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PDF hosted at the Radboud Repository of the Radboud University Nijmegen The following full text is a publisher's version. For additional information about this publication click this link. Please be advised that this information was generated on and may be subject to change. Melanopsin, a unique non-visual photosensitive pigment: A challenge from expression to purification Nazhat Shirzad-Wasei Cover design: Nazhat Shirzad-Wasei Printing: Gildeprint - the Netherlands 2016 Nazhat Shirzad-Wasei The research presented in this thesis was performed at the Department of Biochemistry, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, Nijmegen, the Netherlands. This research was supported by funds to Prof. Wim de Grip from the Council for Chemical Sciences of the Netherlands Organization for Scientific Research (NWO-CW project ) Melanopsin, a unique non-visual photosensitive pigment: A challenge from expression to purification Proefschrift ter verkrijging van de graad van doctor aan de Radboud Universiteit Nijmegen op gezag van de rector magnificus prof. dr. J.H.J.M. van Krieken, volgens besluit van het college van decanen in het openbaar te verdedigen op vrijdag 28 oktober 2016 om uur precies door Nazhat Shirzad-Wasei geboren op 17 oktober 1979 te Kabul, Afghanistan Promotoren Prof. dr. W.J. de Grip (Universiteit Leiden) Prof. dr. R. Brock Copromotor Dr. G.J.C.G.M. Bosman Manuscriptcommissie Prof. dr. B. Wieringa Prof. dr. G.J.M. Pruijn Prof. dr. A.P.IJzerman (Universiteit Leiden) Melanopsin, a unique non-visual photosensitive pigment: A challenge from expression to purification Doctoral Thesis to obtain the degree of doctor from Radboud University Nijmegen on the authority of the Rector Magnificus prof. dr. J.H.J.M. van Krieken, according to the decision of the Council of Deans to be defended in public on Friday, October 28, 2016 at hours by Nazhat Shirzad-Wasei Born on October 17, 1979 in Kabul, Afghanistan Supervisors Prof. dr. W.J. de Grip (Leiden University) Prof. dr. R. Brock Co-supervisor Dr. G.J.C.G.M. Bosman Doctoral Thesis Committee Prof. dr. B. Wieringa Prof. dr. G.J.M. Pruijn Prof. dr. A.P. IJzerman (Leiden University) Table of contents Chapter 1 General introduction 9 Chapter 2 Chapter 3 Chapter 4 Chapter 5 Functional Expression, Targeting and Ca2+ Signaling of a Mouse Melanopsin-eYFP Fusion Protein in a Retinal Pigment Epithelium Cell Line Large scale expression and purification of mouse melanopsin-l in the baculovirus expression system Construction and expression of a chimeric receptor containing the C-terminal tail of melanopsin-l, Rho-C-Mel(His) Rapid transfer of overexpressed integral membrane protein from the host membrane into soluble lipid nanodiscs without previous purification Chatpter 6 General discussion, and Major Perspectives 161 Summary 169 Samenvatting 173 Abbreviations 179 References 181 List of publications 201 Curriculum Vitae 203 Dankwoord/Acknowledgments 205 To a king, Intellect is his glory. So, his wisdom and intelligence Are his strong army. (Asad Tusi) خرد شاه را بهترین افسر است هش و دانشش نیکتر لشکر است )اسدى طوسى( 1 General introduction Part of this chapter is adapted from: Heterologous Expression of Melanopsin: Present, Problems and Prospects Nazhat Shirzad-Wasei and Willem J. de Grip Department of Biochemistry, Radboud University Medical Center, Radboud Institute for Molecular Life Sciences, P.O. Box 9101, NL-6500 HB Nijmegen, The Netherlands Progress in Retinal and Eye Research, 2016, 52: General introduction G-protein-coupled receptors G-protein-coupled receptors (GPCRs) are regarded as the largest superfamily of integral membrane proteins containing seven transmembrane α helices (Tate and Grisshammer, 1996). More than 800 GPCRs have been identified in the human genome, representing 2-3% of the coding sequences (Baneres et al., 2011). They play a crucial role in transmembrane signal transduction by binding extracellular ligands (e.g. neurotransmitters, peptides and hormones), participating in the regulation of major biological processes such as neurotransmission, proliferation, differentiation, chemotaxis or inflammation and are the target of approximately 30% of current pharmacological drugs. Ligand binding is thought to trigger a conformational change in the receptor, resulting in the activation of heterotrimeric guanine nucleotide-binding proteins (G proteins) at the intracellular face of the plasma membrane. Because of their central role in biological systems, it is therefore critical to gain detailed knowledge of their structure and their dynamics to understand their function. A major difficulty encountered in the study of GPCRs is obtaining the target protein in sufficient quantities for functional and structural studies. Membrane proteins are usually present at low levels in their native membrane and thus usually heterologous overexpression is attempted. The main subject of this thesis, melanopsin, is a light-sensitive protein and member of the opsin subfamily of GPCRs. 1 Light-triggered physiology Life on earth has evolved around a predictable light and dark cycle. And, all organisms have an inner clockwork mechanism which allows them to anticipate to predictable changes, thus permitting them to be prepared to respond to future challenges in an optimal way. This endogenous circadian system ( derived from the Latin words circa diem, about a day) is indeed an evolutionarily, highly conserved feature of virtually all light sensitive organisms from cyanobacteria to humans suggesting that such a system functions as an orchestral conductor that arranges the rhythm and synchrony 11 Chapter 1 of the physiological processes in the body. Circadian rhythms are biological cycles that have period of about a day. Many behavioral, physiological and biochemical facets of life are dependent on circadian organization. This internal clock mechanism coordinates biochemical, physiological and behavioral processes to maintain synchrony with the environmental cycles of light, temperature and nutrients. The main task of the circadian timing system can be viewed as the optimization of metabolism and energy utilization for sustaining life processes in the organism. Most mammalian physiology is influenced at least to some extent by the circadian clock (Schibler et al., 2003). For example, rest and activity, heart rate, blood pressure, liver and renal plasma flows, bile and urine production, intestinal peristalsis, secretion of digestive enzymes into the gastrointestinal tract, major endocrine functions and metabolism and other physiological variables exhibit such daily oscillations. In mammals, a master pacemaker resides in the suprachiasmatic nucleus (SCN) of the brain s hypothalamus which drives these circadian rhythms and orchestrates countless clocks in most peripheral cell types (Ralph et al., 1990). The SCN is a self-sustaining oscillator, generating endogenous rhythms whose period length can deviate up to 20 minutes from the 24 h day. This rhythm will slowly drift out of phase from the environmental rhythm, hence must be tuned daily to the day/night cycle, a process known as photoentrainment (Roenneberg and Foster, 1997). This photic information reaches the SCN through a dedicated monosynaptic pathway, the retinohypothalamic tract (RHT) originating in the retina (Figure 1.1) (Gooley et al., 2001;Hattar et al., 2002). The RHT is the projection from only a minor fraction ( 1 %) of the retinal ganglion cells (Provencio et al., 1998). It should be noted that the photic regulation of the circadian system is qualitatively different from the processes involved in generating a visual image. A visual image is formed when light is detected as a measure of brightness in a certain point of space, whilst the circadian photoreceptor acts as an irradiance detector, providing a measure of the gross amount of light in the environment (Foster, 2002). 12 General introduction Figure 1.1. Schematic summary of brain regions and circuits influenced by intrinsically photosensitive retinal ganglion cells (iprgcs). The iprgcs and their axons are shown in dark blue, their principal targets in red. Projections of iprgcs to the suprachiasmatic nucleus (SCN) form the bulk of the retinohypothalamic tract and contribute to photic entrainment of the circadian clock. Another direct target of iprgcs is the olivary pretectal nucleus (OPN), a crucial link in the circuit underlying the pupillary light reflex, shown in light blue (fibers) and purple (nuclei). Other targets of iprgcs include two components of the lateral geniculate nucleus of the thalamus, the ventral division (LGNv) and the intergeniculate leaflet (IGL). This figure is adapted from Berson et al Reproduced with permission of Elsevier Science Ltd. 1 Anatomy of the eye and retina In mammals the eyeball is the only sensory organ for the detection and translation of light into electrochemical signals that are passed to the brain (Figure 1.2). It is surrounded by a protective orbital cavity of the skull. The outermost structures of the eyeball are primarily avascular, providing structural support and a mucous layer to keep the eye moist. The posterior eye is composed of concentric membranous, vascular, and neural layers that convert light into neural signals. The choroid is a highly vascularized membrane that lies between the retina and the sclera, providing nourishment to the eye, especially the retina. The retina receives the focused image formed by the lens and cornea and converts it into neural signals transmitted by the afferent fibers of the optic nerve. The neural signals are transferred via optic tracts to the occipital region of the brain, where visual images are integrated. The anterior eye contains the structures essential for light transmittance: the iris, ocular lens, and ciliary body. A circular opening in the center of the iris, called pupil, 13 Chapter 1 controls the amount of light that enters the eye by means of sphincter and dilator muscles. The ocular lens lies directly behind the pupil and is attached circumferentially to the ciliary body via suspensory ligaments called zonules. The ciliary body changes the shape of the lens for near and far vision and secretes aqueous humor, the main nutrient source for the avascular anterior structures. Figure 1.2. Cross-section of a human eye. Adapted from: The spaces created by the anatomic structures are the anterior chamber, posterior chamber, and vitreous space. The vitreous space, located posterior to the ocular lens, contains vitreous humor, a gel-like substance that holds the lens and retina in place and provides an additional refractive medium for the incoming light. The retina is approximately 0.5 mm thick and lines the back of the eye. It is a highly organized sheet of neural tissue, composed of five classes of neurons (rod and cone photoreceptors, bipolar cells, horizontal cells, amacrine cells and ganglion cells), two types of glial cells (microglia and Müller cells) and vascular endothelium cells (Figure 1.3). The optic nerve contains the ganglion cell axons (the output neurons of the retina) running to the brain and, additionally, blood vessels that branch out into the retina to vascularize the retinal layers and neurons. 14 General introduction A radial section of a portion of the retina reveals that the photoreceptors lie posterior in the retina against the pigment epithelium and choroid. Light must, therefore, travel through the entire retinal layer before striking and activating the rods and cones. Subsequently the absorption of photons by the visual pigment of the photoreceptors is translated into first a biochemical message and then an electrical message that can modulate the activity of all the projected neurons of the retina. 1 Figure 1.3. An overview of the organization of the retina. Courtesy of utah.edu/ Establishment and maintenance of proper cell-cell interaction are essential processes in retinal development and homeostasis. All vertebrate retinas are composed of three layers of nerve cell bodies and two layers of synapses which are rich in interneuronal contacts (Figure 1.3). The outer nuclear layer (ONL) contains cell bodies of the rods and cones, the inner nuclear layer (INL) contains cell bodies of the bipolar, horizontal and amacrine cells and the ganglion cell layer contains cell bodies of ganglion cells and amacrine cells. Separating these nerve cell layers, there are two areas where synaptic contacts occur. The first area is the outer plexiform layer (OPL) where rod and cones are interconnected, and vertically 15 Chapter 1 running bipolar cells and horizontally oriented horizontal cells are situated. The second area is the inner plexiform layer (IPL), and it functions as a relay station for the vertical-information-carrying nerve cells, the bipolar cells, to connect to ganglion cells. In addition, different varieties of horizontally- and vertically-directed amacrine cells somehow interact in further networks to modulate and integrate the ganglion cell signals. The complex neural processing in the retina culminates in the inner plexiform layer where eventually the coded visual image is transmitted to the brain along the optic nerve. Müller cells on the other hand, belong to the class of non-neuronal cells called glia and comprise 90% of the retinal glia. These cells interact with most of the neurons in the retina (Kubrusly et al., 2005).They traverse the entire retina in a radial direction from the ganglion cells to the photoreceptor cell bodies. They are considered to be the principal glial cells of the retina, because of their ability to perform functions that astrocytes (also known collectively as astroglia which are characteristic star-shaped glial cells in the brain and spinal cord) effect in other regions of the central nervous system (Guidry, 2005;Limb et al., 2002). In addition to providing structural support within the retina and blood vessels, Müller cells regulate the control of ionic concentrations, degradation of neurotransmitters, and removal of certain metabolites and may be important for the normal development of photoreceptor cell connections with other retinal neurons and for the maintenance of cone functionality. They also prevent aberrant photoreceptor migration into the subretinal space (Limb et al., 2002). The visual photoreceptor cells The human retina contains approximately 125 million photoreceptors which can be subdivided into two types. They are named rods and cones after their morphology. Approximately 95% of the photoreceptor population consists of rods, which are located in the peripheral retina. The remaining 5% of cones photoreceptors are predominantly found in the foveal area, the center of our visual field (see Figure 1.2 and 1.4). Primates and birds have true foveas (some birds, notably hawks and 16 General introduction eagles, actually possess two foveas per retina!), other species such as squirrels, cats, dogs, deer, etc., have a less pronounced regional specialization, and possess what is called an area centralis or a visual streak. The optic nerve receives all of the signals created in the eye and transfers them to the brain to be processed. The nerve exits in the posterior of the eye, and that place lacks photoreceptors (blind spot). 1 Figure 1.4. Distribution of rods and cones in the human retina. Graph illustrates that cones are present at a low density throughout the retina, with a sharp peak in the center of the fovea. Conversely, rods are present at high density throughout most of the retina, with a sharp decline in the fovea. The increased density of cones in the fovea is accompanied by a striking reduction in the diameter of their outer segments. Source: adapted with permission from Rods are extremely light sensitive and are of greatest importance under dim light conditions where discriminating colours is not of prime importance (scotopic vision). All rods express only one type of visual pigment, rhodopsin (Nathans and Hogness, 1984;Wald, 1968). The cones are about 10 3 fold less photosensitive, hence require higher levels of light to generate a signal (photopic vision). However they offer a much higher spatial and temporal resolution than rods. In addition the cones in a retina usually cover different spectral ranges, and are therefore responsible for colour vision. In man and some other primates, colour vision is mediated by three types of cones (trichromats), each containing a distinct visual pigment absorbing maximally at 420 nm (blue or short wavelength sensitive pigment, SWS), 17 Chapter nm (green sensitive pigment) and 560 nm (red sensitive pigment) (Nathans et al., 1986). The latter two belong to the long wavelength sensitive (LWS) class of cone pigments. Most mammals possess only one LWS pigment in addition to a SWS pigment (dichromats). The rods and cones are the actual sites of transduction of light energy into neuronal signals. They are, in essence, exceptionally specialized bipolar neurons, which have developed some structural features to carry out this task. Rods and cones have a similar structure, but there are some differences. Both photoreceptors are composed of an inner segment and an outer segment, as well as a cell body and synaptic terminal (Figure 1.5). The main difference between rods and cones is in the outer segment, where the visual pigment is located and light absorption and subsequent signal transduction takes place. The capture of individual photons by the photopigment molecules in the disc membranes is what initiates neural signaling. The characteristically shaped outer segment at the distal end of the rod cell is composed of an intracellular stack of numerous flattened membrane disks. In contrast, the shorter cone outer segments are composed of a stack of plasma membrane invaginations called sacs. The disks from the rod cell contain the visual pigment, rhodopsin. In primates the sacs of the cone photoreceptors contain a single one of the above mentioned cone pigments. Disks and sacs are formed by invagination of the plasma membrane at the ciliary region and migrate, while the outer segment is under continuous renewal, through the entire length of the outer segment. They are removed at the distal end of the outer segment through phagocytosis by the retina pigment epithelium cells (Bok, 1985). The photoreceptor cell body harbors the nucleus, support organelles (mitochondria, ribosomes, endoplasmic reticulum, synaptic vesicles, etc.), and the axon terminal which contains neurotransmitter vesicles for signal transduction onto the bipolar and horizontal cells. The outer and inner segments of the photoreceptors are connected by the cilium. This cilium is a primary cilium which has an axonemal structure consisting of a circular array of nine pairs of microtubules (9 + 0 arrangement). They can be found on most eukaryotic cells. Primary cilia, which are usually nonmotile, are thought to have either a chemosensory or mechanosensory function. These cilia often contain high concentrations of receptors and are ideally positioned to interact with their environment, a good starting point for the highly 18 General introduction specialized cone and rod photoreceptors that respond to light (Calvet, 2003). In addition, photoreceptor cells are packed, together with processes of Müller glia cells which serve for structural and metabolic support, in the ONL. The establishment and maintenance of apical basal polarization and cell adhesion is crucial for functionality and survival of the photoreceptors. At the apical site of the ONL, an adhesion belt, named the outer limiting membrane (OLM) contains specialized adherence junctions, which are present between the photoreceptors and Müller glia cells. The adherence junctions consist of multi-protein complexes and are linked to the cell skeleton for the cell shape (Tepass, 2002;van de Pavert et al., 2004). 1 Figure 1.5. Schematic overview o
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