Commentary and Perspective
Welcome to the borderless rhodopsin world
2023 年 20 巻 4 号 論文ID: e200044
詳細
2023 年 20 巻 4 号 論文ID: e200044
Rhodopsin is a general term for photoreceptive proteins that bind the retinal as a chromophore. Rhodopsins are classically classified into two types, animal-type opsins and microbial-type opsins [1]. Although these two types share common structural elements including the seven transmembrane helical domains and a chromophore retinal, they show no sequence similarities with each other, which leads to the diversity of their molecular functions. However, recent accumulation of the molecular properties of rhodopsins has crossed the border between animal-type and microbial-type. Optogenetics is a cutting-edge technology that involves the use of light to control and manipulate the activity of genetically modified cells. The application of various rhodopsins as optogenetics tools has crossed borders between the scientific fields, and greatly contributed to the understanding of the molecular mechanisms underlying the physiological functions in animals [2]. Furthermore, rhodopsin opens a new field in the gene therapy of diseases [3].
To highlight recent progress in this “borderless” research, we organized a symposium during the 61st Annual Meeting of the Biophysical Society of Japan held in Nagoya in November 2023. The symposium was co-organized by the CREST Program of Japan Science and Technology Agency (JST) named “OptoBio”, directed by Prof. Ryoichiro Kageyama (Director, Center for Brain Science, RIKEN). Six researchers covering biophysics, molecular cell biology, neurobiology, and ophthalmology were invited to the talk about their recent achievements. The topics of their talks are categorized into 3 groups and summarized as follows.
Dr. Takahiro Yamashita (Kyoto University, Japan) introduced a unique animal opsin, Opn5L1, whose activity is controlled by a photocyclic reaction upon photoreception [4]. Opn5L1 is a nonvisual animal opsin that binds all-trans retinal as a chromophore. A striking feature of Opn5L1 is that the protein exhibits a photocyclic reaction which is unusual for animal rhodopsins and common for microbial rhodopsins. Cys188 located in close proximity to the retinal is the key to the cyclic reaction. Moreover, bovine rhodopsin acquired the photocyclic and photoreversible properties by the introduction of the cysteine residue at position 188, indicating the importance of regulation of the recovery from the active state to the original dark state [5].
Recent metagenomic analysis has expanded the number of rhodopsin genes. Dr. Masae Konno (University of Tokyo, Japan) introduced a new microbial rhodopsin subfamily called “bestrhodopsin” from marine unicellular algae [6]. One of the surprising features is that bestrhodopsin consists of 1–2 rhodopsin domains fused with the following bestrophin domain. Cryo-EM analysis revealed a megacomplex of the bestrodopsin (~700 kDa) forming a pentameric structure in which rhodopsin domains surround the central bestrophin channel. Bestrodopsin shows a markedly red-shifted absorption maximum wavelength (λmax=661 nm), and changes its structure through photoconversion between two metastable states, and works as a light-gated anion channel. Bestrhodopsin showed a unique photoconversion of the retinal chromophore from the all-trans to 11-cis form which is unusual for microbial rhodopsins and common for animal rhodopsins.
Dr. Keiichi Kojima (Okayama University, Japan) presented a new optical method of inducing apoptosis, a programmed cell death. It has been suggested that intracellular alkalization results in apoptosis. By expressing a proton pump rhodopsin, archaerhodopsin-3 (AR3) in human cell lines, the authors successfully induced the intracellular alkalization by light, triggering the mitochondrial apoptotic signaling pathway, which resulted in cell death accompanied by morphological changes [7]. They also applied the light-induced apoptosis method to amphid sensory neurons in Caenorhabditis elegans. Thus, the method has a high applicability both in vivo and in vitro as an optogenetic tool to selectively eliminate target cells.
Optogenetic manipulation of GPCR signaling is highly valuable. Dr. Mitsumasa Koyanagi (Osaka Metropolitan University, Japan) presented unique molecular features of mosquito Opn3 (MosOpn3) and lamprey parapinopsin (LamPP), and attracted us with optical manipulation of the C. elegans behavior. MosOpn3 activates Gi-type and Go-type G proteins light-dependently, and thus regulating cAMP-related G protein-coupled receptor signaling [8]. LamPP is a UV-sensitive bi-stable opsin that can be photo-regenerated by green light [9]. Unlike vertebrate opsins, invertebrate-derived opsins are bi-stable and do not dissociate a retinal, and thus return to their original state upon light absorption. Thus, these opsins can be used satisfactorily as an optogenetics tool for repetitive optical stimulations. When MosOpn3 was introduced into nociceptor neurons, the C. elegans exhibited ~7,000 times higher sensitivity than ChR2 in the light-induced avoidance behavior. Dual color manipulation was feasible when LamPP was introduced into motor neurons, in which violet light avoided its movement and green light-induced go-motion.
Retinitis pigmentosa (RP), a leading cause of youth blindness in Japan, remains untreatable. RP is caused by the loss of photoreceptor cells in the retina and thus results in blindness. Recent progress in optogenetics inspired researchers to develop optogenetic gene therapy for visual restoration. Many global startups are striving to develop optogenetic gene therapy, furthering advancements in the field [3]. However, it has not yet been approved as a product, partly due to the insufficient light sensitivity. Dr. Yusaku Katada (Keio University and Restore Vision Inc., Japan) presented the current state of the clinical and R&D aspects of the disease. He also introduced a chimeric rhodopsin, a hybrid of a human and a microbial rhodopsin, which exhibits high light sensitivity and its non-bleaching feature, making it an optimal choice for visual restoration [10]. Testing in a model mouse confirmed its efficacy, indicating a promising tool for the therapy. As the CEO of the start-up company as well as a physician-scientist, he emphasized the importance of commercializing academic intellectual property into a product.
Dr. Satoshi Tsunoda (Nagoya Institute of Technology, Japan) also presented the development of optogenetic gene therapy for RP. Channelrhodopsins (ChRs) are light-gated ion channels. Thus, expressing ChRs in bipolar cells or ganglion cells in the retina of a patient would enable these cells to become light-sensitive, thereby restoring visual function. He introduced a high light-sensitive ChR, GtCCR4, exhibiting ~25 times higher sensitivity compared to the well-known ChR (ChR2) in mammalian cells and rat cortical neurons [11,12]. A model mouse of RP restored the photo-sensitivity after expressing GtCCR4 in the retina, an indication of a promising tool of the therapy.
We thank all the speakers for providing attractive talks, all the attendees for fruitful discussion in the symposium, and Prof. Ryoichiro Kageyama for kindly supporting the symposium. This work and symposium are in part supported by JST CREST grant JPMJCR1753 (to T.Y.).
