Dissertations / Theses on the topic 'M2 protein'

The influenza A M2 protein and, in particular, the proposed transmembrane domain, has been implicated in viral infectivity at two stges in the replicative cycle: viral uncoating and assembly. An identical function has been proposed for the protein at both points of interest, that is, that M2 acts as a proton channel. In order to work, this hypothesis assumes that M2 possesses a transmembrane domain, presumably in a α-helical conformation, that this domain orientates in a prescribed manner across the bilayer and that, indeed, M2 is able to translocate protons across the aforementioned bilayer. This thesis examines these assumptions experimentally using a synthetic, 25 amino acid peptide representing the proposed transmembrane domain of M2. The work described herein may be divided into three main areas, each employing a separate biophysical technique. Circular dichroism was employed to assign an α-helical secondary structure to the M2 peptide. Neutron diffraction orientated this region precisely in the bilayer. Electrophysiological techniques observed directly, for the first time in viruses, proton translocation. The effects of amantadine, the only drug prescribed for use against influenza A infections, in each of these structural asnd functional investigations has also been recorded, providing revealing insights into the drug's efficacy. M2 has structural analogs in other enveloped viruses and work such as that reported in this thesis may reveal a common pathway of viral infectivity for groups of enveloped viruses, therefore allowing the possibility of broad-spectrum drug therapies.

Atherosclerosis is an inflammatory disease in which an accumulation of fatty acids and cholesterol occurs to form a plaque in small and large arteries. Monocyte polarization to classic M1 macrophages or alternative M2 macrophages is an important area of research that can determine the severity of disease progression. BMP-7 is a key growth factor responsible for directing differentiation of mesenchymal stem cells into brown fat cells, suggesting a role of BMP-7 in cellular plasticity; however, its role in monocyte polarization is yet to be revealed. In the current study, we hypothesize that monocyte treatment with BMP-7 will significantly result in increased polarization of monocytes into M2 macrophages and increased expression of anti-inflammatory cytokines. To that effect, we have established a stress induced cell culture system with monocytes (THP-1 cells) and apoptotic conditioned medium (ACM), simulating injury, to understand the effects of BMP-7 on M2 macrophage polarization from monocytes. Our data demonstrates that the BMP type 2 receptor (BMPR2) is found on monocytes and its activation is significantly (p<0.05) increased in both monocytes and M2 macrophages following treatment with BMP-7. Furthermore, a significant (p<0.05) increase of M2 macrophages in the BMP-7 treated group was shown following immunostaining with CD206 and arginase-1, two M2 macrophage markers, whereas a significant (p<0.05) decrease of iNOS expression, an M1 macrophage marker, was shown. Moreover, treatment with BMP-7 resulted in significantly (p<0.05) increased expression of IL-10 and IL-1ra, two anti-inflammatory cytokines, but significantly (p<0.05) decreased levels of the pro-inflammatory cytokines, MCP-1, IL-6 and TNF-?. We also hypothesize that polarization of monocytes to M2 macrophages occurs through activation of SMAD1/5/8 and PI3K-Akt-mTOR pathways. Upon BMP-7 binding to its receptor, BMPR2, activation of SMAD1/5/8 occurs which then activates the p85 subunit of PI3K resulting in downstream activation of Akt and mTOR. Our data shows that following treatment with BMP-7, expression of p-SMAD1/5/8, p-PI3K, p-Akt and p-mTOR is significantly (p<0.05) increased compared to controls whereas p-PTEN, an inhibitor of the PI3K pathway, is significantly (p<0.05) decreased in the BMP-7 treated group compared to controls. In conclusion, our data reveals that BMP-7 polarizes monocytes into M2 macrophages and it achieves this through activation of the PI3K-Akt-mTOR pathway, which will have significant applications for atherosclerosis treatment.
M.S.
Masters
Molecular Biology and Microbiology
Medicine
Biotechnology

The M2-1 protein of the important pathogen human respiratory syncytial virus is a transcription antiterminator that is essential for viral gene expression. We present the X-ray crystal structure of full-length M2-1 protein in its native tetrameric form at a resolution of 2.52 Å. The structure reveals M2-1 forms a disk-like assembly with tetramerisation driven by a long helix forming a four-helix bundle at its center, further stabilised by contact between the zinc finger and adjacent protomers. The tetramerisation helix is linked to a core domain responsible for RNA binding activity by a flexible loop on which lie two functionally critical serine residues, 58 and 61, that are phosphorylated during infection. The identity of these residues was confirmed by mass spectrometric analysis of M2-1 protein expressed in baculovirus-assisted insect cell culture. The crystal structure of a phosphomimetic M2-1 variant, S58DS61D revealed altered charge density surrounding this flexible loop, although loop position was unaffected. Structure guided mutagenesis identified residues that contributed to RNA binding and antitermination activity, revealing a strong correlation between these two activities, and further defining the role of phosphorylation in M2-1 antitermination activity. The data presented here identify surfaces critical for M2-1 function that may be targeted by anti-viral compounds, and allow us to propose a possible model for M2-1 function during respiratory syncytial virus transcription.

This thesis describes the expression of three human seven transmembrane receptors: the M2 Muscarinic; H1 Histamine and 5HT2A Serotonin receptors, in the baculovirus/insect cell expression system. Purification trials werre conducted on the M2 Muscarinic and H1 Histamine receptors. Preliminary crystallisation attempts were made with the H1 receptor.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2017.
Cataloged from PDF version of thesis.
Includes bibliographical references.
Solid-state nuclear magnetic resonance (SSNMR) has been frequently used to elucidate the structure and dynamics of membrane proteins and fibrils that are difficult to characterize by Xray crystallography or solution NMR. This thesis focuses on the structure determination and the proton conduction mechanism of the full-length matrix protein 2 (M2) of influenza A virus. The M2 membrane protein can be separated into three domains: an N-terminal ectodomain (1-2 1), an cc-helical transmembrane domain (TM) (22-46) connected to an amphipathic helix (AH) and a Cterminal cytoplasmic tail (63-97). The TM domain of M2 is responsible for proton conduction ant the ectodomain has been the target for vaccine development. The cytoplasmic tail has been implicated in M2 interaction with other viral proteins from mutagenesis studies. Given the importance of both N- and C-termini, it is essential to determine the structure and the dynamics of M2FL. Furthermore, we are interested in how the cytoplasmic tail affects proton conduction and the interaction of the anti-viral drug amantadine with M2 in the presence of the C-terminus. Using uniformly ¹³C, ¹⁵N-labeled M2FL, our water-selected 2D ¹³C-¹³C correlation experiment indicated that N- and C- termini are on the surface of the lipid bilayer moreover combining with chemical shift prediction, we determined that these two domains are mostly disordered. Deleting the ectodomain of M2FL (M2(21-97)) proved that a small [beta]-strand is located at the N-terminus only in the DMPC-bound state. The M2 conformation is found to be cholesterol-dependent since [beta]-strand is not found in cholesterol-rich membranes. M2(21-97) shows cationic histidine at higher pH, in contrast to M2TM, indicating that the cytoplasmic tail shifts the His37 pKa equilibria. Quantification of the ¹⁵N intensities revealed two pKa's as opposed to of four in M2TM suggesting cooperative proton binding. A possible explanation is that the large number of positively charged residues in the cytoplasmic tail facilitates proton conduction. The cytoplasmic tail was also found to restore drug-binding as amantadine no longer binds to M2(21-61) a in virus-mimetic membrane. These results have extended our understanding of the influence of the cytoplasmic domain on the structure and proton conduction of M2.
by Shu-Yu Liao.
Ph. D.

Thesis: Ph. D., Massachusetts Institute of Technology, Department of Chemistry, 2014.
Cataloged from PDF version of thesis. Vita.
Includes bibliographical references.
Determination of the 3D structure of membrane proteins is a frontier that is rapidly being explored due to the importance of membrane proteins in regulating cellular processes and because they are the target of many drugs. In addition, measuring and understanding how these proteins interact with ligands and other molecules is of critical importance to the design of the next generation of therapeutic agents. With this motivation, we report new methods for the structural characterization of proteins using magic angle spinning (MAS) nuclear magnetic resonance (NMR) applied to the membrane protein M2 from Influenza A, a small helical transmembrane proton transporter that is the target of adamantane based inhibitors but which is now drug resistant due primarily to the point mutation S31N. The development of techniques that boost the sensitivity of NMR are key to its extension to larger molecules such as membrane proteins, and two such methods, dynamic nuclear polarization (DNP) and proton detection, are applied herein. We report the measurement of the distance between the amine of the inhibitor rimantadine and the pore of M2 using DNP. We have applied recoupling techniques to assign the spectra and measure internuclear distances in the drug resistant S31N mutant of M2 in lipid bilayers. Attenuation of strong proton dipole couplings with 60 kHz spinning has allowed us to detect well-resolved proton spectra, and with the higher receptivity of protons, we measured interhelical distances with a methyl-methyl 4D spectrum. Synthesis of the information resulted in a high resolution structure of S3 IN M2.
by Loren B. Andreas.
Ph. D.

Human respiratory syncytial virus (HRSV) is a non-segmented negative stranded RNA virus classified within the Mononegavirales order. HRSV is the leading cause of lower respiratory tract illness in young children and the immunocompromised, with over 250,000 annual fatalities worldwide. HRSV-mediated diseases are especially prevalent in developing countries, where no financially viable treatment exists. The M2-1 protein of HRSV represents a promising potential anti-viral target for the treatment of HRSV-mediated diseases. M2-1 is a transcription antiterminator with an essential role in viral gene expression. M2-1 binds both viral RNA and the polymerase co-factor phosphoprotein (P), and these interactions are essential for its anti-termination activity. Here, the crystal structure of M2-1 in complex with a P protein peptide was determined and used in addition to the unbound M2-1 crystal structure as a basis to identify potential anti-viral compounds. Structure based drug design (SBDD) of M2-1s interaction interface with RNA/ P produced hit compounds that were analysed through biophysical, structural and in cellulo methods including the use of a mini-genome and infectious HRSV assay. SBDD identified hit compounds that significantly inhibited the growth of HRSV. Subsequently, synthetic chemistry was used to generate libraries of molecules to establish structure-activity relationships (SAR) as well as assess their pharmacokinetic properties. Further, a novel facility that allowed assessment of fragment binders to HRSV M2-1 by X-ray crystallography was utilised to determine hit binders towards the entire M2-1 protein, including the previously targeted RNA/ P surface and allowed further rapid SAR. The work presented here represents an effective strategy to rationally design anti-viral compounds for the M2-1 protein of HRSV with potential applications for the related virus, bovine respiratory syncytial virus (BRSV).

Thesis: Ph. D. in Physical Chemistry, Massachusetts Institute of Technology, Department of Chemistry, 2017.
Cataloged from PDF version of thesis.
Includes bibliographical references.
Solid-state nuclear magnetic resonance (ssNMR) spectroscopy is a powerful technique that can be used to probe the structure and dynamics of biomolecules that are intractable to study by other structural biology methods. In this thesis, the mechanisms of conduction and gating are investigated in the influenza AM2 and BM2 proton channels bound to native-like lipid bilayers. Using pH-dependent 15N chemical shifts of the conserved Histidine residue, we have found that the proton conduction mechanism is the same in all of the M2 channels (WT AM2, S3 IN AM2, BM2) studied, and that the conduction cycle involves proton shuttling via hydrogen-bonding with water. For the first time, unambiguous structural restraints and sidechain dynamics were measured for the gating residue Trp4l in the AM2 channel. Trp4l was found to come into close contact with His37, close enough to undergo periodic cation-[pi] interactions that ensure unidirectional proton flow. Water-edited ¹H-¹³C correlation experiments were used to investigate the channel hydration of AM2 and BM2 tetramers, and S12 in the C-terminal BM2 channel was found to have the largest pH-dependent spin-diffusion buildup of all residues studied, indicating that the BM2 serine triplet plays an important role in conduction. New ssNMR methods were developed to probe long-range ¹³C-¹³C distances, and to edit and clean-up crowded spectra of peptides and proteins containing aromatic residues. The ASSET technique removes the aliphatic cross peaks of all residues except for Phe, Trp, Tyr, and His in a two-dimensional ¹³C-¹³C correlation experiment, while a two-dimensional ¹³C-¹³C correlation experiment with gated-decoupling retains only non-protonated 3C resonances. The studies in this thesis have provided insight into the functionally important His and Trp residues in the M2 proton channel of the influenza virus, and provide new avenues for studying other peptides and proteins containing aromatic residues.
by Jonathan K. Williams.
Ph. D. in Physical Chemistry

Influenza viruses exploit sophisticated host cell machinery to replicate, causing both seasonal epidemics and unpredictable pandemics. Studying the host cellular factors interacting with conserved domains of viral proteins will help us to identify key host proteins for the virus infection. This will not only strengthen our understanding of the precise mechanisms of the virus life cycle, but also pave new avenues for anti-viral development. The cytoplasmic tail of M2 ion channel (M2/CT) is one of these highly conserved domains. It is fully accessible to the host cell machinery after fusion of the virus envelope with the endosomal membrane and during the trafficking, assembly, and budding processes. I hypothesized that recruitment of host cellular factors by M2/CT may regulate the M2-dependent stages of the virus life cycle. Through a large scale yeast two-hybrid (Y2H) screen with the M2/CT used as bait, the human annexin A6 was identified as a novel host cell interactant and this interaction was further confirmed by both GST pull-down assay on purified proteins and co-immunoprecipitation assay on virus infected cells. A functional characterization of this novel interaction demonstrated that depletion of annexin A6 could enhance the virus production, while its overexpression could reduce the virus propagation, which indicates that annexin A6 is a negative regulator of the virus infection. However, I found that the virus infection could not induce any changes of annexin A6 expression. Therefore, the annexin A6-mediated regulation may depend on the subcellular localization where the interaction with M2/CT occurs. To decipher which step of the virus replication is regulated, we dissected the virus life cycle and found that modulation of annexin A6 expression had no effect on the early stages of the virus life cycle or on viral RNA replication but impaired the release of progeny virus, as suggested by delayed or defective budding events observed at the plasma membrane of virus-infected and annexin A6-overexpressing cells during a transmission electron microscopy study. To further decipher the underlying molecular mechanisms, the contribution of annexin A6-mediated plasma membrane lipid rafts reorganization through cholesterol homeostasis modulation and cortical actin cytoskeleton remodeling was also investigated. In conclusion, here I have identified the human annexin A6 as a novel host cell interactant of M2/CT that negatively modulate the influenza virus infection by impairing the virus budding and release. This work further supports the idea that M2 is a multifunctional protein and is also consistent with the discovery by Rossman et al. that M2/CT mediates the virus budding process (Rossman et al., 2010). This study further emphasizes the importance of host cell interactants of M2/CT in this process. Regarding the biology of annexins, this study also adds a new member of this protein family in the list of regulators of influenza virus infection.
published_or_final_version
Public Health
Doctoral
Doctor of Philosophy

Influenza A virus (IAV) exhibits a high mutation frequency. Mutations in primary viral targets such as hemagglutinin, neuraminidase, and the proton channel M2 have afforded IAV with substantial drug resistance to previously effective drugs such as oseltamivir and amantadine (AMT) along with their analogs. One of the main drug-resistant alterations in the M2 protein that has become ubiquitous is the mutation S31N. Divalent copper has previously been suggested to have anti-IAV properties using in vitro assays involving Xenopus oocytes and SSNMR. In this work, the EC50 of AMT, two AMT analogs, CuCl2 · 2H2O, and four previously published Cu2+ complexes were tested for antiviral activity against the California/07/2009 (H1N1) IAV strain containing the S31N M2 protein in viral mini-plaque assays. AMT, CuCl2 · 2H2O, and two previously published divalent copper complexes were tested for M2 proton-transport blocking efficacy in liposomes using truncated M2 (22-62, S31N). Two novel divalent copper species, NAG101 and NAG107, both derived from AMT analogs were developed and tested for both antiviral activity and M2 blocking efficacy. Cell integrity was maintained at concentrations up to 1 mM (48 hours of exposure) with all compounds except Cu(II) 3. In the viral mini-plaque assay the novel divalent copper complexes NAG101 (EC50 25.7 +/-7.7 µM) and NAG107 (EC50 2.91 +/- 0.29 µM) were 2.5-fold and 21-fold better than AMT (EC50 64.3 +/- 9.3 µM) respectively. In the liposome assay NAG101 (EC50 18.9 +/- 1.5 µM) and NAG107 (EC50 4.5 +/- 0.6 µM) were 2.5-fold and 11-fold better than AMT (EC50 49.3 +/- 2.9 µM respectively. In the viral mini-plaque assay, CuCl2 · 2H2O (EC50 57.2 +/- 10.1 µM) was comparably effective to AMT, but was more than ten times more effective in the liposome assay targeting S31N M2 with an EC50 6.1 +/- 0.8 µM. Divalent copper species possess anti-IAV activity against the ubiquitous S31N M2-containing viral strain California/07/2009 (H1N1) and may prove to be effective against other IAV strains by blocking M2.

Muskarin-cholinerge und adrenerge Rezeptoren des autonomen Nervensystems im Respirationstrakt spielen sowohl bei der physiologischen Regulation der Atemwegsfunktion als auch bei der Entstehung und Behandlung wiederkehrender obstruktiver Atemwegserkrankungen (RAO) des Pferdes eine wichtige Rolle. Im Wesentlichen sorgen diese Rezeptorsysteme für eine Relaxation und bzw. oder Kontraktion der glatten Atemwegsmuskulatur. Bei einer Erkrankung der Atemwege wird eine Störung der Balance beider Rezeptorsysteme vermutet. Neuere Forschungsergebnisse belegen, dass zum einen der β–adrenerge Rezeptor-Gs-Protein-Adenylatzyklase-Signalweg im Respirationstrakt RAO-erkrankter Pferde beeinträchtigt ist. Gleichzeitig war die Verteilung und Dichte der muskarin-cholinergen Rezeptoren unverändert. Die Signaltransduktionswege, insbesondere die des muskarin-cholinergen Rezeptorsystems, wurden jedoch bisher noch nicht ausreichend untersucht. Membranen des Lungenparenchyms sowie des Tracheal- und Bronchialepithels mit der darunter liegenden glatten Muskulatur gesunder und RAO-erkrankter Pferde wurden hinsichtlich der Funktionalität der M2- Rezeptor-Gi-Protein-AC-Kopplungsmechanismen mittels 35S-GTPγS-Bindungsstudien untersucht. Mit verschiedenen muskarin-cholinergen Agonisten und den Substanzen N-Ethylmaleimid (NEM) und Pertussistoxin (PTX) wurde die M2-Rezeptor-Gi-Protein-Kopplung überprüft. Vorher war die Ermittlung der Konzentrationen von Na+ (200 mmol/l), Mg2+ (10 mmol/l) und GDP (10 μmol/l) zur Etablierung einer optimierten 35S-GTPγS-Bindung notwendig. Es konnte gezeigt werden, dass die Basalbindung und die Agonisten-stimulierte 35S-GTPγS-Bindung zeitabhängig ist und eine Inkubationszeit festgelegt werden musste, bei der eine hohe relative Stimulation gemessen werden konnte. Diese Inkubationszeit lag bei 120 min bei einer Temperatur von 30°C. Bis zu diesem Zeitpunkt stieg die relative Stimulation der 35S-GTPγS-Bindung. Die muskarin-cholinergen Agonisten Oxotremorin M, Carbachol und Acetylcholin waren in der Lage die 35S-GTPγS-Bindung in allen untersuchten Segmenten bei gesunden und bei an RAO-erkrankten Pferden zu stimulieren, wobei Oxotremorin M die 35S-GTPγS-Bindung stärker als Carbachol und Acetylcholin stimulierte. Die maximale relative Stimulation lag in den Bronchien bei 126 %, in der Trachea bei 104 % und in der Lunge bei 41 %. Diese segmentabhängige Stimulierbarkeit der 35S-GTPγS-Bindung entspricht der Dichte der M2-muskarin-cholinergen Rezeptoren, die an Gi-Proteine gekoppelt sind. Bei an RAO-erkrankten Pferden konnte eine tendenziell stärkere Agonisten-stimulierte Interaktion zwischen den M2-muskarincholinergen Rezeptoren und Gi-Proteinen gemessen werden (Bronchien 141 % und Lunge 78 %) als bei gesunden Pferden. Dies könnte mit einer erhöhten Anzahl an Bindungsstellen für 35S-GTPγS begründet sein, was eine erhöhte Menge an Gi-Proteinen im erkrankten Gewebe bedeuten würde. Hierfür spricht die Hemmung der 35S-GTPγS-Bindung mit NEM und PTX, die die Agonisten-vermittelte 35S-GTPγS-Bindung im Gewebe von an RAO-erkrankten Pferden schwächer hemmte als im Gewebe von gesunden Pferden. Die indirekte Messung der AC-Aktivität zeigte eine segmentabhängige Reduzierung der cAMP-Produktion von Trachea über Bronchien bis hin zur Lunge. Oxotremorin M hemmte die Forskolin-vermittelte cAMPProduktion, und PTX war in der Lage diese durch Entkopplung der Gi-Proteine vom M2-Rezeptor zu hemmen. Ein weiterer Hinweis auf die erhöhte Menge von Gi-Proteinen im Gewebe RAO-erkrankter Pferde war die gemessene Reduzierung der Forskolin-induzierten cAMP-Produktion. In der vorliegenden Arbeit wurde erstmalig die Methode zur Messung der Rezeptorinteraktion mit korrespondierenden G-Proteinen - die 35S-GTPγS-Bindung - in Geweben des Respirationstrakts von Pferden etabliert. Die Stimulation mit muskarin-cholinergen Agonisten führte zu einer erhöhten 35S-GTPγS-Bindung bei an RAO-erkrankten Pferden. Außerdem konnte bei RAO eine Reduzierung der Forskolin-induzierten cAMP-Produktion beobachtet werden. Diese Ergebnisse deuten auf eine erhöhte Gi-Proteinmenge hin. Somit verschiebt sich bei RAO das Gi/Gs-Gleichgewicht auf Seite des Gi-Protein-vermittelten Signalwegs. Diese Veränderung des M2-Rezeptor-Gi-Protein-Signalwegs trägt, wie auch die Verschiebung des muskarincholinergen/ adrenergen Gleichgewichts durch β-Adrenozeptor-Downregulation, letztendlich zur bronchokonstriktorischen Aktivität bei RAO bei. Die Ergebnisse dieser Arbeit liefern somit die Grundlage für ein Evidenz-basiertes Konzept der Pharmakotherapie der RAO des Pferdes mit Muskarin-M2-selektiven Antagonisten zusätzlich zur etablierten Basistherapie mit β2-Sympathomimetika, insbesondere wenn gegenüber diesen bereits eine Toleranz besteht.

Bien que le Virus Respiratoire Syncytial, responsable de la bronchiolite du nourrisson, soit aujourd’hui un problème de santé publique majeur, il n’existe encore aucun vaccin ou antiviral curatif contre ce pathogène. Le manque de données sur les étapes clés du cycle viral et sur les interactions virus-cellule freine le développement de nouvelles molécules antivirales.Nous avons étudié l’interactome de deux protéines virales : la polymérase L et le facteur de transcription M2-1. Dans ce but, nous avons mis au point un crible s’appuyant à la fois sur des critères d’interactomique et sur des critères fonctionnels.La première étape consistait à identifier des partenaires potentiels de M2-1 et L par des co-immunoprécipitations couplées à une approche de protéomique quantitative. Pour plus de pertinence, ce crible a été réalisé sur cellules infectées, grâce des virus recombinants produits par génétique inverse. Ceci nous a permis d’identifier 45 et 137 partenaires potentiels de L et M2-1 respectivement. Une étude systématique de l’impact de l’inhibition de 15 partenaires potentiels de M2-1 sur la multiplication virale a mis en avant trois candidats : ILF2, PABPN1 et PABPC1.Nous nous sommes par la suite concentrés sur PABPC1. L’inhibition de l’expression de PABPC1 altère la multiplication virale, mais nous n’avons pas pu mettre en évidence un effet spécifique sur la transcription ou la traduction virale. Son interaction avec M2-1 a été confirmée, et le domaine MLLE de PABPC1 a été identifié comme le site de liaison à M2-1. L’interaction entre M2-1 et PABPC1 a été observée à la fois dans le cytoplasme et dans les IBAGs, des sous-structures concentrant les ARNm viraux au sein des corps d’inclusion viraux. Nous avons formulé l’hypothèse que M2-1, liée à PABPC1, accompagne les ARNm viraux après leur sortie des corps d’inclusion. Ceci suggère un rôle de M2-1 dans le devenir des ARNm viraux en aval de leur transcription
Although the Respiratory Syncytial Virus, responsible of bronchiolitis in infants, represents a major public health problem, there are currently no vaccine or curative antiviral directed against it. The lack of information on key steps of its viral cycle and on virus-cell interactions hinders the development of new antiviral molecules.We chose to study the interactome of two viral proteins: the polymerase L and the transcription factor M2-1. To do so, we developed a screen based on interactomic and functional criteria.The first step consisted in identifying potential binding partners of M2-1 and L by co-immunoprecipitations coupled to quantitative proteomics. For better relevance, this screen was realised on infected cells, thanks to recombinant viruses produced by reverse genetics. 45 and 137 potential binding partners of M2-1 and L respectively were thus identified. A systematic study of the inhibition of 15 potential partners of M2-1 and its impact on viral multiplication enabled the selection of three candidates: ILF2, PABPN1 and PABPC1.We chose to concentrate on PABPC1. The inhibition of PABPC1’s expression reduces viral multiplication, but no specific effect on viral transcription or translation was brought to light. Its interaction with M2-1 was confirmed, and the MLLE domain of PABPC1 was identified as the M2-1 binding site. The interaction between M2-1 and PABPC1 was observed both in the cytoplasm and in IBAGs, substructures of viral inclusion bodies where viral mRNA accumulate. We formulated the hypothesis that M2-1, with PABPC1, stays with viral mRNA after leaving inclusion bodies and during their translation. This suggests a role for M2-1 in the fate of viral mRNA downstream of transcription

A novel truncate of Influenza A M2 protein (residues 22-62), incorporated into a uniquely tailored proteoliposome proton uptake assay, demonstrated proton flux more characteristic of an ion transporter than a traditional ion "channel." The liposome paradigm was essential for testing the conductance activity of this M2 truncate at a range of extraphysiological pHs appropriate for channel vs. transport function determination. In addition to transporter-typical proton flux, M2(22-62) showed the key characteristics of functional integrity: selective proton uptake into liposomes and block of uptake by amantadine. Two sets of proteoliposome proton flux assays were carried out, Set 1 at pH values of 6.5, 6.0. 5.5, 5.0, and 4.5; Set 2 at pH values of 6.25, 6.0, 5.75, 5.5, 5.25, 5.0, and 4.75. Observed flux rates followed a proton transport saturation curve similar to that observed in mouse erythroleukemia cells1. Proton transport was maximal at pH 5.5 in Set 1 (139 H+/second/tetramer) and at pH 5.75 in Set 2 (43 H+/second/tetramer). Amantadine block was strongest at pH 5.5 in Set 1 and 6.25 in Set 2, and apparent desensitization of the protein severely reduced proton flux and amantadine sensitivity below pH 5.5 in both sets of experiments. Decreased external pH increased proton uptake with an apparent pKa of 6 (Set 1) or 6.5 (Set 2). These data indicate acid activation of M2(22-62) between pH 5.5-6, optimal amantadine block between pH 5.5-6.25, and a loss of peptide functionality between pH 5.9-4.7.

Die Assemblierung von Influenzaviren erfolgt an Rafts der apikalen Wirtszellplasmamembran mit denen das Hämagglutinin (HA) über Acylierungen im C-Terminus und hydrophobe Aminosäuren seiner Transmembrandomäne (TMD) interagiert. M2 besitzt eine cytoplasmatische amphiphile Helix (AH), die ebenso potenzielle Raft-Motive aufweist: Eine Acylierung und Cholesterol-Bindemotive. In dieser Arbeit wurde per Konfokalmikroskopie an polarisierten Zellen, die fluoreszenzmarkierte M2-Varianten exprimierten, gezeigt, dass diese M2-Motive nicht für den apikalen Transport, der vermutlich durch Raft-ähnliche Vesikel erfolgt, benötigt werden. Messungen des Förster-Resonanzenergietransfers über Fluoreszenz-Lebenszeit-Mikroskopie (FLIM-FRET) in der Plasmamembran lebender Zellen, die fluoreszenzmarkiertes HA und M2 koexprimierten, ergaben, dass diese Motive auch nicht für die Interaktion mit den durch HA, in Abhängigkeit von dessen Raft-Motiven, stabilisierten Raft-Domänen notwendig sind. Mittels reverser Genetik konnten infektiöse WSN-Viren mit fehlender Acylierung am Ende der HA-TMD, nicht jedoch Viren ohne die zwei cytoplasmatischen Acylierungen hergestellt werden. Weiterhin ergaben Wachstumsanalysen, dass die Acylierung von HA und M2 für den gleichen Schritt des viralen Replikationszyklus von Bedeutung sind. Für die M2-AH wurde postuliert, dass sie die Membrankrümmung detektiert und durch Insertion in die Wirtszellmembran die Virusabschnürung bewirkt. Infektiöse Viren ohne M2 oder ohne die AH konnten ebenso wie Viren mit M2 mit einer Helix mit reduzierter Amphiphilität in dieser Arbeit nicht hergestellt werden. Allerdings führte die Substitution der AH durch typische krümmungsdetektierende oder modulierende Helices zu Viren, deren Wachstum um zwei bis vier Titerstufen im Vergleich zum Wildtyp reduziert war. Die Helix-Amphiphilität scheint wichtig zu sein, aber auch die Sequenz oder bestimmte Aminosäuren sind offenbar für eine effiziente Virusreplikation notwendig.
The assembly of influenza virus particles occurs at the apical plasma membrane of the host cell at membrane rafts which the hemagglutinin (HA) interacts with via acylations in its C-terminal region and via hydrophobic amino acids in the transmembrane domain (TMD). M2 possesses a cytoplasmic amphiphilic helix (AH) that also contains potential raft motifs: an acylation and cholesterol-binding motifs. In this work, confocal microscopy of polarised cells, which were expressing fluorescently labelled M2-variants, demonstrated that these motifs of M2 are not required for apical transport, which is assumed to be mediated by raft-like vesicles. Furthermore, FLIM-FRET (Förster resonance energy transfer measured via fluorescence lifetime imaging microscopy) analyses, performed in the plasma membrane of living cells coexpressing fluorescently labelled HA and M2, revealed that these M2-motifs are not required for association with the large coalesced raft phase organised by HA. In contrast, deleting HA’s raft-targeting features clearly reduced clustering with M2. While the removal of the two cytoplasmic acylations prevented the rescue of infectious virus by reverse genetics, a mutant virus without acylation in the HA-TMD could be rescued. Moreover, growth analyses revealed that the acylations of HA and M2 are important for the same step in the viral replication cycle. It has been postulated that the M2-AH detects membrane curvature and accomplishes membrane scission by inserting into the host cell membrane. Viruses without M2, without the M2-AH or with M2 containing a helix with reduced amphiphilicity could not be produced in this work. However, substituting the AH by typical curvature-sensing or -generating helices led to viruses with two to four orders of magnitude reduced growth as compared to wildtype virus. The amphiphilicity of the helix seems to be important, but also the sequence or specific amino acids appear to be necessary for an efficient virus replication.

碩士
國立陽明大學
微生物及免疫學研究所
107
Influenza A virus belongs to Orthomyxoviridae, and is a common pathogen to human. Influenza viruses cause yearly outbreak around the world, resulting in about three to five millions cases of sever illness. Because the virus mutates frequently, we have to predict vaccine strains every year, moreover preexisting immunity cannot provide protection fully. Mutations also render influenza viruses resistance to current antivirals. A better understanding of host-virus interaction may helps us identify potential target for new antiviral drugs. Activation of RIG-I-like Receptors ( RLR ) signaling pathway induces type I interferon production to defend against influenza infection. This signal is transmitted through the aggregation of the adaptor protein MAVS on mitochondria. Previously, we found that activation of RIG-I promoted protein degradation of influenza viral proteins. M2 and PB1-F2 were the most affected, so we try to understand the possible mechanism mediating their degradation. We have found that RNF125, an unbiquitin E3 ligase known to regulate RIG-I signaling negatively, played a role in RIG-I-induced PB1-F2 protein degradation. Here, we extended the study to M2. On the other hand, using different pharmacological inhibitors has previous helped us identified the autophagy may be part of RIG-I- induced viral protein degradation. In this study, we examined the role of autophagy in RNF125-mediated M2 degradation. First, we found the overexpression of RNF125 facilitated the protein degradation of M2. Consistently, knockdown of RNF125 reduced RIG-I-induced M2 degradation. Next, by using pharmacological inhibitors, we found the M2 expression could be partially rescued by autophagy inhibitor including 3-MA and Bafilomycin-A1, suggesting M2 might be degraded through autophagy upon RIG-I activation. Surprisingly, we didn’t observe the proteasome inhibitors, such as MG132 and lactacystin, can reduce degradation. Finally, we investigated that whether RNF125 affects influenza virus ( PR8 ) during infection. We found that in presence of overexpressed RNF125, M2 expression was reduced moderately. This may lead to the reduction of viral tirter we observed. We concluded that RNF125 may play a role in RIG-I-mediatied antivral activity through enhancing viral protein degradation.

碩士
國立陽明大學
微生物及免疫學研究所
105
RIG-I like receptors (RLR) signaling activation induces type I interferon response to defend against influenza virus infection. RIG-I and MAVS overexpression also induces autophagy to maintain mitochondrial homeostasis. In our previous studies, we observed that activation of RIG-I signaling led to PB1-F2 derived from A/Puerto Rico/34/8/H1N1 (PR8) degradation. As a result, we investigated whether activation of RIG-I signaling induces other influenza viral proteins degradation. We found that PR8 PB1, PA, HA and NP expression level decreased slightly when N-RIG, an active form of RIG-I, was overexpressed, while PR8 PB1-F2, M2 and NS2 viral proteins reduced more significantly. After comparing the expression level of different influenza viral proteins under N-RIG overexpression, we chose viral protein M2 as our model to study the degradation mechanisms. M2 is responsible for virus releasing to nucleus and is known to help virus to evade infection-induced autophagy by blocking autophagosome fusion with lysosome, thus M2 plays an important role in the life cycle of influenza virus. To investigate how RIG-I mediated M2 degradation, cells were treated with different pharmacological inhibitors in the presence of RLR signaling activation. We observed that M2 expression level could be rescued by a proteasome inhibitor, lactacystin, and three autophagy-related inhibitors, 3-MA, Bafilomycin-A1 and Leupeptin, indicating that M2 might be degraded via both proteasomel and lysosomal pathways. In the study of autophagy and lysosomal pathways, we further observed that M2 could be degraded when cells were transfected with plasmids expressing EGFP-LC3 or treated with CCCP to induce autophagy. It showed that autophagy and lysosomal pathway indeed participated in RIG-I mediated M2 degradation. In the study of proteasome degradation pathway, we also investigated whether E3 ligases involved in RLR signaling led to M2 degradation. We found that several E3 ligases, such as RNF125, RNF135 and Parkin, could further enhance M2 degradation in the presence of RIG-I signaling, suggesting that E3 ligases might also play a role in RIG-I mediated M2 degradation. However, the detailed mechanisms remain to be deduced.

碩士
國立臺灣大學
醫事技術學研究所
90
Due to the antigenic variation of the viral surface proteins, hemagglutinin (HA) and neuraminidase (NA), the conventional influenza vaccine has to be evaluated every year. In order to improve the defectiveness, we chose another surface protein, M2 protein, to study and analyze the M2 protein specific immune response to understand if it is suitable as a vaccine component. This thesis could be divided into two parts. The first part was to study the M2 antibody response after vaccination a commercial influenza vaccine, Pasteur vaccine. The inactivated vaccine was inoculated to human. Paired serum were collected and the M2 antibody was detected by Western blotting using recombinant M2 protein. The M2 specific antibody could not be detected in all of the serum. It was possible that the M2 protein present in the vaccine was too low to induce antibody response. High dose of the inactivated vaccine was inoculated into mice, no M2 specific antibody could be detected. The second part is to study the immune response of M2 DNA vaccine. The mice were inoculated with three different M2 plasmids, and the M2 specific antibody was detected. It was found that the plasmid containing M2 (full) gene could induce M2 specific antibody six weeks after inoculation, and the highest antibody titer could be detected at the eighth week. To analyze the antibody subtype, we found that the major subtype was IgG2a. This implied a Th1 immune response. In the experiment to detect cellular immune response, the IFN-γ secreting cells were detected by ELISPOT. It was found that all of the three different M2 plasmids could induce cellular immune response. The neutralization assay showed that the M2 antibody could not neutralize the influenza virus. Our results showed that no M2 specific antibody could be detected after inoculating inactivated influenza vaccine. However, the M2 specific antibody and cellular immune response could be detected after immunization with M2 (full) DNA vaccine. Further study of the protection efficiency in animals will be required to understand what the M2 protein could be one of the suitable vaccine components.

碩士
國立陽明大學
生醫光電研究所
104
Ion channels are pore-forming proteins in charge of permeability of specific ions on lipid bilayer by electrochemical gradients. They exist not only in every cellular membrane but in most of viruses and reveal the key part in how cells and viruses work. To investigate how ion channel proteins interact with each other from the aspect of biophysics, there is a program built for helping the user to generate the whole energy profile. This program of ion channel assembly (PICA) has 5 degrees of freedom (DOFs), namely, D, θ r, θ t, N and h, in order to describe each of the ion channel structures. PICA screens every point of DOF in the range the user is setting. Unlike other existing docking program or software that they search space all around the target proteins for proper conformation, PICA only search the space around the side surface of trans-membrane domain (TMD) of protein to find the its possible pore structure. Thus, it is more like a 2 dimensional search method rather than 3 dimensional search algorithm like other programs for globular proteins. The situation of PICA is different from those of the others tools. PICA is built under the assumption that pore-forming proteins are assembled on lipid bilayer of endoplasmic reticulum; that also is, these pore-forming proteins don’t need to be tilted at big angles and is assembled together under the lipid environment whose dielectric constant is nearly 2. An important feature of PICA is that the displacement of the structure includes the Cα atoms and sidechain. It turns out that the Root-Mean-Square Deviation (RMSD) values of top 10 structures are large; however, all of top 10 structures form proper orientation of residues to function as channels. In this research, I also performed other docking software and programs to assemble M2 proton channel of influenza A virus. These software and programs are Molecular Operating Environment (MOE), Sam, M-ZDOCK and Rosetta. In order to see how well it is on predicting pore structures of membrane proteins, RMSD comparison of backbones between original structure and prediction is applied. I also checked the top 10 prediction structures which these software and programs provided to user. Most of these prediction results failed to reproduce the actual structure of the M2 proton channel, and even though RMSD values of some of the predictions are small enough that they seem promising. In the case of M2 proton channel, the pore structure of the M2 channel must have four His and four Trp positioned inside the pore region in order to be proton conducting. The His of channel function as a pH sensor and the Trp forms the ion gate of the channel3. Most of the prediction results of existing software and programs have presented incorrect orientation of His and Trp. The final comparison tells the users that PICA provided ten proper structures to the user, and Sam provided the two, and M-ZDOCK provided the one and both MOE and Rosetta failed to provide any proper structures on the docking of M2 proton channel of influenza A virus.

碩士
國立臺灣大學
醫事技術學研究所
89
Influenza virus is one of the most widespread viral infectious agent known. The virus mainly induces acute respiratory tract infection in man, and often causes severe morbidity and even death. Because of the rapid transmission of influenza virus and circulating epidemics of the whole world, it is very important to prevent and control the influenza virus infection. The conventional influenza vaccine is an inactivated vaccine and composed of two A influenza viruses and one B influenza virus. However, due to the antigenic variation of two viral surface glycoprotein hemagglutinin (HA) and neuraminidase (NA), the virus strains incorporated into influenza vaccine must be evaluated every year. DNA vaccine is a new direction in the vaccine development. By gene gun or direct muscle injection, plasmid DNA can be delivered to the host cells and induces strong and persistent cellular and humoral immune responses to the antigen encoded by the plasmid. M2 protein is an integral membrane protein of the influenza virus. Compared with HA and NA, M2 is a minor protein component of influenza virion. However, M2 protein is expressed abundantly in virus-infected cells and is a more structurally conserved viral surface protein. According to the prior studies, vaccination of mice with M2 protein has been shown to induce high antibody titer and provide protection from lethal viral protection. All these studies have shown the potential of M2 protein as a vaccine component. In order to understand the M2 protein specific immune response after influenza virus infection, the first part of this study was trying to express M2 protein in E.coli system. In order to express M2 protein in E.coli effectively, we constructed three M2 protein genes, M2(full), M2(26-43), M2(25-55), and subcloned into three different expression vectors. The recombinant protein MBP-M2(25-55) showed the best expression quality and quantity, so the it was further purified and used in the detection of M2 specific antibody. The second part of this study was trying to apply M2 protein gene in DNA vaccine. First we subcloned the three M2 constructs into a mammalian expression vector pcDNA3 and then transfected 293 cells with the three different constructs. And we found that only the construct with full length M2 gene could express protein in the mammalian cells. Vaccination of mice with the three M2 constructs by intramuscular injection resulted in similar response. Analysis of the M2 protein specific antibody titer of mice indicated that only the pcDNA3-M2(full) expression plasmid could induce moderate humoral immune response. In this study, we have successfully expressed and purified recombinant protein MBP-M2(25-55) in E.coli system, and used it to detect M2 specific antibody in mice. However, the M2 portion of the recombinant protein should be cleaved and further purified for the detection of human M2 specific antibody in order to understand the role of M2 protein in immune response. Furthermore, in this study we applied M2 protein gene in DNA vaccine and found that it induced immune response in mice. These results showed the potential of M2 protein DNA in vaccine development, and further studies are needed to understand the mechanism of M2 DNA vaccine and the protection efficiency.

碩士
國立嘉義大學
生物農業科技學系研究所
106
Avian influenza（Avian Influenza；AI）is commonly known as avian flu. It is one kind of animal infectious diseases caused by flu viruses. AI viruses can be grouped into highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI). The mortality of the poultry infected by HPAI can be higher than 80%. In this study, H1N1 viral M2 gene segment was constructed into the E. coli protein expression vector for expressing the M2 gene 's fusion protein with 8X-Histidine tag. The serum samples of vaccinated mice were assayed by dot blotting and ELISA. The specific antibody against the overexpressed M2 gene's protein produced in the vaccinated mice was determined by dot blotting assay. The M2 gene's fusion protein was also shown to have better immune response in association with the commercial adjuvant. These studies have laid a strong foundation for developing an effective AI subunit vaccine.

G protein-coupled receptors (GPCRs) are known to exist as oligomers, but there is much uncertainty over the oligomeric size, the number of interacting G proteins and the stability of that interaction. The present approach to these questions has been threefold. Monomers of the M2 muscarinic receptor were purified from Spodoptera frugiperda (Sf9) cells and reconstituted in phospholipid vesicles, where they spontaneously formed tetramers. The size of the reconstituted complex was determined from its electrophoretic mobility after cross-linking and inferred from a quantitative, model-based assessment of cooperative effects in the binding of two muscarinic antagonists: N-methylscopolamine and quinuclidinylbenzilate. Binding of the agonist oxotremorine-M to receptor reconstituted with purified G proteins revealed at least three classes of sites that interconverted from higher to lower affinity upon the addition of guanylylimidotriphosphate (GMP-PNP). The binding properties resemble those of muscarinic receptors in myocardial preparations, thereby implying the existence of tetramers in native tissues. G proteins that copurify with the M2 receptor from cardiac membranes also were found to exist as oligomers, some of which contain both alpha(o) and alpha(i2), and the purified complexes contained receptor and G protein in near-equal amounts. A tetrameric receptor implies a tetramer of G proteins, a conclusion that is supported by the distribution of sites between different states identified in the binding of [35S]GTPgammaS to the purified complex. Covalent adducts of a GPCR fused to a Galpha-subunit provide a model system in which the relationship between receptor and G protein complex is defined with respect to stability and composition. Such a fusion of the M2 receptor and Galpha(i1) underwent a cleavage near the amino terminus of the alpha-subunit, however, flagging the likelihood of similar effects in other such adducts. Truncation of the amino terminus prior to fusion generated a stable product that revealed GMP-PNP-sensitive, biphasic binding of oxotremorine-M and noncompetitive interactions between N-methylscopolamine and quinuclidinylbenzilate. A covalent RG complex therefore exhibits the functional properties of M2 receptors in native systems. These observations are consistent with the notion that signaling through the M2 receptor occurs via cooperative interactions within a stable complex that comprises four receptors and four G proteins.

碩士
國立臺灣大學
醫事技術學研究所
93
Influenza virus causes acute respiratory infection in humans. Epidemics occur annually. It can cause severe mortality especially in the elderly and children. The influenza vaccine being used now is composed of two inactivated Influenza A virus and one inactivated Influenza B virus. Due to the antigenic variation of viral surface proteins, hemagglutinin (HA) and nuraminidase (NA), the vaccine has to be evaluated and adjusted every year. In order to improve the situation, this study choses another virus surface protein, M2, which is highly conserved, to study. Trying to observe and analyze the specific immune response of recombinant M2 protein and to evaluate the potential of the plasmid DNA expressing recombinant M2 protein as influenza DNA vaccine. The previous studies of our lab showed that administrating mice with plasmid containing the sequence of M2 gene（pcDNA-Mfull, pMfull）could induce antibody response. In addition, our lab also constructed the plasmid pcDNA3-MdFc（pMdFc）. The Md portion was formed by deleting the transmembrne domain of M2 protein and a linker contains 5 amino acid（G-G-G-G-S）was added between extracellular and intracellular domains. The secreting signal peptide of Staphylococcus aureus protein A was added to the N-terminus of Md and human IgG Fc fragment was added to the C-terminus of Md to form MdFc. The muce immunized with pMdFc and plasmid containing mouse IL-5 sequence（pIL-5）as the adjuvant were also induced high antibody response. In this study, we continued previous studies and tried to enhance the expression and the immune responses of recombinant M2 protein. The different lengths of linker between Md and Fc was added, to construct a series of plasmids. On the other hand, the human IgG Fc fragment was moved to the N-terminus of Md, linkers with different length was added between Fc and Md to construct pFcMd group. Then we compared these two groups of plasmids with pMfull after tranfecting cell and administrating mice. The mRNA of recombinant M2 protein could be detected after transfecting cells, however, there was no detectable recombinant M2 protein. The animal experiments showed that pMfull, pMdFc group, and pFcMd group all could induce high antibody responses, and the titers from high to low were in the order of pMfull, pMdFc group, and pFcMd group. Furthermore, immunization mice with pIL-5 as the adjuvant resulted in higher antibody responses than those without pIL-5. Analyzing the IgG subtype of induced antibody showed that pIL-5 not only could increase the amount of total antibodies but also significantly raise the amount of IgG1 antibodies. The result of ELISPOT, which detected the cellular immune responses, corresponded well to that of antibody response. According to the study above, though there was still much left to be studied about M2 protein in the future, pMfull with appropriate adjuvant may be a potential DNA vaccine for influenza.

Zahlreiche experimentelle Befunde lassen vermuten, dass G-Protein gekoppelte Rezeptoren (GPCR) nach ihrer Aktivierung einer ligandenselektiven Änderung der Rezeptorkonformation unterliegen. Ziel der vorliegenden Arbeit war es dieses Phänomen am Subtyp 2 der muskarinischen Acetylcholinrezeptoren (M2 AChR) zu untersuchen. Muskarinische Acetylcholinrezeptoren (mAChR) können in fünf Subtypen (M1-M5) unterschieden werden. Durch die Beteiligung der mAChR an zahlreichen physiologischen Prozessen stellen sie wichtige Zielstrukturen pharmakologischer Therapien dar. Da die orthosterische Ligandenbindestelle (= Bindestelle des endogenen Liganden) in allen fünf Subtypen hoch konserviert ist, wird ihr pharmakologischer Nutzen derzeit allerdings durch die unselektive Rezeptormodulation und dem damit verbundenen Auftreten unerwünschter Arzneimittelwirkungen stark limitiert. Ein Ansatz zur Erzielung subtypselektiver Effekte besteht in der Verwendung allosterischer Modulatoren. Da die allosterische Bindestelle der mAChR eine geringere Sequenzhomologie aufweist, können so gezielt einzelne Subtypen der mAChR reguliert werden. Der M2 AChR stellt hinsichtlich allosterischer Modulation ein gut charakterisiertes Modellsystem dar. Für ihn wurde bereits eine Vielzahl allosterischer Liganden entwickelt. Auch bitopische Liganden, die sowohl einen allosterischen als auch einen orthosterischen Anteil enthalten, wurden für den M2 AChR bereits beschrieben. Im ersten Teil der vorliegenden Arbeit wurden verschiedene FRET-Sensoren des M2 AChR generiert und charakterisiert. Als FRET-Paar wurden das cyan fluoreszierende Protein (CFP) und der niedermolekulare fluorescein-basierte Fluorophor FlAsH (fluorescein arsenical hairpin binder) gewählt. CFP wurde in den Sensoren am Ende des C-Terminus angefügt. Die zur Markierung mit FlAsH nötige Tetracysteinsequenz wurde in verschiedenen Bereichen der dritten intrazellulären Rezeptorschleife (IL) eingebracht. Die auf diese Weise erstellten Re-zeptorsensoren trugen das Tetracysteinmotiv in der N terminalen (M2i3-N) bzw. in der C terminalen Region (M2i3-C) von IL 3. Die Charakterisierung der Rezeptorsensoren bezüglich Ligandenbindung, Gi-Protein Aktivierung und β-Arrestin2 Translokation ergab keine signifikanten Unterschiede zwischen M2i3-N, M2i3 C und M2CFP oder Wildtyp M2 AChR. Zunächst wurden sowohl unterschiedliche orthosterische, als auch allosterische Liganden hinsichtlich ihrer mittleren effektiven Konzentration und ihrer maximalen Wirkstärke an den Rezeptorsensoren untersucht. Mit Hilfe von FRET-Messungen konnte ein superago-nistisches Verhalten des orthosterischen Testliganden Iperoxo gezeigt werden. Die Eigenschaften der allosterischen Substanzen wurden durch Messung der Rezeptordeakti-vierungskinetik und des maximalen Hemmeffekts auf einen vorstimulierten Rezeptor charakterisiert. Alle allosterischen Liganden deaktivierten den vorstimulierten M2 AChR mit einer schnelleren Kinetik als Atropin. Die EC50-Werte der unterschiedlichen Substanzen waren unabhängig von der Markierungsposition im verwendeten Rezeptorsensor vergleich-bar. Ausnahmen bildeten die allosterischen Liganden JK 289, JK 338, ½ W84 und EHW 477, die liganden- und sensorabhängig unterschiedliche mittlere effektive Konzentrationen aufwie-sen. Bei der Untersuchung der Konformationsänderung des M2 AChR konnte kein liganden-selektiver Unterschied zwischen den FRET-Signalen für 19 getestete orthosterische Liganden beobachtet werden. Dies deutet darauf hin, dass alle orthosterischen Testliganden eine dem Acetylcholin (ACh) vergleichbare Änderung der M2 AChR Konformation induzier-ten. Um zu untersuchen, ob für die orthosterischen Testliganden eine Korrelation zwischen ihrer maximalen Wirkstärke hinsichtlich Rezeptoraktivierung in FRET-Experimenten und der Aktivierung nachgeschalteter Signalwege besteht, wurde die orthosterisch-vermittelte Translokation von β-Arrestin2 mit Hilfe der Konfokalmikroskopie bestimmt. Bis auf 5-Methyl-furmethiodid translozierten alle orthosterischen Liganden β-Arrestin2 in einem Ausmaß, das mit der maximalen Rezeptoraktivierung vergleichbar war. Dagegen rief 5 Methylfurmethiodid verglichen mit dem endogenen Liganden ACh zwar eine ca. halbmaximale Rezeptorakti-vierung, aber nur eine äußerst geringe β-Arrestin2 Translokation hervor. Im zweiten Teil der Arbeit wurde der Einfluss verschiedener Allostere auf eine ligandenselektive Konformationsänderung des M2 AChR untersucht. Die allosterischen Liganden JK 337 und Seminaph beeinflussten den M2i3-C Sensor signifikant stärker, als das M2i3-N Konstrukt. Dagegen zeigte EHW 477 eine stärkere Beeinflussung der Rezeptorkon-formation des M2i3-N-, als des M2i3-C Sensors. Dies erlaubt die Vermutung, dass JK 337 und Seminaph eine stärkere Bewegung unterhalb von Transmembrandomäne (TM) 6, als unterhalb von TM 5 hervorriefen. Die Ergebnisse für EHW 477 legen nahe, dass TM 5 eine größere Bewegung eingeht, als TM 6. FRET-basierte Untersuchungen der Einflüsse der allosterischen Testliganden auf nachgeschaltete Signalwege ergaben, dass sowohl die Akti-vierung des Gi Proteins, als auch die β-Arrestin2 Translokation selektiv durch einzelne allosterische Liganden beeinflusst werden. Auch ein Zusammenhang zwischen Rezeptor-aktivierung und der Regulation nachgeschalteter Signalwege war erkennbar. Allerdings waren auf Grund der Versuchsbedingungen keine quantitativen Aussagen möglich. Im Folgenden wurden die bitopischen Liganden Hybrid 1 und 2 (H 1, H 2) hinsichtlich ihres Effekts auf die Konformationsänderung des M2 AChR untersucht. Während eine Stimulation mit H 1 vergleichbare FRET-Signale an beiden Sensoren ergab, konnte mit H 2 weder am M2i3-N-, noch an M2i3-C Sensor eine FRET-Änderung detektiert werden. Um den mangeln-den Effekt der Hybridsubstanzen in FRET-mikroskopischen Untersuchungen aufzuklären, wurden verschiedene Ansätze gewählt: Mit kettenverlängerten Derivaten der Hybridsubstanzen konnte in FRET-Messungen eine Änderung des FRET-Signals detektiert werden. Die Entfernung des allosterischen Bausteins führte in FRET-Experimenten zu einer verglichen mit dem endogenen Liganden ACh etwa halbmaximalen Aktivierung beider Sensoren. Dagegen resultierte die Mutation der alloste-rischen Bindestelle in nachfolgenden FRET-Untersuchungen mit H 1 und H 2 in keiner Signaländerung des FRET-Ratio. Diese Beobachtungen führten zu der Annahme, dass die Linkerkette, die orthosterischen und allosterischen Baustein der Hybride miteinander verbindet, zu kurz war um eine gleichzeitige Bindung an die allosterische und orthosterische Bindestelle zu ermöglichen. Ein anderer Erklärungsansatz besteht darin, dass nach Bindung des Orthosters der Kanal zwischen orthosterischer und allosterischer Bindestelle durch die Konformationsänderung des Rezeptors verschlossen wird, weshalb keine dauerhafte, dualsterische Bindung der Hybridsubstanzen an den M2 AChR möglich ist. Im Rahmen der vorliegenden Arbeit ist es gelungen mittels FRET-Experimenten die Existenz einer ligandenselektiven Rezeptorkonformation des M2 AChR mit allosterischen Liganden nachzuweisen. Darüber hinaus konnte auch ein Bezug zum Auftreten einer funktionellen Selektivität mit allosterischen Liganden hergestellt werden. Die Untersuchung von 19 orthosterischen Liganden hinsichtlich ihres Einflusses auf die Rezeptorkonformation des M2 AChR ergab keinen Hinweis auf eine ligandenselektive Konformationsänderung. Die Betrachtung der orthosterisch-vermittelten Translokation von β-Arrestin2 zeigte, dass zwischen der Effizienz der orthosterischen Testliganden, den M2 AChR zu aktivieren und dem Ausmaß, in dem sie eine β Arrestin2 Translokation induzierten eine direkte Korrelation besteht. Lediglich 5-Methylfurmethiodid rief eine ungleich geringere β-Arrestin2 Translokation hervor, verglichen mit dem Ausmaß an Rezeptoraktivierung. Diese Beobachtung deutet auf die Existenz eines signaling-bias für diesen Liganden hin. Die Untersuchung der dualsterischen Liganden H 1 und 2 bezüglich ihrer Fähigkeit zur Rezeptoraktivierung ergab, dass erst durch eine Verlängerung der Linkerkette, durch die orthosterischer und alloste-rischer Baustein miteinander verbunden sind eine Konformationsänderung des M2 AChR hin zu einer aktiven Konformation erreicht werden kann. Es kann somit angenommen werden, dass in den ursprünglichen Hybridsubstanzen H 1 und H 2 eine zu kurze Linkerkette, durch die keine dualsterische Bindung der Hybride an die allosterische und orthosterische Bindestelle möglich ist, ursächlich für die mangelnde Rezeptoraktivierung des M2 AChR war
A large body of experimental evidence suggests that upon receptor activation G-protein coupled receptors are subject to ligandspecific changes of receptor conformation. The aim of this study was to investigate this phenomenon using the muscarinic M2 acetylcholine receptor (M2 AChR). Muscarinic acetylcholine receptors (mAChR) can be subdivided into five different subtypes (M1-M5). Their involvement in various physiological processes makes them an important target of pharma-cological therapies. With the orthosteric binding site (= binding site of the endogenous ligand) being highly conserved across all five mAChR subtypes, the unselective receptor modulation can lead to severe side effects. Thus the clinical use of drugs modulating muscarinic receptors is currently limited. Allosteric modulation is one attempt to achieve subtype-selective receptor regulation. Since the allosteric binding site of mAChR is less well conserved, it is possible to selectively target one mAChR subtype. As far as allosteric modulation is concerned, the M2 AChR represents a well characterized model with a large number of allosteric modulators being available. For the M2 AChR bitopic ligands which contain an allosteric as well as an orthosteric binding block have been developed as well. In the first part of this study several FRET-sensors of the M2 AChR were designed and characterized. The cyan fluorescent protein (CFP) was fused to the C-terminus of both sensors while the FlAsH (fluorescein arsenical hairpin binder) binding site was inserted into the N-terminal (M2i3-N) or the C terminal (M2i3-C) region of the third interacellular loop (IL). The receptor sensors were characterized concerning ligand affinity, activation of the Gi protein and -arrestin2 translocation and did not display any significant differences compared to the wildtype M2 or the M2 CFP receptor. Various orthosteric as well as allosteric ligands were investigated regarding their affinity and efficacy at both sensors. Using FRET-measurements iperoxo was proven to behave as a superagonist. The characteristics of the allosteric ligands were investigated by measuring the receptor deactivation kinetics and their maximum inhibitory effect on a pre-stimulated receptor. All allosteric test substances displayed faster deactivation kinetics compared to the antagonist atropine and similar EC50 values at both receptor sensors. When investigating the change of receptor conformation of the M2 AChR upon ligand binding there were no ligand selective differences in the FRET-signal detected for either of the 19 orthosteric ligands at both M2 sensors. This data suggest that all orthosteric ligands induced a change in receptor conformation comparable to acetylcholine (ACh). In order to correlate the efficacy of various orthosteric ligands to activate the M2 AChR in FRET-experiments with their effect on downstream signaling pathways, the translocation of  arrestin2 upon receptor activation with orthosteric ligands was investigated using confocal microscopy. Except for 5 methylfurmethiodide all orthosteric ligands induced -arrestin2 translocation to an extent which was comparable to the maximal receptor activation observed with each other ligand, respectively. In contrast 5-methylfurmethiodide evoked a half maximal receptor activation compared to the endogenous ligand ACh while only a minimal translocation of -arrestin2 was observed. The second aim of this study was to investigate the effects of allosteric ligands on the change of receptor conformation of the M2 AChR. The allosteric ligands JK 337 and seminaph more strongly influenced the M2i3-C than the M2i3-N, whilst EHW 477 behaved just the opposite way. This data suggest that the orthosteric ligands induce a conformation of the M2 AChR comparable to ACh. JK 337 and seminaph seem to evoke a greater movement underneath TM 6 compared to TM 5 whereas EHW 477 probably induces a larger movement beneath TM 5. The allosteric ligands were tested via FRET-measurements concerning their ability to activate the Gi protein and to translocate  arrestin2. The activation of the Gi protein as well as the -arrestin2 translocation were selectively influenced by all allosteric ligands. However, due to the experimental setup, a quantification of the effects was not possible. Furthermore the bitopic ligands hybrid 1 and 2 (H 1, H 2) were tested regarding their effect on the receptor conformation of the M2 AChR. While stimulation with H 1 induced FRET signals that were comparable for both receptor sensors, it wasn’t possible to detect any change in the FRET ratio neither of the M2i3-N nor of the M2i3-C with H 2. The lack of effect of H 1 and H 2 in the FRET-experiments was explored using two different approaches: Derivatives of H 1 and H 2, in which the carbon linker between the allosteric and the orthosteric building block had been elongated, were able to induce changes in the FRET ratio. Upon the removal of the allosteric building block a half-maximal activation of both receptor sensors could be detected. However, the mutation of the allosteric binding site did not result in any change of the FRET-signals upon stimulation of the receptor mutants with H 1 or H 2. These data suggest that the carbon linker, which connects the allosteric and the orthosteric building block, is not long enough to enable a simultaneous binding to the allosteric and the orthosteric binding site. Another explanation would be that upon binding of an orthoster the channel between the orthosteric and the allosteric binding site of the M2 AChR is closed because of the change in receptor conformation, hence a stable, dual-steric binding of the hybrid substances to the M2 AChR would not be possible. In the course of this study it was possible to prove the existence of a ligand selective receptor conformation of the M2 AChR with allosteric ligands using FRET-experiments. In addition a connection was found to the occurrence of a functional selctivity with allosteric ligands. The investigation of 19 orthosteric ligands regarding their influence on the receptor conformation of the M2 AChR did not reveal any evidence of the existence of a ligand selective change of the receptor conformation. Regarding the translocation of β arrestin2 induced by orthosteric ligands there was a direct correlation between the efficency of the orthosteric ligands to activate the receptor and the extend of β-arrestin2 translocation observed. With the only exception being 5-methylfurmethiodide which induced far less β arrestin2 translocation compared to the magnitude of the conformational change of the receptor. This data suggest the existence of a signaling bias for this ligand. The analysis of the dualsteric ligands H 1 and H 2 concerning their ability to activate the M2 AChR revealed that an activation of the M2 AChR could just be observed upon elongation of the linker which connects the orthosteric with the allosteric building block. This suggests that the short linker chain of the original hybrid substances inhibited a dualsteric binding to the orthosteric and the allosteric binding site and thus caused the difficency of H 1 and H 2 to activate the M2 AChR

Thesis (Ph. D.)--Florida State University, 2005.
Advisor: Dr. Timothy A. Cross, Florida State University, College of Arts and Sciences, Dept. of Chemistry and Biochemistry. Title and description from dissertation home page (viewed June 13, 2005). Document formatted into pages; contains xiv,160 pages. Includes bibliographical references.