Structure of the archaeal chemotaxis protein CheY in a domain-swapped dimeric conformation

Archaea are motile by the rotation of the archaellum. The archaellum switches between clockwise and counterclockwise rotation, and movement along a chemical gradient is possible by modulation of the switching frequency. This modulation involves the response regulator CheY and the archaellum adaptor protein CheF. In this study, two new crystal forms and protein structures of CheY are reported. In both crystal forms, CheY is arranged in a domain-swapped conformation. CheF, the protein bridging the chemotaxis signal transduction system and the motility apparatus, was recombinantly expressed, purified and subjected to X-ray data collection

A glassy contribution to the heat capacity of hcp 4^4He solids

We model the low-temperature specific heat of solid $^4$He in the hexagonal closed packed structure by invoking two-level tunneling states in addition to the usual phonon contribution of a Debye crystal for temperatures far below the Debye temperature, $T < \Theta_D/50$. By introducing a cutoff energy in the two-level tunneling density of states, we can describe the excess specific heat observed in solid hcp $^4$He, as well as the low-temperature linear term in the specific heat. Agreement is found with recent measurements of the temperature behavior of both specific heat and pressure. These results suggest the presence of a very small fraction, at the parts-per-million (ppm) level, of two-level tunneling systems in solid $^4$He, irrespective of the existence of supersolidity.Comment: 11 pages, 4 figure

microRNAs in nociceptive circuits as predictors of future clinical applications

Neuro-immune alterations in the peripheral and central nervous system play a role in the pathophysiology of chronic pain, and non-coding RNAs - and microRNAs (miRNAs) in particular - regulate both immune and neuronal processes. Specifically, miRNAs control macromolecular complexes in neurons, glia and immune cells and regulate signals used for neuro-immune communication in the pain pathway. Therefore, miRNAs may be hypothesized as critically important master switches modulating chronic pain. In particular, understanding the concerted function of miRNA in the regulation of nociception and endogenous analgesia and defining the importance of miRNAs in the circuitries and cognitive, emotional and behavioral components involved in pain is expected to shed new light on the enigmatic pathophysiology of neuropathic pain, migraine and complex regional pain syndrome. Specific miRNAs may evolve as new druggable molecular targets for pain prevention and relief. Furthermore, predisposing miRNA expression patterns and inter-individual variations and polymorphisms in miRNAs and/or their binding sites may serve as biomarkers for pain and help to predict individual risks for certain types of pain and responsiveness to analgesic drugs. miRNA-based diagnostics are expected to develop into hands-on tools that allow better patient stratification, improved mechanism-based treatment, and targeted prevention strategies for high risk individuals. \ua9 2013 Kress, H\ufcttenhofer, Landry, Kuner, Favereaux, Greenberg, Bednarik, Heppenstall, Kronenberg, Malcangio, Rittner, c\ue7eyler, Trajanoski, Mouritzen, Birklein, Sommer and Soreq

Peripheral non-viral MIDGE vector-driven delivery of β-endorphin in inflammatory pain

<p>Abstract</p> <p>Background</p> <p>Leukocytes infiltrating inflamed tissue produce and release opioid peptides such as β-endorphin, which activate opioid receptors on peripheral terminals of sensory nerves resulting in analgesia. Gene therapy is an attractive strategy to enhance continuous production of endogenous opioids. However, classical viral and plasmid vectors for gene delivery are hampered by immunogenicity, recombination, oncogene activation, anti-bacterial antibody production or changes in physiological gene expression. Non-viral, non-plasmid minimalistic, immunologically defined gene expression (MIDGE) vectors may overcome these problems as they carry only elements needed for gene transfer. Here, we investigated the effects of a nuclear localization sequence (NLS)-coupled MIDGE encoding the β-endorphin precursor proopiomelanocortin (POMC) on complete Freund's adjuvant-induced inflammatory pain in rats.</p> <p>Results</p> <p>POMC-MIDGE-NLS injected into inflamed paws appeared to be taken up by leukocytes resulting in higher concentrations of β-endorphin in these cells. POMC-MIDGE-NLS treatment reversed enhanced mechanical sensitivity compared with control MIDGE-NLS. However, both effects were moderate, not always statistically significant or directly correlated with each other. Also, the anti-hyperalgesic actions could not be increased by enhancing β-endorphin secretion or by modifying POMC-MIDGE-NLS to code for multiple copies of β-endorphin.</p> <p>Conclusion</p> <p>Although MIDGE vectors circumvent side-effects associated with classical viral and plasmid vectors, the current POMC-MIDGE-NLS did not result in reliable analgesic effectiveness in our pain model. This was possibly associated with insufficient and variable efficacy in transfection and/or β-endorphin production. Our data point at the importance of the reproducibility of gene therapy strategies for the control of chronic pain.</p

Mycobacteria Attenuate Nociceptive Responses by Formyl Peptide Receptor Triggered Opioid Peptide Release from Neutrophils

In inflammation, pain is regulated by a balance of pro- and analgesic mediators. Analgesic mediators include opioid peptides which are secreted by neutrophils at the site of inflammation, leading to activation of opioid receptors on peripheral sensory neurons. In humans, local opioids and opioid peptides significantly downregulate postoperative as well as arthritic pain. In rats, inflammatory pain is induced by intraplantar injection of heat inactivated Mycobacterium butyricum, a component of complete Freund's adjuvant. We hypothesized that mycobacterially derived formyl peptide receptor (FPR) and/or toll like receptor (TLR) agonists could activate neutrophils, leading to opioid peptide release and inhibition of inflammatory pain. In complete Freund's adjuvant-induced inflammation, thermal and mechanical nociceptive thresholds of the paw were quantified (Hargreaves and Randall-Selitto methods, respectively). Withdrawal time to heat was decreased following systemic neutrophil depletion as well as local injection of opioid receptor antagonists or anti-opioid peptide (i.e. Met-enkephalin, β-endorphin) antibodies indicating an increase in pain. In vitro, opioid peptide release from human and rat neutrophils was measured by radioimmunoassay. Met-enkephalin release was triggered by Mycobacterium butyricum and formyl peptides but not by TLR-2 or TLR-4 agonists. Mycobacterium butyricum induced a rise in intracellular calcium as determined by FURA loading and calcium imaging. Opioid peptide release was blocked by intracellular calcium chelation as well as phosphoinositol-3-kinase inhibition. The FPR antagonists Boc-FLFLF and cyclosporine H reduced opioid peptide release in vitro and increased inflammatory pain in vivo while TLR 2/4 did not appear to be involved. In summary, mycobacteria activate FPR on neutrophils, resulting in tonic secretion of opioid peptides from neutrophils and in a decrease in inflammatory pain. Future therapeutic strategies may aim at selective FPR agonists to boost endogenous analgesia

Linear epitopes of Paracoccidioides brasiliensis and Other Fungal Agents of Human Systemic Mycoses As vaccine Candidates

Dimorphic fungi are agents of systemic mycoses associated with significant morbidity and frequent lethality in the Americas. Among the pathogenic species are Paracoccidioides brasiliensis and Paracoccidioides lutzii, which predominate in South AmericaHistoplasma capsulatum, Coccidioides posadasii, and Coccidioides immitis, and the Sporothrix spp. complex are other important pathogens. Associated with dimorphic fungi other important infections are caused by yeast such as Candida spp. and Cryptococcus spp. or mold such as Aspergillus spp., which are also fungal agents of deadly infections. Nowadays, the actual tendency of therapy is the development of a pan-fungal vaccine. This is, however, not easy because of the complexity of eukaryotic cells and the particularities of different species and isolates. Albeit there are several experimental vaccines being studied, we will focus mainly on peptide vaccines or epitopes of T-cell receptors inducing protective fungal responses. These peptides can be carried by antibody inducing beta-( 1,3)-glucan oligo or polysaccharides, or be mixed with them for administration. The present review discusses the efficacy of linear peptide epitopes in the context of antifungal immunization and vaccine proposition.FAPESPCAPESUniv Fed Sao Paulo, Dept Microbiol Immunol & Parasitol, Sao Paulo, BrazilUniv Fed Sao Paulo, Inst Biomed Sci, Dept Microbiol, Sao Paulo, BrazilUniv Fed Sao Paulo, Lab Med Mycol IMTSP HCFMUSP LIM53, Sao Paulo, BrazilUniv Fed Sao Paulo, Dept Microbiol Immunol & Parasitol, Sao Paulo, BrazilUniv Fed Sao Paulo, Inst Biomed Sci, Dept Microbiol, Sao Paulo, BrazilUniv Fed Sao Paulo, Lab Med Mycol IMTSP HCFMUSP LIM53, Sao Paulo, BrazilFAPESP: 2016/08730-6FAPESP: 2010/51423-0Web of Scienc

Engineering animal fatty acid synthase towards polyketide synthases

Naturstoffe sind wertvolle Quellen für biologisch aktive Verbindungen, die als Arzneimittel verwendet werden können. Dabei basiert die Produktion rein auf Biokatalyse, die oft von sogenannten Megaenzymen durchgeführt wird. Ein wichtiger biosynthetischer Weg ist der Acetatweg, der die Polyketid- und Fettsäuresynthese einschließt, und eine der größten Klassen chemisch unterschiedlicher Naturstoffe hervorbringt. Diese haben aufgrund ihrer antibakteriellen, antimykotischen, anthelmintischen, immunsuppressiven und antitumoralen Eigenschaften medizinische Relevanz. Aufgrund der hohen strukturellen und funktionellen Ähnlichkeit zwischen Polyketidsynthasen und Typ I Tierfettsäure-Synthasen (FASs) kann die FAS als Paradigma für die gesamte Klasse von multifunktionellen Enzymen dienen. Um das biosynthetische Potential von FASs voll ausschöpfen zu können, ist ein guter Zugang zum Enzym von wesentlicher Bedeutung. In dieser Hinsicht bleibt Escherichia coli ein unangefochtener heterologer Wirt aufgrund niedriger Kulturkosten, schneller Mutagenesezyklen und relativ einfacher Handhabung. Überraschenderweise wurde noch keine effiziente Expressionsstrategie für eine Tier-FAS in E. coli berichtet, da sich herausgestellt hat, dass die einzige, publizierte Strategie nicht reproduzierbar ist. Wir begannen unsere Analyse mit der Suche nach einem geeigneten FAS-Homolog, das unsere Anforderungen an hohe Proteinqualität, ausreichende Ausbeute und garantierte Funktionalität erfüllt. Nach einem umfangreichen Screening verschiedener Varianten, Kultivierungsbedingungen und Koexpressionsstrategien identifizierten wir die murine FAS (mFAS) als unser Protein der Wahl. Unsere etablierte Aufreinigungsstrategie, die auf der Verwendung von kurzen künstlichen Peptidsequenzen an beiden Termini basiert, gewährleistete einen reproduzierbaren und ausreichenden Zugang zum Protein in ausgezeichneter Qualität. Das Enzym wurde weiter biochemisch charakterisiert, einschließlich einer enzymkinetischen Analyse der Fettsäuresynthese und einer Untersuchung, ob verschiedene Acyl-CoA Substrate als Startermoleküle fungieren können. Damit wurde unser Repertoire von handhabbaren Megaenzymen um die mFAS erweitert, was den Weg zu einer Nutzung der katalytischen Effizienz in Bezug auf mikrobielle Synthese von maßgeschneiderten Verbindungen ebnet. Mit dem Fokus, das Verständnis für die Funktionsweise solcher Megaenzyme zu vertiefen, anstatt entsprechende Biosyntheseprodukte zu analysieren, haben wir uns mit der Frage beschäftigt, ob mFAS selbst in eine PKSs umgewandelt werden kann oder ob Eigenschaften von mFAS für die Entwicklung von PKSs genutzt werden können. Dieser Ansatz wurde auf drei Komplexitätsebenen von der Funktion einzelner Domänen über die Organisation von Domänen zu Modulen bis hin zum Zusammenspiel zweier Module in bimodularen Konstrukten durchgeführt. Die Fettsäuresynthese beginnt mit der Beladung der FAS mit Acyleinheiten, die von einer Domäne namens Malonyl-/Acetyltransferase (MAT) durchgeführt wird. Diese Domäne wurde aufgrund ihrer wichtigen Rolle bei der Auswahl der Substrate, die das Kohlenstoffgerüst des finalen Produktes mitbestimmen, detailiert charakterisiert. Unsere Analyse umfasste strukturelle und funktionelle Aspekte, wie z. B. das Lösen von Kristallstrukturen in zwei unterschiedlichen Acyl-gebundenen Zuständen und eine enzymkinetische Beschreibung der Hydrolyse- und Transacylierungsreaktionen für zwölf exemplarische CoA-Ester. Zu diesem Zweck wurde ein kontinuierlicher fluorometrischer Assay etabliert, der unter Verwendung der α-Ketoglutaratdehydrogenase als ein gekoppeltes Enzym freies Coenzym A in Nicotinamidadenindinukleotid umwandelt. Diese Daten enthüllten eine ausgedehnte Substratambiguität der MAT-Domäne, die zuvor nicht in diesem Ausmaß beschrieben worden war. Weiterhin konnten wir durch Expression der Domäne in verschiedenen strukturellen Anordnungen (Robustheit) und durch Veränderung der Substratspezifität innerhalb einer Mutagenesestudie (Plastizität) zeigen, dass MAT beide Kriterien für Proteinevolvabilität erfüllt. Daher sind wir davon überzeugt, dass diese Domäne als ein vielseitiges Werkzeug für PKS-Engineering in potenziellen FAS/PKS Hybridsystemen eingesetzt werden kann. Auf der höheren Komplexitätsebene untersuchten wir die architektonische Variabilität der mFAS-Faltungseinheit, was eine fundamentale Basis für eine breitere biosynthetische Anwendung darstellt. Wir konnten alle vier Modultypen rekonstruieren, die in typischen modularen PKSs auftreten, was einen hohen Grad an Modularität innerhalb der Faltungseinheit bestätigt. Nicht nur die strukturelle, sondern auch die funktionelle Integrität dieser Module wurde unter Verwendung der Triacetsäurelactonbildung und der Ketoreduktaseaktivität validiert. Insbesondere letztere Analyse ermöglichte es, Effekte des Engineerings innerhalb des prozessierenden Teils durch entsprechende enzymkinetische Parameter zu quantifizieren. Danach haben wir unseren Fokus über ein einzelnes Modul hinaus erweitert und die mFAS-Faltungseinheit zur Konstruktion von bis zu 380 kDa großen bimodularen Konstrukten verwendet. Bei diesem Ansatz wurde eine N-terminale Ladedidomäne angehängt, die eine zusätzliche MAT- und Acyl-Carrier-Protein-Domäne enthielt. Zwei Konstrukte konnten in exzellenter Qualität exprimiert und gereinigt werden, mit deren Hilfe der Einfluss einer veränderten Gesamtarchitektur auf die Fettsäuresynthese untersucht wurde. Durch einen Vergleich mit entsprechenden Kontrollen konnte in der Tat ein funktioneller Effekt des zusätzlichen Lademoduls nachgewiesen werden. Diese Konstrukte erlauben in der Zukunft eine umfassende Analyse des zugrunde liegenden molekularen Mechanismus und dienen daher als potentielles Modellsystem zur Untersuchung des Übergangs von einer iterativen zu einer vektoriellen Polyketidsynthese in vitro.Natural products are valuable sources for biologically active compounds, which can be utilized as pharmaceuticals. Thereby, the synthesis is based purely on biosynthetic grounds often conducted by so-called megaenzymes. One major biosynthetic pathway is the acetate pathway including polyketide and fatty acid synthesis, which encompass one of the largest classes of chemically diverse natural products. These have medicinal relevance due to their antibacterial, antifungal, anthelmintic, immunosuppressive and antitumor properties. Due to the high structural and functional similarity between polyketide synthases and type I animal fatty acid synthases (FASs), FAS can serve as a paradigm for the whole class of multifunctional enzymes. To fully exploit the biosynthetic potential of FASs, a good access to the enzyme is of essential importance. In this regard, Escherichia coli remains an unchallenged heterologous host due to low culturing costs, particularly fast mutagenesis cycles and relatively easy handling. Surprisingly, no sufficient expression strategy for an animal FAS in E. coli has yet been reported, as it turned out that the only approach was not reproducible. We commenced our analysis with searching for an appropriate FAS homolog that fulfills our requirements of high protein quality, sufficient yield and ensured functionality. After extensive screening of different variants, culturing conditions and co-expression strategies, we identified the murine FAS (mFAS) as our protein of choice. The established purification strategy using tags at both termini led to a reproducible and sufficient access to the protein in excellent quality. The enzyme was further biochemically characterized including an enzyme kinetic investigation of fatty acid synthesis and an examination whether different acyl-CoA substrates can serve as priming units. This adds mFAS to our repertoire of manageable megaenzymes paving the way to exploit the catalytic efficiency in regards of microbial custom-compound synthesis. With a strong focus on deepening our understanding of the working mode of such megaenzymes, rather than analyzing respective biosynthetic products, we have addressed the question whether mFAS itself can be engineered towards PKSs or whether properties of mFAS can be exploited to engineer PKSs. This approach was conducted on three levels of complexity from function of individual domains via organization of domains to form modules to the interplay of two modules in bimodular constructs. Fatty acid synthesis begins with the loading of acyl moieties onto the FAS, which is conducted by a domain called malonyl-/acetyltransferase (MAT). This domain was in-depth characterized due to its important role of choosing the substrates that are built in the final compound. Our analysis comprised structural and functional aspects providing crystal structures of two different acyl-bound states and kinetic parameters for the hydrolysis and transacylation reaction using twelve exemplary CoA-esters. For this purpose, we have successfully established a continuous fluorometric assay using the α-ketoglutarate dehydrogenase as a coupled enzyme, which converts the liberated coenzyme A into Nicotinamide adenine dinucleotide. These data revealed an extensive substrate ambiguity of the MAT domain, which had not been reported to that extent before. Further, we could demonstrate that the fold fulfills both criteria for the evolvability of an enzyme by expressing MAT in different structural arrangements (robustness) and by altering the substrate ambiguity within a mutagenesis study (plasticity). Taken these aspects together, we are persuaded that the MAT domain can serve as a versatile tool for PKSs engineering in potential FAS/PKS hybrid systems. On the higher level of complexity, we investigated the architectural variability of the mFAS fold, which constitutes a fundamental basis for a broader biosynthetic application. We could rebuild all four module types occurring in typical modular PKSs confirming a high degree of modularity within the fold. Not only structural, but also functional integrity of these modules was validated by using triacetic acid lactone formation and ketoreductase activity. Especially the latter analysis, made it possible to quantify effects of the engineering within the processing part by respective enzyme kinetic parameters. Expanding our focus beyond a singular module, we have utilized the mFAS fold for designing up to 380 kDa large bimodular constructs. In this approach, a loading didomain was attached N-terminally containing an additional MAT and acyl carrier protein (ACP) domain. Two constructs could be expressed and purified in excellent quality to investigate the influence of an altered overall architecture on fatty acid synthesis. By comparison with appropriate controls, a functional effect of the additional loading module could indeed be proven in the bimodular systems. Those constructs allow a comprehensive analysis of the underlying molecular mechanism in the future and serve as a potential model system to study the transition from iterative to vectorial polyketide synthesis in vitro

CXCL10 Controls Inflammatory Pain via Opioid Peptide- Containing Macrophages in Electroacupuncture

Acupuncture is widely used for pain treatment in patients with osteoarthritis or low back pain, but molecular mechanisms remain largely enigmatic. In the early phase of inflammation neutrophilic chemokines direct opioid-containing neutrophils in the inflamed tissue and stimulate opioid peptide release and antinociception. In this study the molecular pathway and neuroimmune connections in complete Freund's adjuvant (CFA)-induced hind paw inflammation and electroacupuncture for peripheral pain control were analyzed. Free moving Wistar rats with hind paw inflammation were treated twice with electroacupuncture at GB30 (Huan Tiao - gall bladder meridian) (day 0 and 1) and analyzed for mechanical and thermal nociceptive thresholds. The cytokine profiles as well as the expression of opioid peptides were quantified in the inflamed paw. Electroacupuncture elicited long-term antinociception blocked by local injection of anti-opioid peptide antibodies (beta-endorphin, met-enkephalin, dynorphin A). The treatment altered the cytokine profile towards an anti-inflammatory pattern but augmented interferon (IFN)-gamma and the chemokine CXCL10 (IP-10: interferon gamma-inducible protein) protein and mRNA expression with concomitant increased numbers of opioid peptide-containing CXCR3+ macrophages. In rats with CFA hind paw inflammation without acupuncture repeated injection of CXCL10 triggered opioid-mediated antinociception and increase opioid-containing macrophages. Conversely, neutralization of CXCL10 time-dependently decreased electroacupuncture-induced antinociception and the number of infiltrating opioid peptide-expressing CXCR3+ macrophages. In summary, we describe a novel function of the chemokine CXCL10 - as a regulator for an increase of opioid-containing macrophages and antinociceptive mediator in inflammatory pain and as a key chemokine regulated by electroacupuncture

Pain Control by Targeting Oxidized Phospholipids: Functions, Mechanisms, Perspectives

Within the lipidome oxidized phospholipids (OxPL) form a class of chemically highly reactive metabolites. OxPL are acutely produced in inflamed tissue and act as endogenous, proalgesic (pain-inducing) metabolites. They excite sensory, nociceptive neurons by activating transient receptor potential ion channels, specifically TRPA1 and TRPV1. Under inflammatory conditions, OxPL-mediated receptor potentials even potentiate the action potential firing rate of nociceptors. Targeting OxPL with D-4F, an apolipoprotein A-I mimetic peptide or antibodies like E06, specifically binding oxidized headgroups of phospholipids, can be used to control acute, inflammatory pain syndromes, at least in rodents. With a focus on proalgesic specificities of OxPL, this article discusses, how targeting defined substances of the epilipidome can contribute to mechanism-based therapies against primary and secondary chronic inflammatory or possibly also neuropathic pain