RPE65

Last updated
RPE65
Protein RPE65 PDB 3FSN.png
Identifiers
Aliases RPE65 , BCO3, LCA2, RP20, mrd12, sretinal pigment epithelium-specific protein 65kDa, retinal pigment epithelium specific protein 65, retinoid isomerohydrolase, p63, retinoid isomerohydrolase RPE65
External IDs OMIM: 180069; MGI: 98001; HomoloGene: 20108; GeneCards: RPE65; OMA:RPE65 - orthologs
Orthologs
SpeciesHumanMouse
Entrez

6121

19892

Ensembl

ENSG00000116745

ENSMUSG00000028174

UniProt

Q16518

Q91ZQ5

RefSeq (mRNA)

NM_000329

NM_029987

RefSeq (protein)

NP_000320

NP_084263

Location (UCSC) Chr 1: 68.43 – 68.45 Mb Chr 3: 159.3 – 159.33 Mb
PubMed search [3] [4]
Wikidata
View/Edit Human View/Edit Mouse

Retinal pigment epithelium-specific 65 kDa protein (also known as RPE65) is a retinoid isomerohydrolase enzyme of the vertebrate visual cycle. [5] [6] RPE65 is expressed in the retinal pigment epithelium (RPE, a layer of epithelial cells that nourish the photoreceptor cells) and is responsible for the conversion of all-trans-retinyl esters to 11-cis-retinol during phototransduction. [7] 11-cis-retinol is then used in visual pigment regeneration in photoreceptor cells. [8] [9] RPE65 belongs to the carotenoid oxygenase family of enzymes. [8]

Contents

Function

RPE65 is a critical enzyme in the vertebrate visual cycle found in the retinal pigmented epithelium. It is also found in rods and cones. [10] The photoisomerization of 11-cis-retinal to all-trans-retinal initiates the phototransduction pathway through which the brain detects light. All-trans-retinol is not photoactive and therefore must be reconverted to 11-cis-retinal before it can recombine with opsin to form an active visual pigment. [8] [11] RPE65 reverses the photoisomerization by converting an all-trans-retinyl ester to 11-cis-retinol. Most commonly, the ester substrate is retinyl palmitate. The other enzymes of the visual cycle complete the reactions necessary to oxidize and esterify all-trans-retinol to a retinyl ester (RPE65's substrate) and to oxidize 11-cis-retinol to 11-cis-retinal (the required photoactive visual pigment component). [8] [9]

The reaction completed by RPE65 in the retinoid cycle. RPE65 Reaction.png
The reaction completed by RPE65 in the retinoid cycle.

RPE65 is also referred to as retinol isomerase or retinoid isomerase, owing to past debates about the enzyme's substrate and whether it was involved in ester hydrolysis. [9]

Structure

RPE65 is a dimer of two symmetrical, enzymatically independent subunits. The active site of each subunit has a seven-bladed beta-propeller structure with four histidines that hold an iron(II) cofactor. [9] [12] This structural motif is common across the studied members of the carotenoid oxygenase family of enzymes. RPE65 is strongly associated with the membrane of the smooth endoplasmic reticulum in RPE cells. [8]

Active site structure

The active site of each RPE65 active site contains an Fe(II) cofactor bound by four histidines (His180, His241, His313, and His527), each contributed by a separate blade on the beta-propeller structure. Three of the four histidines are coordinated to nearby glutamic acid residues (Glu148, Glu417, and Glu469), which are thought to help position the histidines to bind the iron cofactor in an octahedral geometry. [13] Phe103, Thr147, and Glu148 surround the active site where they help stabilize the carbocation intermediate and increase the stereoselectivity of RPE65 for 11-cis-retinol over 13-cis-retinol. [9]

The RPE65 iron(II) cofactor, showing its coordination with 4 histidine residues and 3 glutamic acid residues. RPE65 Active Site.png
The RPE65 iron(II) cofactor, showing its coordination with 4 histidine residues and 3 glutamic acid residues.

Reactants and products likely enter and leave the active site through a hydrophobic tunnel which is thought to open into the lipid membrane for direct lipid substrate absorption. A second, smaller tunnel also reaches the active site and may serve as a pathway for water, but is too narrow to transport the retinoid reactants and products. [9] [13]

Membrane interactions

RPE65 is strongly associated with the membrane of the sER. sER is abnormally abundant in RPE cells due to their role in processing lipidic retinoids. Structural studies indicate that RPE65 is partially imbedded in the sER membrane via interactions between its hydrophobic face and the interior of the lipid membrane. This is supported by the need for detergent to solubilize RPE65. A major portion of RPE65's hydrophobic face, residues 109–126, forms an amphipathic alpha helix that likely contributes to the protein's membrane affinity. Additionally, Cys112 is palmitoylated in native RPE65, further supporting the theory that the hydrophobic face of RPE65 is imbedded in the membrane. [13]

The hydrophobic face contains the entrance to the large tunnel that leads to the enzyme's active site. The presence of this channel on the hydrophobic face combined with RPE65's demonstrated ability to absorb substrate direction from the lipid bilayer is consistent with RPE65 being partially embedded in the membrane. [8]

Conservation

RPE65 has been isolated from a wide range of vertebrates including zebra fish, chicken, mice, frogs, and humans. [8] [14] [15] Its structure is highly conserved between species, particularly in the beta-propeller and likely membrane bound regions. The amino acid sequences of human and bovine RPE65 differ by less than 1%. [13] The histidine residues of the beta-propeller structure and the bound iron(II) cofactor are 100% conserved across studied RPE65 orthologs and other members of the carotenoid oxygenase family. [9]

Soluble RPE65 (sRPE65)

Previously, it was proposed that RPE65 exists in two, interconverted forms: membrane bound mRPE65 and soluble sRPE65. This theory suggested that the reversible conversion of sRPE65 to mRPE65 by palmitoylation at Cys231, Cys329, and Cys330 played a role in regulating the retinoid cycle and endowing mRPE65 with its membrane affinity. [16] However, crystallographic studies of RPE65 have demonstrated that these residues are neither palmitoylated nor surface facing. New studies have also failed to confirm the presence of abundant soluble RPE65. Thus, this theory has been largely abandoned. [8] [13]

Mechanism

The proposed RPE65 O-alkyl cleavage mechanism. The residues shown are, clockwise from top left - Phe , Thr , His , His , His , His , and Glu . RPE65 Mechanism Updated.png
The proposed RPE65 O-alkyl cleavage mechanism. The residues shown are, clockwise from top left - Phe , Thr , His , His , His , His , and Glu .

RPE65 catalyzes the conversion of all-trans-retinyl ester to 11-cis-retinol through a proposed SN1 O-alkyl bond cleavage. RPE65's combination of an O-alkyl ester cleavage, geometric isomerization, and water addition is currently thought to be unique in biology. However, O-alkyl ester cleavage reactions with similarly stabilized carbocation intermediates are used by organic chemists. [9] [17]

O-Alkyl cleavage

The O-alkyl cleavage of the ester bond, assisted by an Fe(II) cofactor, creates a carbocation intermediate that is stabilized by the conjugated polyene chain. The delocalization of the carbocation reduces the bond order of the polyene chain, thereby reducing the activation energy of the trans-to-cis isomerization. Phe103 and Thr178 additionally stabilize the isomerized carbocation and are thought to be responsible for the stereoselectivity of the enzyme. After isomerization, a nucleophilic attack by water at C15 restores the conjugation of the polyene chain and completes the ester bond cleavage. [9] [13]

Alternate SN2 mechanism

Nearly all other biochemical ester hydrolysis reactions occur through an addition-elimination reaction at the acyl carbon. However, isotope labeling studies have demonstrated that the oxygen on the final 11-cis-retinol product of RPE65 originates from the solvent rather than the reacting ester, supporting the O-alkyl cleavage mechanism. [13] Another possible mechanism would begin with a nucleophilic attack at C11, but such an attack would rely on some nucleophile - most likely a cystine residue - to complete the isomerization portion of the reaction. Not only is the polyenyl ester probably not electron-poor enough to allow this reaction, but the active site region is lacking cystine residues to act as the nucleophile. [8] [9]

Clinical significance

Mutations in this gene have been associated with Leber's congenital amaurosis type 2 (LCA2) and retinitis pigmentosa (RP). [6] [18] RPE65 mutations are the most commonly detected mutations in LCA patients in Denmark. [19] The vast majority of RPE65 mutations in patients with LCA2 and RP occur in the beta-propeller regime and are believed to inhibit proper protein folding and iron cofactor binding. Particularly common propeller mutation sites are Tyr368 and His182. Substitution at Arg91 is also common and have been shown to impact RPE65 membrane interactions and substrate uptake. [13]

Though complete loss of function is associated with diseases such as LCA and RP, partial inhibition of RPE65 has been proposed as a treatment for age-related macular degeneration (AMD). All-trans-retinylamine (Ret-NH2) and emixustat have both been shown to competitively inhibit RPE65. [9] Emixustat is currently undergoing FDA phase 3 clinical trials as a therapy for AMD. [9] [20]

Jean Bennett and Katherine A. High's work with the RPE65 mutation has reversed an inherited form of blindness. They received the first FDA approval of a gene therapy for a genetic disease, which is called Voretigene neparvovec.[ citation needed ]

See also

Related Research Articles

<span class="mw-page-title-main">Vitamin A</span> Essential nutrient

Vitamin A is a fat-soluble vitamin and an essential nutrient for animals. The term "vitamin A" encompasses a group of chemically related organic compounds that includes retinol, retinal, retinoic acid, and several provitamin (precursor) carotenoids, most notably beta-carotene. Vitamin A has multiple functions: it is essential for embryo development and growth, for maintenance of the immune system, and for vision, where it combines with the protein opsin to form rhodopsin – the light-absorbing molecule necessary for both low-light and color vision.

<span class="mw-page-title-main">Retinol</span> Chemical compound

Retinol, also called vitamin A1, is a fat-soluble vitamin in the vitamin A family that is found in food and used as a dietary supplement. Retinol or other forms of vitamin A are needed for vision, cellular development, maintenance of skin and mucous membranes, immune function and reproductive development. Dietary sources include fish, dairy products, and meat. As a supplement it is used to treat and prevent vitamin A deficiency, especially that which results in xerophthalmia. It is taken by mouth or by injection into a muscle. As an ingredient in skin-care products, it is used to reduce wrinkles and other effects of skin aging.

<span class="mw-page-title-main">Retinal</span> Chemical compound

Retinal is a polyene chromophore. Retinal, bound to proteins called opsins, is the chemical basis of visual phototransduction, the light-detection stage of visual perception (vision).

<span class="mw-page-title-main">Retinoid</span> Group of tetraterpenes

The retinoids are a class of chemical compounds that are vitamers of vitamin A or are chemically related to it. Retinoids have found use in medicine where they regulate epithelial cell growth.

<span class="mw-page-title-main">Choroideremia</span> Medical condition

Choroideremia is a rare, X-linked recessive form of hereditary retinal degeneration that affects roughly 1 in 50,000 males. The disease causes a gradual loss of vision, starting with childhood night blindness, followed by peripheral vision loss and progressing to loss of central vision later in life. Progression continues throughout the individual's life, but both the rate of change and the degree of visual loss are variable among those affected, even within the same family.

<span class="mw-page-title-main">Carotenoid oxygenase</span>

Carotenoid oxygenases are a family of enzymes involved in the cleavage of carotenoids to produce, for example, retinol, commonly known as vitamin A. This family includes an enzyme known as RPE65 which is abundantly expressed in the retinal pigment epithelium where it catalyzed the formation of 11-cis-retinol from all-trans-retinyl esters.

The visual cycle is a process in the retina that replenishes the molecule retinal for its use in vision. Retinal is the chromophore of most visual opsins, meaning it captures the photons to begin the phototransduction cascade. When the photon is absorbed, the 11-cis retinal photoisomerizes into all-trans retinal as it is ejected from the opsin protein. Each molecule of retinal must travel from the photoreceptor cell to the RPE and back in order to be refreshed and combined with another opsin. This closed enzymatic pathway of 11-cis retinal is sometimes called Wald's visual cycle after George Wald (1906–1997), who received the Nobel Prize in 1967 for his work towards its discovery.

In enzymology, a retinol dehydrogenase (RDH) (EC 1.1.1.105) is an enzyme that catalyzes the chemical reaction

In enzymology, a retinol isomerase is an enzyme that catalyzes the chemical reaction

<span class="mw-page-title-main">Retinal G protein coupled receptor</span> Protein-coding gene in the species Homo sapiens

RPE-retinal G protein-coupled receptor also known as RGR-opsin is a protein that in humans is encoded by the RGR gene. RGR-opsin is a member of the rhodopsin-like receptor subfamily of GPCR. Like other opsins which bind retinaldehyde, it contains a conserved lysine residue in the seventh transmembrane domain. RGR-opsin comes in different isoforms produced by alternative splicing.

<span class="mw-page-title-main">Retinaldehyde-binding protein 1</span> Protein-coding gene in the species Homo sapiens

Retinaldehyde-binding protein 1 (RLBP1) also known as cellular retinaldehyde-binding protein (CRALBP) is a 36-kD water-soluble protein that in humans is encoded by the RLBP1 gene.

<span class="mw-page-title-main">RDH12</span> Protein-coding gene in humans

Retinol dehydrogenase 12 is an enzyme that in humans is encoded by the RDH12 gene.

Krzysztof Palczewski is a Polish-American biochemist working at the University of California, Irvine.

Gene therapy using lentiviral vectors was being explored in early stage trials as of 2009.

<span class="mw-page-title-main">Lecithin retinol acyltransferase</span> Mammalian protein found in Homo sapiens

Lecithin retinol acyltransferase is an enzyme that in humans is encoded by the LRAT gene.

Retinal gene therapy holds a promise in treating different forms of non-inherited and inherited blindness.

<span class="mw-page-title-main">RDH13</span> Protein-coding gene in humans

Retinol dehydrogenase 13 (all-trans/9-cis) is a protein that in humans is encoded by the RDH13 gene. This gene encodes a mitochondrial short-chain dehydrogenase/reductase, which catalyzes the reduction and oxidation of retinoids. The encoded enzyme may function in retinoic acid production and may also protect the mitochondria against oxidative stress. Alternatively spliced transcript variants have been described.

<span class="mw-page-title-main">Emixustat</span> Chemical compound

Emixustat is a small molecule notable for its establishment of a new class of compounds known as visual cycle modulators (VCMs). Formulated as the hydrochloride salt, emixustat hydrochloride, it is the first synthetic medicinal compound shown to affect retinal disease processes when taken by mouth. Emixustat was invented by the British-American chemist, Ian L. Scott, and is currently in Phase 3 trials for dry, age-related macular degeneration (AMD).

Christian Hamel was a French Professor at the Institute for Neurosciences of Montpellier, Hôpital Saint Eloi (INM) research unit INSERM 583 of the University. He studied transduction, integration and disorders of sensory and motor systems with the ultimate goal of finding treatments for degeneration of the retina and optic nerve.

<span class="mw-page-title-main">Congenital blindness</span>

Congenital blindness refers to blindness present at birth. Congenital blindness is sometimes used interchangeably with "Childhood Blindness." However, current literature has various definitions of both terms. Childhood blindness encompasses multiple diseases and conditions present in ages up to 16 years old, which can result in permanent blindness or severe visual impairment over time. Congenital blindness is a hereditary disease and can be treated by gene therapy. Visual loss in children or infants can occur either at the prenatal stage or postnatal stage. There are multiple possible causes of congenital blindness. In general, 60% of congenital blindness cases are contributed from prenatal stage and 40% are contributed from inherited disease. However, most of the congenital blindness cases show that it can be avoidable or preventable with early treatment.

References

  1. 1 2 3 GRCh38: Ensembl release 89: ENSG00000116745 – Ensembl, May 2017
  2. 1 2 3 GRCm38: Ensembl release 89: ENSMUSG00000028174 – Ensembl, May 2017
  3. ↑ "Human PubMed Reference:". National Center for Biotechnology Information, U.S. National Library of Medicine.
  4. ↑ "Mouse PubMed Reference:". National Center for Biotechnology Information, U.S. National Library of Medicine.
  5. ↑ Hamel CP, Tsilou E, Pfeffer BA, Hooks JJ, Detrick B, Redmond TM (Jul 1993). "Molecular cloning and expression of RPE65, a novel retinal pigment epithelium-specific microsomal protein that is post-transcriptionally regulated in vitro". The Journal of Biological Chemistry. 268 (21): 15751–7. doi: 10.1016/S0021-9258(18)82319-5 . PMID   8340400.
  6. 1 2 "Entrez Gene: RPE65 retinal pigment epithelium-specific protein 65kDa".
  7. ↑ Wolf G (Mar 2005). "Function of the protein RPE65 in the visual cycle". Nutrition Reviews. 63 (3): 97–100. doi: 10.1111/j.1753-4887.2005.tb00127.x . PMID   15825812.
  8. 1 2 3 4 5 6 7 8 9 Kiser PD, Palczewski K (Sep 2010). "Membrane-binding and enzymatic properties of RPE65". Progress in Retinal and Eye Research. 29 (5): 428–42. doi:10.1016/j.preteyeres.2010.03.002. PMC   2903629 . PMID   20304090.
  9. 1 2 3 4 5 6 7 8 9 10 11 12 Kiser PD, Zhang J, Badiee M, Li Q, Shi W, Sui X, Golczak M, Tochtrop GP, Palczewski K (Jun 2015). "Catalytic mechanism of a retinoid isomerase essential for vertebrate vision". Nature Chemical Biology. 11 (6): 409–15. doi:10.1038/nchembio.1799. PMC   4433804 . PMID   25894083.
  10. ↑ Tang, Peter H.; Buhusi, Mona C.; Ma, Jian-Xing; Crouch, Rosalie K. (2011-12-14). "RPE65 is present in human green/red cones and promotes photopigment regeneration in an in vitro cone cell model". The Journal of Neuroscience. 31 (50): 18618–18626. doi:10.1523/JNEUROSCI.4265-11.2011. ISSN   1529-2401. PMC   3297673 . PMID   22171060.
  11. ↑ Kiser PD, Golczak M, Palczewski K (Jan 2014). "Chemistry of the retinoid (visual) cycle". Chemical Reviews. 114 (1): 194–232. doi:10.1021/cr400107q. PMC   3858459 . PMID   23905688.
  12. ↑ Orban T, Jastrzebska B, Palczewski K (Apr 2014). "Structural approaches to understanding retinal proteins needed for vision". Current Opinion in Cell Biology. 27: 32–43. doi:10.1016/j.ceb.2013.11.001. PMC   3971393 . PMID   24680428.
  13. 1 2 3 4 5 6 7 8 Kiser PD, Golczak M, Lodowski DT, Chance MR, Palczewski K (Oct 2009). "Crystal structure of native RPE65, the retinoid isomerase of the visual cycle". Proceedings of the National Academy of Sciences of the United States of America. 106 (41): 17325–30. doi: 10.1073/pnas.0906600106 . PMC   2765077 . PMID   19805034.
  14. ↑ Takahashi, Yusuke; Moiseyev, Gennadiy; Ma, Jian-xing (2014-09-26). "Identification of key residues determining isomerohydrolase activity of human RPE65". The Journal of Biological Chemistry. 289 (39): 26743–26751. doi: 10.1074/jbc.M114.558619 . ISSN   1083-351X. PMC   4175317 . PMID   25112876.
  15. ↑ Jin M, Li S, Moghrabi WN, Sun H, Travis GH (Aug 2005). "Rpe65 is the retinoid isomerase in bovine retinal pigment epithelium". Cell. 122 (3): 449–59. doi:10.1016/j.cell.2005.06.042. PMC   2748856 . PMID   16096063.
  16. ↑ Ma J, Zhang J, Othersen KL, Moiseyev G, Ablonczy Z, Redmond TM, Chen Y, Crouch RK (Jun 2001). "Expression, purification, and MALDI analysis of RPE65". Investigative Ophthalmology & Visual Science. 42 (7): 1429–35. PMID   11381042.
  17. ↑ Redmond, T. Michael; Poliakov, Eugenia; Kuo, Stephanie; Chander, Preethi; Gentleman, Susan (2010-01-15). "RPE65, visual cycle retinol isomerase, is not inherently 11-cis-specific: support for a carbocation mechanism of retinol isomerization". The Journal of Biological Chemistry. 285 (3): 1919–1927. doi: 10.1074/jbc.M109.027458 . ISSN   1083-351X. PMC   2804350 . PMID   19920137.
  18. ↑ Bowne SJ, Humphries MM, Sullivan LS, Kenna PF, Tam LC, Kiang AS, Campbell M, Weinstock GM, Koboldt DC, Ding L, Fulton RS, Sodergren EJ, Allman D, Millington-Ward S, Palfi A, McKee A, Blanton SH, Slifer S, Konidari I, Farrar GJ, Daiger SP, Humphries P (Oct 2011). "A dominant mutation in RPE65 identified by whole-exome sequencing causes retinitis pigmentosa with choroidal involvement". European Journal of Human Genetics. 19 (10): 1074–81. doi:10.1038/ejhg.2011.86. PMC   3190249 . PMID   21654732.
  19. ↑ Astuti GD, Bertelsen M, Preising MN, Ajmal M, Lorenz B, Faradz SM, Qamar R, Collin RW, Rosenberg T, Cremers FP (Dec 2015). "Comprehensive genotyping reveals RPE65 as the most frequently mutated gene in Leber congenital amaurosis in Denmark". European Journal of Human Genetics. 24 (7): 1071–1079. doi:10.1038/ejhg.2015.241. PMC   5070892 . PMID   26626312.
  20. ↑ "Acucela - Retinal Diseases". acucela.com. Retrieved 2016-03-01.

Further reading

Protein Structure and Function
Clinical and Genetic Studies
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