRN) upon acidification, as well as removal of sodium chloride along the spinning duct. Clustered residues D39, E76 and E81 are the most highly conserved residues of the five-helix bundle, and they are hypothesized to be key residues for switching between a monomeric and a dimeric conformation. Simultaneous replacement of these residues by their non-titratable analogues results in variant D39N/E76Q/E81Q, which is supposed to fold into an intermediate conformation between that of the monomeric and the dimeric state at neutral pH. Here we report the resonance assignment of Latrodectus hesperus NRN variant D39N/E76Q/E81Q at pH 7.2 obtained by high-resolution triple resonance NMR spectroscopy.

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.

Determination of RNA polymerase binding surfaces of transcription factors by NMR spectroscopy.

In bacteria, RNA polymerase (RNAP), the central enzyme of transcription, is regulated by N-utilization substance (Nus) transcription factors. Several of these factors interact directly, and only transiently, with RNAP to modulate its function. As details of these interactions are largely unknown, we probed the RNAP binding surfaces of Escherichia coli (E. coli) Nus factors by nuclear magnetic resonance (NMR) spectroscopy. Perdeuterated factors with [(1)H,(13)C]-labeled methyl groups of Val, Leu, and Ile residues were titrated with protonated RNAP. After verification of this approach with the N-terminal domain (NTD) of NusG and RNAP we determined the RNAP binding site of NusE. It overlaps with the NusE interaction surface for the NusG C-terminal domain, indicating that RNAP and NusG compete for NusE and suggesting possible roles for the NusE:RNAP interaction, e.g. in antitermination and direct transcription:translation coupling. We solved the solution structure of NusA-NTD by NMR spectroscopy, identified its RNAP binding site with the same approach we used for NusG-NTD, and here present a detailed model of the NusA-NTD:RNAP:RNA complex.

Folded or Not? Tracking Bet v 1 Conformation in Recombinant Allergen Preparations.

BACKGROUND: Recombinant Bet v 1a (rBet v 1a) has been used in allergy research for more than three decades, including clinical application of so-called hypoallergens. Quantitative IgE binding to rBet v 1a depends on its native protein conformation, which might be compromised upon heterologous expression, purification, or mutational engineering of rBet v 1a. OBJECTIVE: To correlate experimental/theoretical comparisons of IgE binding of defined molar ratios of folded/misfolded recombinant Bet v 1a variants and to determine accuracy and precision of immuno- and physicochemical assays routinely used to assess the quality of recombinant allergen preparations. METHODS: rBet v 1a and its misfolded variant rBet v 1aS112P/R145P were heterologously expressed and purified from Escherichia coli. Structural integrities and oligomerisation of the recombinant allergens were evaluated by 1H-nuclear magnetic resonance (1H-NMR), circular dichroism (CD) spectroscopy, and dynamic light scattering (DLS). IgE binding of defined combinations of rBet v 1a and rBet v 1aS112P/R145P was assessed using immunoblotting (IB), enzyme-linked immunosorbent assay (ELISA) and mediator release (MR) of humanized rat basophilic leukemia cells sensitized with serum IgE of subjects allergic to birch pollen. Experimental and theoretically expected results of the analyses were compared. RESULTS: 1H-NMR spectra of rBet v 1a and rBet v 1aS112P/R145P demonstrate a native and highly disordered protein conformations, respectively. The CD spectra suggested typical alpha-helical and beta-sheet secondary structure content of rBet v 1a and random coil for rBet v 1aS112P/R145P. The hydrodynamic radii (RH) of 2.49 ± 0.39 nm (rBet v 1a) and 3.1 ± 0.56 nm (rBet v 1aS112P/R145P) showed monomeric dispersion of both allergens in solution. Serum IgE of birch pollen allergic subjects bound to 0.1% rBet v 1a in the presence of 99.9% of non-IgE binding rBet v 1aS112P/R145P. Immunoblot analysis overestimated, whereas ELISA and mediator release assay underestimated the actual quantity of IgE-reactive rBet v 1a in mixtures of rBet v 1a/rBet v 1aS112P/R145P with a molar ratio of rBet v 1a ≤ 10%. CONCLUSION: Valid conclusions on quantitative IgE binding of recombinant Bet v 1a preparations depend on the accuracy and precision of physico- and immunochemical assays with which natively folded allergen is detected.

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.