0115 966 7955 Today's Opening Times 10:00 - 20:00 (BST)

Analysis of Peripherin-2 Cone Mutation V268I

Published: Last Edited:

Disclaimer: This essay has been submitted by a student. This is not an example of the work written by our professional essay writers. You can view samples of our professional work here.

Any opinions, findings, conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of UK Essays.

  1. Introduction:

1.1 Composition of Retina:

Eye an organ which is known for its sensory function - Vision, serves as a basis for perception of the world outside. Three layers of tissues enclose the eye, outermost layer sclera, middle layer choroid and inner layer retina. Central macular region of the retina called as fovea consist of photoreceptor cells, where maximum visual acuity is possible due to minimal light scattering. Iris found in region between the cornea and lens acts like an aperture controlling the amount of light that could enter and pass through retina (Siegel and Sapru.2011).

Retina consists of following layers: The outer most primary epithelium layer with pigmented melanin cells absorbs uncaptured light preventing reflection back to rest of retina. The later parts of retina layers involved in sensing and processing of light stimulus are the layers with photoreceptor cells- light sensitive region with rod and cone cells, external limiting membrane, outer nuclear layer, outer plexiform layer, inner nuclear layer, inner plexiform layer and inner most layer with ganglion cell axon fibers. Optically as light when imaged onto retinal photoreceptor layer, light passing through this layer with rods and cones converts light stimuli to electric signal through horizontal cells, further processed through bipolar, amacrine before they reach ganglion cells. From the axons of Central Nervous System (CNS) ganglion cells information is passed in the form of action potential to optic disc and optic nerve signal is received by brain’s visual center. (Ayoub, 2008).

1.2 Composition of Photoreceptors:

Retina is made up of two different types of photo receptors- Rods and cones, the ratio of these photoreceptors vary in different species depending on its habitat being diurnal or nocturnal, in human’s rod cells are in majority with cone cells in a ratio of 20:1 (Mustafi et al., 2009). Rods and cones consist of outer segment, inner segment and synaptic terminal. Towards outer surface retina outer segments are located, these segments contain membranous discs arranged in the form of a stack, and these regions are involved in photo transduction by detecting photons and converting them into electrical signals. Inner segment consist of nucleus, both segments are integrated by a stalk containing microtubules. The synaptic terminal carries out synaptic contact with other cells (Siegel and Sapru. 2011). Retinal pigment epithelial cells phagocytize the aged disks which are shed from distal end when new a new disk is added to the base (Young. 1967).

Fig: 1 Structure of rod and cone cell.

In rods outer segment disc membrane is separated by plasma membrane, disks consist of enclosing lipid bilayer compartment called as disk lumen or intra distal space. Lamellar region of the disk is joined by curved rim region, lamellar region consist of photoreceptor protein, Rhodopsin – an integral membrane protein, and curved rim regions consist of filaments extending to cytoplasmic regions of adjoining disks maintaining structure of outer segment. Rim regions also contain membrane proteins Rim-1, a high molecular weight protein and two other lower molecular weight proteins Peripherin/rds and Rom-1. In cones discs are not completely formed, they are adjoined and are continuous with plasma membrane. (Molday. 1994). These two proteins Peripherin/rds and Rom-1 are homologous in structure, peripherin/rds is expressed both in rods and cones while Rom-1 is only found in rods, they can interact to form dimers and tetramers. (Elizabeth. 2005)

Cones are known to be responsible for vision during daylight and mediate color vision, while rods do not. Axial structure of cones makes them capture light efficiently during day, with fast responsive membrane potential with less integration time. There are three types of cones L, M, S cones which are specific to a region of visible spectrum to which they are found to be sensitive. L cones are sensitive to ÊŽmax~ 553-565, M cones are sensitive to ÊŽmax~ 530-537, S cones are sensitive to ÊŽmax~ 415-430, frequency of photons. (Mustafi et al., 2009). Rods are specialized to work under dim light conditions, rhodopsin is the most abundant protein found in the rods constituting about 85 % of total protein in rod outer segments. (Elizabeth. 2005). Circuit transmitting information to ganglion cells is different in rods and cones, each rod bipolar cell is connected to many rod cells, and many rod bipolar cells are connected by amacrine cell. More convergence make rods better detector of light but reduces spatial resolution. In cones it’s less convergent as each ganglion cell receives input from only one cone bipolar cell, which is connected to single cone cell (Mustafi et al., 2009).

  1. Signal transduction in Photoreceptors:

The signaling cascade in photoreceptors involves from light absorption till signal generation by membrane polarization, resulting in controlling the rate of release of neurotransmitter through synaptic terminal.

In Dark light condition gyanylate cyclases (GCs) actively produce high Cyclic guanosine monophosphate (cGMP) levels, There by the cGMP sensitive cyclic nucleotide-gated (CNG) channel is in its open state and gives rise to the influx of Na+ and Ca+2. Rods and cones are depolarized to ~35 to 45 mV, where outer segment membrane channels are open permeable to Na+ by effluxing k+ balancing cations. This constitutes a circuit known as “Dark or circulating current”. In this condition with the channel opened for influx of ca+2 initiates the release of neurotransmitter from synaptic terminal. In light conditions cation channel in outer membrane is closed, thereby membrane hyperpolarizes towards equilibrium potential for k+, causing halt in neurotransmitter release (Jindrova. 1998).

When photon is absorbed by Rhodopsin- a 40 kDa protein belonging to G protein coupled receptors, consist of a protein opsin, a single polypeptide with seven transmembrane helical segments forming three cytoplasmic loops and a chromophore 11-cis retinal, which binds to lysine, 296 residue on opsin’s seventh helix. In rods and cones photon absorption leads to isomerization of 11-cis retinal form to all trans retinal confirmation and in the surrounding opsin repositioning of cytoplasmic loops occur which in turn activates the protein to Metarhodopsin II (R*) state, which stimulates the heterotrimeric G protein transducin (Hargrave et al.1993; Scot and John . 2008).

Preceding from here all the steps occur in dark state, Transducin stimulates cGMP phosphodiesterase (PDE6) to hydrolyze cGMP to 5’GMP by dissociation of γ subunit from PDE αβ subunits, resulting a vast increase of 5’GMP and decrease in cGMP concentration leading to the closing of the CNG channel. As a consequence, the outer membrane hyperpolarises to ~-70 mV and release of neurotransmitter is halted to bipolar cells, finally light signal information is sent by termination of transmitter release to the brain.

For regeneration of dark current, the Rod cell needs to restore back the concentration of cGMP which is regulated by concentration of Ca+2 and by gyanylate cyclases (GC), Ca+2 molecules are bound by gyanylate activating proteins. In light conditions, where the CNG channels are closed and Ca+2 levels are low, the low level of calcium activates gyanylate activating proteins, which in turn stimulates gyanylate cyclases leading to production of cGMP. Increased level of cGMP causes to open CNG channel and dark current is restored (Jindrova. 1998).

Additional mechanisms are found to be involved to restore the molecules from active to inactive state. Inactivation of Rh* by phosphorylation, catalyzed by rhodopsin kinase (Chenet al.1999). Phosphorylated rhodopsin is blocked by the protein arrestin by binding to it preventing activation of transducin, resulting in breakdown of activated rhodopsin (Xu et al,. 1997). The all trans retinal disassociates from opsin, diffuses to cytosol transported to outersegment then into pigmental epithelium where it is reconverted to 11- cis retinal, then the recycled 11 -cis retinal is transported back to outer segments.

  1. Peripherin- 2:

This membrane protein is named on basis of localization studies done in 1987, which revealed that this protein was found localized around the periphery region of the outer segments, disks in rods and lamellae or disks in cones. (Molday et al,. 1987). In humans peripherin/rds is encoded by cDNA Peripherin/rds with open reading frame of 346 amino acids, weighing 39.3 KDa (Travis et al., 1989). Peripherin is an integral membrane glycoprotein having four trans membrane domains. When photoreceptor cells fail to develop in outer segments it results in retinal disorder named retinal degeneration slow (rds). Amino acid sequence of peripherin in bovine photoreceptor cell is 92.5% identical to protein encoded by rds gene in wild type mouse. Localization of these proteins was also found to be in rod outer segment (ROS) membranes, implying the role of peripherin-2 as a cause in retinal disorder. (Connell et al, .1991). Peripherin protein is also known and addressed with many other names like photoreceptor peripherin, peripherin-2, peripherin/rds, rds/peripherin and rds.

The two dimensional structure proposed consisted of four membrane spanning domains, with its N and C terminals located intracellular in extradiscal space, two extra cellular loops D1 and D2 ( EC1 and EC2) are localized with each containing a N-linked glycan in intradiscal space. The D2 loop among four species mouse, rat, human and bovine is found to be 92% identical, predicted that evolutionary drift in this loop could be a reason for its involvement in protein -protein interactions. (Connell and Molday, 1990; Travis et al., 1991).

Fig: 2 Structure of Peripherin-2.

D2 loop in intradiskal region is found to be key in protein folding and tetrameric subunit formation, changes in amino acids in this loop resulted in either protein misfolding or reduction in sediment coefficient of protein or even both, seven cysteine residues found in this loop to be important for intramolecular disulphide bonds, one of the cysteine residue is responsible for polymerization of tetramers (Loewen and Molday, 2000). When seven conserved cysteine residues were replaced they showed abnormalities, C214S linked mutant for adRP was not normally folding and interacting with rom-1 protein to form tetramer. While in C150S mutant failed to form intra molecular disulphide bonds, these results revealed that cysteine residues were crucial for folding of protein and subunit assembly, failure of these functions are linked to retinal disorders like adRP. (Goldberg et al., 1997).

Human rom-1 protein with 351 amino acids exhibiting 35% identity to peripherin-2, hydropathy profiles revealed that rom-1 and peripherin-2 have a similar topology with four transmembrane domains, one of differences noted in the both the proteins were consensus sequence for N-linked glycosylation is absent in the rom-1 (Molday. 1994). Peripherin homologous protein rom-1 forms disulfide-linked dimers with each other (Bascom et al.,1992). Peripherin-2/rom-1 core native complex was found to exist as a tetramer, membrane curvature was found to induce with tetramer formation when they were reconstructed in lipid vesicles (Kevany et al,. 2013). Peripherin-2 associates with itself and also with its homologue rom-1 in forming homo and heterotetramers core, these tetramers when linked intramolecularly together by disulphide bonding forms octamers and higher order oligomers (Loewen and Molday. 2000). A highly conserved region within the C-terminal domain of peripherin/rds was found to be important for membrane fusion, it forms a complex with melanoregulin (MREG) onto the last five residues of the C-terminus (Gln341-Gly346), membrane fusion is important in organelle biogenesis, disc morphogenesis and disc shedding. (Boesze-Battaglia et al., 2007). Peripherin/rds plays a major role in rod and cone outer segment morphogenesis. In mice, absence of peripherin/rds leads to develop normal photoreceptor inner segments but they fail to form outer segments, these outer segments undergo apoptosis (Nir and Papermaster. 1986).

Peripherin- 2 is found to have differential roles in rod and cone cells with respect to their binding partners. Peripherin 2 found to be binding with the Glutamic acid rich protein (GARP) portion of β subunit of rod CNG channel. This interaction is predicted to anchorage disc rim and rod plasma membrane, but in cone CNG channel such interaction was not observed, as GARP is not expressed in cone CNG channel or in other from. (Conley et al,.2010).

Peripherin-2 links CNGB1a - CNG channel subunit to rhodopsin, these three forms a complex localizing its contact between disk rims and plasma membrane regions. FRET experiments revealed that transmembrane -4 (TM4) of periperin-2 is key for rhodopsin interaction. In peripherin-2, G266D mutation specific to TM4 region, eliminated the specific binding capacity of peripherin-2 with rhodopsin (Becirovic et al,. 2014).

Peripherin-2 being a multifunctional protein and mutations in peripherin/RDS gene results in a broad spectrum of retinal disorders like macular dystrophies, cone and cone-rod dystrophies and retinitis pigmentosa. Studying Peripherin/RDS mutations and protein structure elucidates pathophysiological mechanisms underlying these retinal disorders for effective therapeutic intervention (C.J.F. Boon et al,. 2008).

  1. Zscan29:

At present, very less information is known and available regarding the function, structure and localization of this protein Zscan29. mRNA specific for this protein were found to be expressed in retina through RT- PCR experiments from mice different tissues.

In mice 5 different isoforms of this protein were found, with longest isoform having 869 amino acids (aa), and other isoforms were of 834 aa, 548 aa,265 aa,206 aa. The isoform with 265 aa was found to interact with the C- terminus of CNGB1a - sub unit of CNG channel. In long isoforms with 869aa and 834aa, a SCAN domain, a DUF2 domain, 2 GT-1 domains and 6 zinc finger domains were found. The scan domain is predicted to be a DNA binding domain and involved in transcription regulation, as this domain in other proteins were also found to be associated as transcription factors. DUF-2 Domain function is unclear and yet to be known, Two GT-1 domains of 79 aa each, were predicted to play a major role in light sensing mechanisms by binding GT trihelix transcription factors. (___citation__). In Arabidopsis GT-1 is predicted to respond to light signals via calcium dependent phosphorylaton, after GT-1 binding to GT cis element of light inducing gene (Bauer et al).

Fig: 3 Isoforms of Zscan29 protein with its domains.

  1. Aims of this work:
  1. Analysis of Peripherin-2 cone mutation V268I:

Verification of the interaction of WT peripherin-2 and peripherin-2 with mutation at V268I position with S-opsin, M-opsin and Rhodopsin via co immunoprecipitation from HEK293 cells.

Interaction and localization of Peripherin-2, V268I mutation in mouse retina.

  1. Analysis of Peripherin-2 mutations at position P210:

Verification of the interaction of peripherin-2 protein with mutations at P210 position with wild type Peripherin-2 and its homolog Rom-1 via coimmunoprecipitation from HEK293 cells.

In vitro imaging of HEK293 cells expressing peripherin-2 protein with mutation at P210 position.

Expression and localization of Peripherin-2, P210L and P210R mutations in mouse retina.

  1. Affinity purification of Anti-Zscan29 antibody and checking antibody efficiency.

To export a reference to this article please select a referencing stye below:

Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.
Reference Copied to Clipboard.

Request Removal

If you are the original writer of this essay and no longer wish to have the essay published on the UK Essays website then please click on the link below to request removal:

More from UK Essays

We can help with your essay
Find out more
Build Time: 0.0024 Seconds