Analyse diurnaler Rhythmen in humanen post-mortalen Geweben

Rhythmic changes in environmental lighting conditions have ever been the most reliable environmental cue for life on earth. Nature has therefore selected a genetically encrypted endogenous clock very early in evolution, as it provided cells and subsequently organisms with the ability to anticipate persevering periods of light and darkness. Rhythm generation within the mammalian circadian system is achieved by clock genes and their protein products. The mammalian endogenous master clock, which synchronizes the body to environmental time, is located in the suprachiasmatic nucleus (SCN) of the hypothalamus. As an integral part of the time-coding system, the pineal gland serves the need to tune the body to the temporal environment by the rhythmic nocturnal synthesis and immediate release of the hormone melatonin. In contrast to the transcriptional regulation of melatonin synthesis in rodents, a post-translational shaping is indicated in the human pineal gland. Another important mediator of circadian time and seasonality to the body is the pituitary gland. The aim of this work was to elucidate regulation of melatonin synthesis in the human pineal gland. Furthermore, presence and regulation of clock genes in the human pineal and pituitary gland, and in the SCN were analyzed. Therefore, human tissue, taken from regular autopsies, was analyzed simultaneously for different parameters involved in melatonin biosynthesis and circadian rhythm generation. Presented data demonstrate that post-mortem brain tissue can be used to detect the remnant profile of pre-mortem adaptive changes in neuronal activity. In particular, our results give strong experimental support for the idea that transcriptional mechanisms are not dominant for the generation of rhythmic melatonin synthesis in the human pineal gland. Together with data obtained for clock genes and their protein products in the pituitary, data presented here offer 1) a new working hypothesis for post-translational regulation of melatonin biosynthesis in the human pineal gland, and 2) a novel twist in the molecular competence of clock gene proteins, achieved by nucleo-cytoplasmic shuttling in neuronal and neuroendocrine human tissue. Furthermore, in this study, oscillations in abundance of clock gene proteins were demonstrated for the first time in the human SCN.Für alle auf der Erdoberfläche lebenden Organismen bedeutet der regelmäßige Wechsel zwischen Licht und Dunkelheit eine fundamentale Veränderung ihrer Lebensbedingungen und prägt damit ihre interne zeitliche Organisation. Durch die Generierung eines endogenen, zirkadianen Rhythmus werden in Antizipation die tageszeitlichen Veränderungen der Umwelt abgebildet. Bei Säugern ist dieser zentrale endogene Oszillator der zirkadianen Rhythmogenese im Nucleus suprachiasmaticus (SCN) des Hypothalamus lokalisiert. Über einen spezifischen neuronalen Schaltkreis, dem sogenannten photoneuroendokrinen System (PNS), erfolgt die Synchronisation des SCN mit der Umwelt und die Weitergabe der Zeitinformation an den Körper. Ein essenzieller Bestandteil des PNS ist neben der Retina, dem retino-hypothalamischen Trakt und dem SCN das Pinealorgan, in welchem die Melatoninproduktion die Länge der Dunkelphase in Abhängigkeit von Signalen des SCN kodiert. Das Schlüsselenzym der Melatoninsynthese ist die Arylalkylamin N-acetyltransferase (AANAT), deren Regulation artspezifisch ist und bei Nagern auf der transkriptionalen, bei allen bislang untersuchten Ungulaten und Primaten jedoch auf der post-translationalen Ebene erfolgt. Um das Enzym vor dem Abbau durch proteasomale Proteolyse zu schützen, wird es im Zytosol durch Komplexierung mit einem 14-3-3-Dimer gebunden. Die Generierung eines endogenen Rhythmus mit einer Periodenlänge von zirka 24 h erfolgt im SCN durch die sogenannten Uhrengene (Period, Cryptochrome, Clock, Bmal1). Diese konnten nicht nur im SCN selbst, sondern auch in allen bislang untersuchten peripheren Geweben und Organen nachgewiesen werden, darunter auch im Pinealorgan sowie der Hypophyse von Nagetieren. Dabei ist die Rolle der Uhrengene in peripheren Organen, welche keinen endogenen Oszillator besitzen und damit abhängig von Signalen des SCN sind, bislang nicht eindeutig geklärt. Ziel dieser Arbeit war die Untersuchung der Mechanismen, die an der molekularen Synchronisation rhythmischer Vorgänge im humanen Pinealorgan, der Hypophyse und dem SCN beteiligt sind. Zur Beantwortung dieser Aufgabenstellung wurden humane Pinealorgane, Hypophysen und Hypothalami vom Zentrum der Rechtsmedizin aquiriert. Die Gewebe wurden in vier tageszeitliche Gruppen (Tag, Abend, Nacht, Morgen) entsprechend dem Todeszeitpunkt der Person eingeordnet. Mittels RT-PCR und real-time PCR, Enzymaktivitätsmessungen, ELISA, Immunoblotting und Immunofluoreszenz, sowie Co-Immunopräzipitation, wurden die humanen Gewebe simultan prozessiert. In der vorliegenden Arbeit wurden zum ersten Mal die Komponenten der Melatoninbiosynthese auf RNA- und Proteinebene beim Menschen untersucht. Dabei konnte gezeigt werden, dass eine post-translationale Kontrolle der AANAT vorliegt. Dabei ist das AANAT Protein im Menschen unabhängig von der Tageszeit in konstanter Menge vorhanden, und liegt zur Verhinderung der proteasomalen Proteolyse im Komplex mit einem 14-3-3-Dimer vor. Darüber hinaus konnte gezeigt werden, dass auch das letzte Enzym der Melatoninsynthese (HIOMT) Bestandteil dieses Komplexes ist, der überraschenderweise in zellularen Fortsätzen der Pinealozyten lokalisiert ist. Weiterhin konnten in der vorliegenden Arbeit zum ersten Mal Uhrengenproteine in humanem Nervengewebe nachgewiesen werden. Beim Menschen scheint, wie auch beim Schaf, keine transkriptionale Kontrolle der Uhrengene im Pinealorgan vorzuliegen. Stattdessen konnte hier eine Translokation ihrer Genprodukte in Abhängigkeit von der Tageszeit in humanen Pinealozyten und Hypophysenzellen gezeigt werden, das sogenannte ‚nucleo-cytoplasmic shuttling’. Außerdem gelang es erstmalig, Mengenunterschiede der Uhrengenproteine PER1 und CLOCK im humanen SCN im Tagesgang nachzuweisen. Sowohl bezüglich der AANAT als auch der Uhrengenproteine erfordern die in dieser Arbeit gefundenen Regulationsmechanismen keine de novo Proteinsynthese, um auf sich ändernde Umgebungsreize zu reagieren. Dadurch sind sie nicht nur zeitsparender, sondern auch weitaus energieeffizienter als die bei Nagern beobachteten Mechanismen. Es scheint, dass vor allem die schnelle und effiziente Reaktion auf sich verändernde Umweltbedingungen einen Selektionsvorteil für Ungulaten und Primaten bedeutet hat. Die in dieser Arbeit erhobenen Datensätze liefern neue Einblicke in die Regulation der Melatoninbiosynthese und der zirkadianen Rhythmik beim Menschen, und dienen dadurch dem besseren Verständnis von Regulationsmechanismen in komplexeren neuronalen und neuroendokrinen Systemen

Melatonin synthesis in the human pineal gland

Poster presentation: The mammalian pineal organ is a peripheral oscillator, depending on afferent information from the so-called master clock in the suprachiasmatic nuclei of the hypothalamus. One of the best studied outputs of the pineal gland is the small and hydrophobic molecule melatonin. In all vertebrates, melatonin is synthesized rhythmically with high levels at night, signalling the body the duration of the dark period. Changes or disruptions of melatonin rhythms in humans are related to a number of pathophysiological disorders, like Alzheimer's disease, seasonal affective disorder or the Smith-Magenis-Syndrome. To use melatonin in preventive or curative interferences with the human circadian system, a complete understanding of the generation of the rhythmic melatonin signal in the human pineal gland is essential. Melatonin biosynthesis is best studied in the rodent pineal gland, where the activity of the penultimate and rate-limiting enzyme, the arylalkylamine N-acetyltransferase (AA-NAT), is regulated on the transcriptional level, whereas the regulatory role of the ultimate enzymatic step, achieved by the hydroxyindole O-methyltransferase (HIOMT), is still under debate. In rodents, Aa-nat mRNA is about 100-fold elevated during the night in response to adrenergic stimulation of the cAMP-signalling pathway, with AA-NAT protein levels closely following this dynamics. In contrast, in all ungulates studied so far (cow, sheep), a post-transcriptional regulation of the AA-NAT is central to determine rhythmic melatonin synthesis. AA-NAT mRNA levels are constantly elevated, and lead to a constitutive up-regulation of AA-NAT protein, which is, however, rapidly degraded via proteasomal proteolysis during the day. AA-NAT proteolysis is only terminated upon the nocturnal increase in cAMP levels. Similar to ungulates, a post-transcriptional control of this enzyme seems evident in the pineal gland of the primate Macaca mulatta. Studies on the molecular basis of melatonin synthesis in the human being are sparse and almost exclusively based on phenomenological data, derived from non-invasive investigations. Yet the molecular mechanisms underlying the generation of the hormonal message of darkness can currently only be deciphered using autoptic material. We therefore analyzed in human post-mortem pineal tissue Aa-nat and Hiomt mRNA levels, AA-NAT and HIOMT enzyme activity, and melatonin levels for the first time simultaneously within tissue samples of the same specimen. Here presented data show the feasibility of this approach. Our results depict a clear diurnal rhythm in AA-NAT activity and melatonin content, despite constant values for Aa-nat and Hiomt mRNA, and for HIOMT activity. Notably, the here elevated AA-NAT activity during the dusk period does not correspond to a simultaneous elevation in melatonin content. It is currently unclear whether this finding may suggest a more important role of the ultimate enzyme in melatonin synthesis, the HIOMT, for rate-limiting the melatonin rhythm, as reported recently for the rodent pineal gland. Thus, our data support for the first time experimentally that post-transcriptional mechanisms are responsible for the generation of rhythmic melatonin synthesis in the human pineal gland

Spectroscopically orthogonal labelling to disentangle site-specific nitroxide label distributions

Funding: The authors acknowledge support by the Wellcome Trust (204821/Z/16/Z), the Leverhulme Trust (RPG-2018-397), and the EPSRC (EP/X016455/1). BEB acknowledges equipment funding by BBSRC (BB/R013780/1 and BB/T017740/1).Biomolecular applications of pulse dipolar electron paramagnetic resonance spectroscopy (PDS) are becoming increasingly valuable in structural biology. Site-directed spin labelling of proteins is routinely performed using nitroxides, with paramagnetic metal ions and other organic radicals gaining popularity as alternative spin centres. Spectroscopically orthogonal spin labelling using different types of labels potentially increases the information content available from a single sample. When analysing experimental distance distributions between two nitroxide spin labels, the site-specific rotamer information has been projected into the distance and is not readily available, and the contributions of individual labelling sites to the width of the distance distribution are not obvious from the PDS data. Here, we exploit the exquisite precision of labelling double-histidine (dHis) motifs with CuII chelate complexes. The contribution of this label to the distance distribution widths in model protein GB1 has been shown to be negligible. By combining a dHis CuII labelling site with cysteine-specific nitroxide labelling, we gather insights on the label rotamers at two distinct sites, comparing their contributions to distance distributions based on different in silico modelling approaches and structural models. From this study, it seems advisable to consider discrepancies between different in silico modelling approaches when selecting labelling sites for PDS studies.Publisher PDFPeer reviewe

Pulse dipolar electron paramagnetic resonance spectroscopy distance measurements at low nanomolar concentrations : the CuII-trityl case

Funding: To meet institutional and research funder open access requirements, any accepted manuscript arising shall be open access under a Creative Commons Attribution (CC BY) reuse licence with zero embargo. The authors acknowledge support by a University of St Andrews-University of Bonn Collaborative Research Grant, by the Wellcome Trust (204821/Z/16/Z), and by the EPSRC (EP/X016455/1). B.E.B. acknowledges equipment funding by BBSRC (BB/R013780/1 and BB/T017740/1). O.S. thanks the DFG for funding (420322655). C.A.H. thanks the DAAD for a travel and research scholarship. The authors thank the StAnD (St Andrews and Dundee) EPR grouping for long-standing support and the St Andrews mass spectrometry and proteomics facility for equipment access.Recent sensitivity enhancements in pulse dipolar EPR spectroscopy (PDS) have afforded distance measurements at submicromolar spin concentrations. This development opens the path for new science, as more biomolecular systems can be investigated at their respective physiological concentrations. Here, we demonstrate that the combination of orthogonal spin labelling using CuII ions and trityl yields a more than 3-fold sensitivity increase compared to the established CuII-nitroxide labelling strategy. Application of the recently developed variable-time RIDME method yields a further approximately 2.5-fold increase compared to the commonly used constant-time RIDME. This overall increase in sensitivity of almost an order of magnitude makes distance measurements in the range of 3 nm with protein concentrations as low as 10 nM feasible, more than two times lower than previously reported. We expect that experiments at single digit nanomolar concentrations are imminent, which has the potential to transform biological PDS applications.Publisher PDFPeer reviewe

Binding dynamics of a monomeric SSB protein to DNA : a single-molecule multi-process approach

People Programme of the European Union’s Seventh Framework Programme [REA 334496 to B.E.B.]; Leonardo da Vinci European Union Programme (to M.F.G.); Wellcome Trust [099149/Z/12/Z, 091825/Z/10/Z]. Funding for open access charge: Wellcome Trust; University of St Andrews.Single-stranded DNA binding proteins (SSBs) are ubiquitous across all organisms and are characterized by the presence of an OB (oligonucleotide/oligosaccharide/oligopeptide) binding motif to recognize single-stranded DNA (ssDNA). Despite their critical role in genome maintenance, our knowledge about SSB function is limited to proteins containing multiple OB-domains and little is known about single OB-folds interacting with ssDNA. Sulfolobus solfataricus SSB (SsoSSB) contains a single OB-fold and being the simplest representative of the SSB-family may serve as a model to understand fundamental aspects of SSB:DNA interactions. Here, we introduce a novel approach based on the competition between Förster resonance energy transfer (FRET), protein-induced fluorescence enhancement (PIFE) and quenching to dissect SsoSSB binding dynamics at single monomer resolution. We demonstrate that SsoSSB follows a monomer-by-monomer binding mechanism that involves a positive-cooperativity component between adjacent monomers. We found that SsoSSB dynamic behaviour is closer to that of Replication Protein A than to Escherichia coli SSB; a feature that might be inherited from the structural analogies of their DNA-binding domains. We hypothesize that SsoSSB has developed a balance between highdensity binding and a highly dynamic interaction with ssDNA to ensure efficient protection of the genome but still allow access to ssDNA during vital cellular processes.Publisher PDFPeer reviewe

Pulse EPR distance measurements to study multimers and multimerisation

This work was supported by funding from the European Union (Marie Curie Actions REA 334496), the Carnegie Trust (70098), the EPSRC (EP/M024660/1) and a Wellcome Trust multi-user equipment grant (099149/Z/12/Z).Pulse dipolar electron paramagnetic resonance (PD-EPR) has become a powerful tool for structural biology determining distances on the nanometre scale. Recent advances in hardware, methodology, and data analysis have widened the scope to complex biological systems. PD-EPR can be applied to systems containing lowly populated conformers or displaying large intrinsic flexibility, making them all but intractable for cryo-electron microscopy and crystallography. Membrane protein applications are of particular interest due to the intrinsic difficulties for obtaining high-resolution structures of all relevant conformations. Many drug targets involved in critical cell functions are multimeric channels or transporters. Here, common approaches for introducing spin labels for PD-EPR cause the presence of more than two electron spins per multimeric complex. This requires careful experimental design to overcome detrimental multi-spin effects and to secure sufficient distance resolution in presence of multiple distances. In addition to obtaining mere distances, PD-EPR can also provide information on multimerisation degrees allowing to study binding equilibria and to determine dissociation constants.PostprintPeer reviewe

Sparse labeling PELDOR spectroscopy on multimeric mechanosensitive membrane channels

BEB is grateful for funding from the European Union (Marie Curie Actions REA 334496). This work was supported by the EPSRC (EP/M024660/1) and the Wellcome Trust (099149/Z/12/Z). CP is a Royal Society of Edinburgh (RSE) Personal Research Fellow, funded by the Scottish Government.Pulse EPR is being applied to ever more complex biological systems comprising multiple subunits. Membrane channel proteins are of great interest as pulse EPR reports on functionally significant but distinct conformational states in a native environment without the need for crystallization. Pulse EPR, in the form of pulsed electron-electron double resonance (PELDOR), using site-directed spin labeling is most commonly employed to accurately determine distances (in the nanometer range) between different regions of the structure. However, PELDOR data analysis is more challenging in systems containing more than two spins (e.g. homo-multimers) due to distorting multi-spin effects. Without suppression of these effects much of the information contained in PELDOR data cannot be reliably retrieved. Thus, it is of utmost importance for future PELDOR applications in structural biology to develop suitable approaches that can overcome the multi-spin problem.Here, two different appro aches for suppressing multi-spin effects in PELDOR, sparse labeling of the protein (reducing the labeling efficiency f) and reducing the excitation probability of spins (λ), are compared on two distinct bacterial mechanosensitive channels. For both, the pentameric channel of large conductance (MscL) and the heptameric channel of small conductance (MscS) of E. coli, mutants containing a spin label in the cytosolic or the transmembrane region were tested. Data demonstrate that distance distributions can be significantly improved with either approach compared to the standard PELDOR measurement, and confirm that λ < 1/(n−1) is needed to sufficiently suppress multi-spin effects (with n being the number of spins in the system). A clear advantage of the sparse labeling approach is demonstrated for the cytosolic mutants due to a significantly smaller loss in sensitivity. For the transmembrane mutants, this advantage is less pronounced but still useful for MscS, but performance is inferior for MscL possibly due to structural perturbations by the bulkier diamagnetic spin label analogue.Publisher PDFPeer reviewe

A general model to optimise CuII labelling efficiency of double-histidine motifs for pulse dipolar EPR applications

JLW is supported by the BBSRC DTP Eastbio. We thank the Leverhulme Trust for support (RPG-2018-397). This work was supported by equipment funding through the Wellcome Trust (099149/Z/12/Z) and BBSRC (BB/R013780/1). We gratefully acknowledge ISSF support to the University of St Andrews from the Wellcome Trust.Electron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to studies of biomolecules underpinning health and disease by providing highly accurate and precise geometric constraints. Combining double-histidine (dH) motifs with CuII spin labels shows promise for further increasing the precision of distance measurements, and for investigating subtle conformational changes. However, non-covalent coordination-based spin labelling is vulnerable to low binding affinity. Dissociation constants of dH motifs for CuII-nitrilotriacetic acid were previously investigated via relaxation induced dipolar modulation enhancement (RIDME), and demonstrated the feasibility of exploiting the double histidine motif for EPR applications at sub-μM protein concentrations. Herein, the feasibility of using modulation depth quantitation in CuII-CuII RIDME to simultaneously estimate a pair of non-identical independent KD values in such a tetra-histidine model protein is addressed. Furthermore, we develop a general speciation model to optimise CuII labelling efficiency, in dependence of pairs of identical or disparate KD values and total CuII label concentration. We find the dissociation constant estimates are in excellent agreement with previously determined values, and empirical modulation depths support the proposed model.Publisher PDFPeer reviewe

Pulse dipolar electron paramagnetic resonance spectroscopy reveals buffer modulated cooperativity of metal templated protein dimerization

Funding: Leverhulme Trust - RPG-2018397; Biotechnology and Biological Sciences Research Council - BB/M010996/1; Engineering and Physical Sciences Research Council - EP/N509759/1; Wellcome Trust - 204821/Z/16/Z.Self-assembly of protein monomers directed by metal ion coordination constitutes a promising strategy for designing supramolecular architectures complicated by the noncovalent interaction between monomers. Herein, two pulse dipolar electron paramagnetic resonance spectroscopy (PDS) techniques, pulse electron–electron double resonance and relaxation-induced dipolar modulation enhancement, were simultaneously employed to study the CuII-templated dimerization behavior of a model protein (Streptococcus sp. group G, protein G B1 domain) in both phosphate and Tris-HCl buffers. A cooperative binding model could simultaneously fit all data and demonstrate that the cooperativity of protein dimerization across α-helical double-histidine motifs in the presence of CuII is strongly modulated by the buffer, representing a platform for highly tunable buffer-switchable templated dimerization. Hence, PDS enriches the family of techniques for monitoring binding processes, supporting the development of novel strategies for bioengineering structures and stable architectures assembled by an initial metal-templated dimerization.Publisher PDFPeer reviewe