published_or_final_version / Anatomy / Master / Master of Philosophy

Solid-state NMR spectroscopy was used to site-specifically investigate the protein-water interface of a seven alpha-helical transmembrane protein, Anabaena sensory rhodopsin (ASR). Water-edited experiments, which employ a T2-filter to select for mobile protons, provided a means to detect residues which appear to be in close contact to water molecules, and to gain insights about the water-protein interface of ASR. First, through the application of Lee-Goldburg homonuclear decoupling, it was determined that polarization transfer across this interface is dominated by through-space interaction mechanisms, as opposed to chemical exchange. A series of two-dimensional experiments were also performed to detect polarization transfer along the backbone and to the sidechains of the protein. Residues located in solvent-accessible regions of the protein, such as the B-C loop, were found to obtain polarization quickly, as expected, and in agreement with previous H/D exchange data. Residues known to be in contact with bound crystal water molecules were also detected. In addition to these, we found new residues which appear to be in contact with water, indicating additional HN-H2O interactions, or additional contacts with bound water molecules. Most of these residues were located beside exchangeable regions of ASR. Sidechains of residues located in the cytoplasmic side of helix F were seen to be in close contact with mobile water molecules, supporting evidence of a hydrophilic chain along the cytoplasmic half of the protein, which is suggested to cause a functional outward tilt of the cytoplasmic half of helix F upon light-activation.

While nocturnal basal snakes have rod-dominant retinae, diurnal garter snakes have all-cone retinae. Previous work from the Chang lab identified three visual pigments expressed in the photoreceptors of Thamnophis proximus: SWS1, LWS and RH1. I further characterized T. proximus photoreceptors using electron microscopy, immunohistochemistry, and in vitro protein expression. T. proximus have four types of morphological cones: double cones, large single cones, small single cones, and very small single cones. Some small single cones have rod-like features, such as rod-like outer-segment membranes and a lack of micro-droplets. Immunohistochemistry showed that rod-specific transducin is expressed in some T. proximus photoreceptors. In vitro expression of T. proximus RH1 produced a functional rhodopsin with λmax at 485nm, which corresponds to microspectrophotometry measurement from some small single cones. Current results suggest that small single cones of T. proximus may have evolved from ancestral rods, and secondarily acquired a cone-like morphology as adaptation to diurnality.

<p>Rod photoreceptors are specialized neurons responsible for capturing photons and translating visual information into electrical signals. Visual signal transduction in rods is confined to the unique outer segment organelle, a modified primary cilium consisting of a stack of hundreds of flattened disc membranes enveloped by a single plasma membrane. By concentrating important signaling molecules on disc membranes, the outer segment provides an ideal biochemical environment for the production of vision with high sensitivity and temporal resolution.</p><p>This dissertation focuses primarily on a molecular dissection of two multifunctional outer segment proteins, R9AP and rhodopsin, and also reassesses the localization of Glut1, a third protein formerly believed to reside in the outer segment. All three experimental lines relied on in vivo expression of novel protein constructs in vertebrate rods using several gene delivery strategies: conventional transgenics, retinal electroporation, and retinal infection with recombinant adeno-associated virus.</p><p>The tail-anchored protein R9AP, in conjunction with RGS9-1 and G-beta5, comprises the transducin GTPase activating complex, which catalyzes the rate-limiting step in rod photoresponse recovery. In addition to maximizing the enzymatic activity of the complex, R9AP is responsible for both the post-translational stability and outer segment targeting of RGS9-1-G-beta5. We investigated the mechanism behind R9AP's poorly understood function in protecting RGS9-1-G-beta5 from proteolysis and found that it is performed simply by recruiting the complex to cellular membranes and can be entirely dissociated from R9AP's outer segment targeting function. Furthermore, we demonstrated that replacement of R9AP's transmembrane domain with a lipid anchor preserves the ability of the GTPase activating complex to function in outer segments.</p><p>Rhodopsin, the visual pigment of rods, has a second important, yet poorly defined, function as a rod outer segment building block: outer segments disc membranes fail to form in the absence of rhodopsin. Our goal was to identify the molecular features of rhodopsin mechanistically involved in outer segment morphogenesis by designing artificial membrane proteins that could fully substitute for rhodopsin in performing this function. We observed that rhodopsin's C-terminal VXPX outer segment targeting motif is unnecessary for outer segment disc formation since it could be replaced with a targeting motif from an unrelated protein, peripherin. Furthermore, we obtained surprising evidence that rhodopsin's role in this process is limited to providing an abundance of transmembrane protein material to disc membranes.</p><p>Finally, while attempting to find a targeting motif to substitute for the VXPX motif of rhodopsin, we made an unexpected observation that the facilitative glucose transporter Glut1, long thought to reside in the outer segment, is actively excluded from this organelle. This revises our understanding of the energy flow in rods by showing that the outer segment is entirely dependent on the inner segment for its energy supply.</p> / Dissertation

G protein-coupled receptors (GPCRs) are signal mediators that have a prominent role in the regulation of physiological processes and they make up the targets for 30-45% of all drugs. Papers I and II describe the discovery of new human GPCRs belonging to the Rhodopsin family, a family which contains many common drug targets. The new receptors have only weak relationships to previously known GPCRs. However, they have been evolutionary conserved in several species and most of them display distinct expression patterns. In paper III we identified new human GPCRs belonging to the Adhesion family, which is characterised by very long N-termini containing conserved domains. The different compositions of conserved domains as well as the expression patterns suggest that the Adhesions can have several different functions. In paper IV we revealed remarkable species variations in the repertoires of Trace Amine-Associated Receptors (TAARs), which are relatives of the biogenic amine receptors. The human, mouse and rat TAAR genes are located in only one locus and are therefore most likely the result of gene tandem duplications. 47 of the 57 zebrafish TAARs were mapped to nine different loci on six chromosomes containing from 1 to 27 genes each. This study suggests that the TAARs arose through several different mechanisms involving tetraploidisation, block duplications, and local duplication events. Papers V and VI are overall analyses of the repertoires of GPCRs in humans, mice and rats; which contain approximately 800, 1800 and 1900 members, respectively. The repertoires were compared to distinguish between species-specific and common (orthologous) members, something which is important for example when predicting drug effects from experiments in rodents. The Glutamate, Adhesion, Frizzled and Secretin families show no or very little variation between human and rodents, whereas the repertoires of olfactory, vomeronasal and Taste2 receptors display large differences between all three species.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [123]-141).

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 91-102).

Thesis (Ph. D.)--Michigan State University. Dept. of Chemistry, 2008. / Title from PDF t.p. (viewed on Aug. 20, 2009) Includes bibliographical references (p. 203-206). Also issued in print.

Στην παρούσα εργασία μελετήθηκε η γενετική δομή και οι φυλογενετικές σχέσεις είκοσι τριών πληθυσμών της A.boyeri που προέρχονταν από λίμνες/λιμνοθάλασσες και θαλάσσιες περιοχές του ελλαδικού χώρου, καθώς και ενός πληθυσμού από τη λίμνη Iznik της Τουρκίας. Πιο συγκεκριμένα, προσδιορίστηκε μέρος της αλληλουχίας των μιτοχονδριακών γενετικών τόπων της υπομονάδας I της κυτοχρωματικής οξειδάσης (COI), του βρόγχου εκτόπισης (D-loop) και του κυτοχρώματος b (Cytb). Επιπλέον, προσδιορίστηκε η αλληλουχία ενός πυρηνικού γενετικού τόπου, της ροδοψίνης (Rh). Πραγματοποιήθηκε φυλογενετική ανάλυση των αλληλουχιών και κατασκευάστηκαν φυλογενετικά δένδρα με τις μεθόδους της Σύνδεσης Γειτόνων (NJ), Μέγιστης Φειδωλότητας (ΜΡ) και Μπεϊσιανής Συμπερασματολογίας (ΒΙ). Παρά τη διαφορετική τοπολογία των δένδρων που προέκυψαν, γεγονός που δεν κατέστησε εφικτό τον προσδιορισμό των φυλογενετικών σχέσεων μεταξύ των υπό μελέτη πληθυσμών, η ύπαρξη τριών διαφορετικών τύπων πληθυσμών διαπιστώθηκε τόσο από την ανεξάρτητη όσο και από τη συνδυασμένη ανάλυση των αλληλουχιών. Ο πρώτος τύπος πληθυσμού, ο «λιμναίος», περιελάμβανε τους πληθυσμούς από τις λίμνες/λιμνοθάλασσες καθώς και τους πληθυσμούς από τις θαλάσσιες περιοχές της Καλύμνου, της Κύμης και των Ν. Μουδανιών. Ο δεύτερος τύπος περιελάμβανε όλους τους θαλάσσιους πληθυσμούς (πρώτος «θαλάσσιος» τύπος), με εξαίρεση του πληθυσμούς από την Πρέβεζα, την Εύβοια και την Κω που συνιστούσαν τον τρίτο τύπο πληθυσμού (δεύτερος «θαλάσσιος» τύπος). Επισημαίνεται ιδιαίτερα ότι ο πυρηνικός μοριακός δείκτης που χρησιμοποιήθηκε έφερε αρκετή ποικιλότητα ώστε να επιβεβαιώσει τα παραπάνω αποτελέσματα. Οι «λιμναίοι» πληθυσμοί εμφάνισαν σαφές φυλογεωγραφικό πρότυπο, διακρίνοντας τους πληθυσμούς του Αιγαίου και της Τουρκίας από τους λιμναίους/λιμνοθαλάσσιους του Ιονίου Πελάγους. Επιπλέον, διαπιστώθηκε πως ο πληθυσμός από τον Αμβρακικό διαφοροποιείται αρκετά από τους υπόλοιπους «λιμναίους» πληθυσμούς, ενώ αξιοσημείωτη είναι και η συσχέτιση που βρέθηκε ανάμεσα σε αυτόν τον πληθυσμό και πληθυσμών από την Αδριατική. Όσον αφορά τους θαλάσσιους πληθυσμούς, κανένας από τους δύο τύπους δεν εμφάνισε συγκεκριμένο φυλογεωγραφικό πρότυπο. Επιπλέον, διαπιστώθηκε πως οι πληθυσμοί από την Πρέβεζα, την Εύβοια και την Κω αντιστοιχούν στον εστιγμένο (punctuated) τύπο πληθυσμού, όπως έχει περιγραφεί, ο οποίος δεν έχει έως σήμερα αναφερθεί στην ανατολική Μεσόγειο. Ωστόσο, σύμφωνα με τα αποτελέσματα μας, τα μαύρα στίγματα κάτω από την πλευρική γραμμή δεν μπορούν να αποτελέσουν διαγνωστικό στοιχείο για τη διάκριση των δύο τύπων θαλάσσιων πληθυσμών (punctuated, nonpunctuated), καθώς στον τύπο αυτό εντάσσονται άτομα που είτε φέρουν μαύρα στίγματα είτε όχι, και το αντίστροφο. Από τον πολλαπλασιασμό με τη μέθοδο της αλυσιδωτής αντίδρασης πολυμεράσης (PCR) του μιτοχονδριακού τμήματος Cytb και τον προσδιορισμό της αλληλουχίας του, διαπιστώθηκε η ύπαρξη μίας ένθεσης ανάμεσα στο tRNA του γλουταμινικού οξέος και το Cytb. Η ένθεση αυτή είχε μέγεθος περίπου 200bp στα «λιμναία» άτομα και λίγο μικρότερο (~150bp) στα άτομα που εμφάνισαν το πρότυπο punctuated (εστιγμένα) (Πρέβεζα, Εύβοια, Κως), ενώ απουσίαζε από τους υπόλοιπους θαλάσσιους πληθυσμούς. Βάσει των παρόντων αποτελεσμάτων και σε συνδυασμό με αντίστοιχα αποτελέσματα προηγούμενων μελετών επιβεβαιώνεται η άποψη σύμφωνα με την οποία η παρουσία ή απουσία της συγκεκριμένης ένθεσης μπορεί να χρησιμοποιηθεί ως διαγνωστικός χαρακτήρας για τη διάκριση των τριών διαφορετικών τύπων πληθυσμών της A.boyeri. / The genetic differentiation and the phylogenetic relationships of twenty three Atherina boyeri populations originating from lakes/lagoons and marine sites of Greece, as well as from a population from lake Iznik in Turkey were investigated. More specifically, the partial sequence of the mitochondrial genes cytochrome oxidase I (COI), cytochrome b (cytb) and control region were determined. The partial sequence of the nuclear proteincoding gene rhodopsin (Rh) was determined, as well. Phylogenetic analysis of the sequences was conducted using the methods: Neighbor-Joining (NJ), Maximum Parsimony (MP) and Bayesian Inference (BI). Even though trees presented different topologies, and thus the phylogenetic relationships among populations were not able to be resolved, the presence of three main population forms was observed both from the separate and combined analyses of datasets. The first population type (‘lagoon’ type) contains all the lagoon/lake populations, as well as three populations (Kymi, Kalymnos and Nea Moudania) originating from marine environment. The second type includes nearly all marine populations (first ‘marine’ type), excluding specimens collected from three geographical distant areas (Preveza, Evvoia and Kos), which form the third A. boyeri population type (second ‘marine’ type). It is worth mentioning that the nuclear marker used was proved to carry sufficient diversity in order to confirm the above results. The ‘lagoon’ type populations presented a clear phylogeographic pattern, with the populations from Aegean Sea and Turkey forming a distinct group from the populations from the Ionian Sea. Moreover, the population from Amvrakikos appeared to be distinct from the rest ‘lagoon’ type populations and a remarkable correlation of this population and populations from Adriatic Sea was found. On the other hand, none of the two ‘marine’ type populations presented a specific phylogeographic pattern. Additionally, it was found that the populations from Preveza, Evvoia and Kos correspond to the ‘punctuated’ marine type, as has been described, which was not previously reported in eastern Mediterranean Sea. However, the fact that in the present study a population (Evvoia) without black spots on the flanks is clustered with the punctuated form and vice versa (specimens from Limnos presented black spots but was clustered with the non-punctuated ‘marine’ type populations), suggests that differences in color pattern do not seem to be a sufficient marker for discriminating the two marine types. From the PCR amplification of cytb and determination of its sequence the presence of approximately 200bp intergenic spacer between tRNA glutamic acid (tRNAGlu) and cyt b genes was observed among the ‘lagoon’ type specimens and a smaller one among the specimens from Preveza, Evvoia and Kos. The intergenic spacer was absent from the rest marine populations. Thus, our results support the suggestion that this character is conserved and capable to be used for distinguishing the different forms.

Rhodopsin-linked retinitis pigmentosa (RP) is the most common form of autosomal dominant RP, an inherited retinal degeneration, in which rod degeneration is followed by secondary cone loss leading to loss of vision and blindness. The overall objective of this work was to develop an optimized gene replacement therapy, delivering the rhodopsin gene for rhodopsin- linked RP and establish whether secondary cone loss can be delayed. A fast-acting single mutant serotype 8 self-complementary adeno-associated virus vector was produced containing the human rhodopsin promoter and the human rhodopsin coding sequence. In vivo studies in rhodopsin knockout mouse showed that the vector administration led to widespread and robust expression of the transgene. Subretinal injection of the vector into three-week pups of rhodopsin knockout mice with cones expressing green fluorescent protein showed restoration of rod-derived electroretinogram (ERG) responses, and preservation of cone- driven ERG responses three months after injection. Similarly, the longitudinal follow-up with confocal scanning ophthalmoscopy found preservation of fluorescent cones up to three months after injection. Overall, these data provided evidence that the designed vector resulted in significant benefit to rod photoreceptors as well as in delay in secondary cone degeneration and built a basis for future use of this vector on dominant models of RP.