Molecular cloning, genomic structure, and protein characterization of mouse optineurin

doi:10.1016/j.ygeno.2004.10.011

Genomics

Volume 85, Issue 1, January 2005, Pages 131-138

https://doi.org/10.1016/j.ygeno.2004.10.011 Get rights and content

Abstract

We recently identified optineurin (OPTN) as a novel gene for glaucoma and determined its mRNA and protein expression patterns in different human tissues. Herein, we describe the cloning, mapping, genomic organization, and mRNA and protein expression patterns for murine optineurin (Optn). We mapped Optn to chromosome 2, within a region that is syntenic to human 10p14. Optn has 13 coding exons and its exon–intron boundaries are evolutionarily conserved with human. Optn encodes an 884-amino-acid protein and shows 78% identity to OPTN. Northern blot analysis revealed three mRNA transcripts with highest expression in adult liver, heart, and testis and with earliest detectable message in 7-day-old embryos. In situ hybridization showed prominent ocular expression during mouse embryonic development. Optn colocalizes with vesicular structures near the nucleus and is expressed in anterior segment, retina, and optic nerve blood vessels. Gene and protein ocular profiling of Optn is a prerequisite for developing a mouse model for glaucoma.

Section snippets

Dummy introduction

Glaucoma represents a group of eye disorders with a prevalence of approximately 2% in the Caucasian population over the age of 40 years. Glaucoma is the second most common cause of blindness and accounts for 12–14% of all reported cases [1], [2], [3], [4]. Primary open-angle glaucoma (POAG) accounts for more than half of all cases. Glaucoma is a disease of slow onset, usually bilateral but unequal in degree, that is characterized by atrophy of the fibers of the optic nerve as they pass through

Isolation and characterization of the mouse optineurin gene

The human OPTN cDNA sequence was used to search the mouse expressed sequence tag (EST) database at the National Center for Biotechnology Information (NCBI). Two potential clones with high homology (82–86%) to human OPTN were identified. These EST clones were purchased (Invitrogen, Inc.) and fully sequenced. The obtained sequences from both ends of these clones were used to design new primers for PCR amplification of the entire mouse Optn gene. A 2-kb fragment was amplified from mouse 11-day

Discussion

We have previously reported that mutations in the human optineurin gene are responsible for adult-onset primary open angle glaucoma [19], [20]. Development of appropriate animal models can help us study the molecular mechanisms of optineurin gene expression and function that eventually lead to the glaucoma phenotype. As the first step, we cloned, mapped, and characterized the mouse Optn gene and its protein products. Mouse and human optineurin genes have 13 coding exons and identical

Cell culture

Mouse BALB/c dermal fibroblasts (ATCC) were used for immunocytochemistry, total RNA extraction, and cDNA synthesis. The cells were maintained in DMEM-F12 (Gibco) medium, supplemented with 10% FBS, 1% antimycotic (Gibco), and 2 mM l-glutamine in an incubator at 37°C containing 5% CO₂.

cDNA cloning and sequence analysis of mouse optineurin

Identification of the mouse optineurin gene was achieved by homology searches of the mouse EST database of GenBank (http://www.ncbi.nlm.nih.gov) using the human optineurin cDNA sequence (GenBank Accession No. AF420371

Acknowledgments

We would like to thank Ivaylo Stoilov (for his help with in situ hybridization), Nilanjana Maulik (for her help in Northern blotting), The Jackson Laboratory (for graphical presentation of Figure 3), Nancy Ryan at the Histology Core Services and Susan Krueger at the Confocal Microscopy Facility, University of Connecticut Health Center (for their technical assistance). This work was funded by grants from the National Eye Institute (EY-09947 and EY-14959), the International Glaucoma Association

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Cited by (47)

Roles and mechanisms of optineurin in bone metabolism
2024, Biomedicine and Pharmacotherapy
Optineurin (OPTN) is a widely expressed multifunctional articulatory protein that participates in cellular or mitochondrial autophagy, vesicular transport, and endoplasmic reticulum (ER) stress via interactions with various proteins. Skeletal development is a complex biological process that requires the participation of various osteoblasts, such as bone marrow mesenchymal stem cells (BMSCs), and osteogenic, osteoclastic, and chondrogenic cells. OPTN was recently found to be involved in the regulation of osteoblast activity, which affects bone metabolism. OPTN inhibits osteoclastogenesis via signaling pathways, including NF-κB, IFN-β, and NRF2. OPTN can promote the differentiation of BMSCs toward osteogenesis and inhibit lipogenic differentiation by delaying BMSC senescence and autophagy. These effects are closely related to the development of bone metabolism disorders, such as Paget's disease of bone, rheumatoid arthritis, and osteoporosis. Therefore, this review aims to explore the role and mechanism of OPTN in the regulation of bone metabolism and related bone metabolic diseases. Our findings will provide new targets and strategies for the prevention and treatment of bone metabolic diseases.
SUMOylation of optineurin is critical for inhibiting interferon β production
2022, Biochemical and Biophysical Research Communications
Targeting the vital kinase protein TBK1, optineurin (OPTN) performs as a suppressor involved in controlling RNA virus-induced interferon β (IFN-β) production. It has been determined that OPTN is altered by phosphorylation and ubiquitination, which are crucial for the generation of IFN-β and mitophagy. In this study, endogenous OPTN was first discovered in human cells at a molecular mass of about 115 kD. The SUMOylation band was verified to be the higher molecular mass band of the OPTN. Additionally, the OPTN SUMOylation band was seen in cells from several species, including mouse, rabbit, bovine, porcine, etc., but not in cells from avian animals (chicken and duck). This finding suggests that OPTN SUMOylation is well conserved in mammals but not in avian animals. Additionally, it was determined that some lysine residues in the human OPTN had SUMOylation sites that followed the consensus motif. LPS, VSV infection, starvation, and RNA virus infection are a few of the stimuli that encourage endogens OPTN SUMOylation. OPTN SUMOylation is essential for OPTN biological activity, as evidenced by the stark differences in the cellular distribution of mutant OPTN SUMOylation sites from wild type (WT) OPTN. Additionally, we discovered that non-SUMOylated OPTN lost its ability to block both IFN-β production. Our findings offer a preliminary understanding of how OPTN SUMOylation regulates IFN-β production.
Optineurin deletion disrupts metabotropic glutamate receptor 5-mediated regulation of ERK1/2, GSK3β/ZBTB16, mTOR/ULK1 signaling in autophagy
2021, Biochemical Pharmacology
Optineurin (OPTN) is a multifunctional protein that mediates a network of cellular processes regulating membrane trafficking, inflammatory responses and autophagy. The OPTN-rich interactome includes Group I metabotropic glutamate receptors (mGluR1 and 5), members of the Gα_q/11 protein receptor family. Recent evidence has shown that mGluR5, in addition to its canonical Gα_q/11 protein-coupled signaling, regulates autophagic machinery via mTOR/ULK1 and GSK3β/ZBTB16 pathways in both Alzheimer’s and Huntington’s disease mouse models. Despite its potential involvement, the role of OPTN in mediating mGluR5 downstream signaling cascades remains largely unknown. Here, we employed a CRISPR/Cas9 OPTN-deficient STHdh^Q7/Q7 striatal cell line and global OPTN knockout mice to investigate whether Optn gene deletion alters both mGluR5 canonical and noncanonical signaling. We find that OPTN is required for mGluR5-activated Ca²⁺ flux and ERK1/2 signaling following receptor activation in STHdh^Q7/Q7 cells and acute hippocampal slices. Deletion of OPTN impairs both GSK3β/ZBTB16 and mTOR/ULK1 autophagic signaling in STHdh^Q7/Q7 cells. Furthermore, mGluR5-dependent regulation of GSK3β/ZBTB16 and mTOR/ULK1 autophagic signaling is impaired in hippocampal slices of OPTN knockout mice. Overall, we show that the crosstalk between OPTN and mGluR5 can have major implication on receptor signaling and therefore potentially contribute to the pathophysiology of neurodegenerative diseases.
Mendelian genes in primary open angle glaucoma
2019, Experimental Eye Research
Citation Excerpt :
OPTN is also expressed in the adult eye and systemically in many tissues, including heart, liver, testes, kidney, brain, and skeletal muscle (Rezaie and Sarfarazi, 2005). In the eye, OPTN is expressed in the non-pigmented ciliary epithelium, trabecular meshwork, and most highly, in the retinal ganglion cells (Rezaie and Sarfarazi, 2005). OPTN has been further localized within the cytoplasm, perinuclear region, golgi apparatus, and trans-golgi network (De Marco et al., 2006).
Mutations in each of three genes, myocilin (MYOC), optineurin (OPTN), and TANK binding kinase 1 (TBK1), may cause primary open-angle glaucoma (POAG) that is inherited as a Mendelian trait. MYOC mutations cause 3–4% of POAG cases with IOP >21 mmHg, while mutations in OPTN, TBK1, and MYOC each cause ∼1% of POAG with IOP ≤21 mmHg, i.e. normal tension glaucoma. Identification of these disease-causing genes has provided insights into glaucoma pathogenesis. Mutations in MYOC cause a cascade of abnormalities in the trabecular meshwork including intracellular retention of MYOC protein, decreased aqueous outflow, higher intraocular pressure, and glaucoma. Investigation of MYOC mutations demonstrated that abnormal retention of intracellular MYOC and stimulation of endoplasmic reticular (ER) stress may be important steps in the development of MYOC-associated glaucoma. Mutations in OPTN and TBK1 cause a dysregulation of autophagy which may directly cause retinal ganglion cell damage and normal tension glaucoma. Discovery of these Mendelian causes of glaucoma has also provided a new set of potential therapeutic targets that may ultimately lead to novel, gene-directed glaucoma treatments.
Identification of a splice variant of optineurin which is defective in autophagy and phosphorylation
2018, Biochimica et Biophysica Acta - Molecular Cell Research
Citation Excerpt :
Optineurin is a cytoplasmic protein, expressed at high levels in many tissues like brain, retina, muscle, heart, liver and testis [1–3].
Optineurin (Optn) is an autophagy receptor that performs various functions in cargo-selective and non-selective autophagy. Here, we have identified and characterized a splice variant of mouse optineurin mRNA, which produces a truncated protein lacking N-terminal 157 amino acids (d157mOptn). This mRNA and protein are expressed in several tissues and cells. d157mOptn has an intact LC3-interacting region and a serine (S187) in it. However, unlike normal optineurin, the d157mOptn was not phosphorylated at this site when expressed in mammalian cells, and showed reduced interaction with TBK1 (tank binding kinase) that mediates phosphorylation at S187 (S177 in human OPTN). This phosphorylation of Optn required intact N-terminal sequence as well as functional C-terminal ubiquitin-binding domain. Unlike normal optineurin, d157mOptn was unable to promote autophagosome and autolysosome formation upon expression in Optn-deficient cells. d157mOptn was recruited to mutant huntingtin aggregates, but unlike wild type optineurin, it was unable to clear these aggregates by autophagy in neuronal NSC-34 cells. Phospho-TBK1 was seen around mutant Huntingtin aggregates in Optn overexpressing cells but it was reduced in cells overexpressing d157mOptn. Thus, we have identified an isoform of mouse optineurin which is defective in cargo-selective and non-selective autophagy possibly due to loss of phosphorylation and impaired interaction with TBK1. This isoform, which inhibits autophagosome formation in neuronal cells, might be involved in selectively modulating some of the functions of Optn, such as autophagy. Our results provide an insight into the role of N-terminal domain of Optn in various autophagic functions.
Cellular Functions of Optineurin in Health and Disease
2016, Trends in Immunology
Citation Excerpt :
Mouse OPTN shows 78% sequence similarity to the human protein, and is also encoded by 13 exons, generating a full-length protein of similar size. Alternative splicing can generate five different isoforms [5]. OPTN is a multidomain protein mediating several functions by interacting with many different proteins.
Optineurin (OPTN) was initially identified as a regulator of NF-κB and interferon signaling, but attracted most attention because of its association with various human disorders such as glaucoma, Paget disease of bone, and amyotrophic lateral sclerosis. Importantly, OPTN has recently been identified as an autophagy receptor important for the autophagic removal of pathogens, damaged mitochondria, and protein aggregates. This activity is most likely compromised in patients carrying OPTN mutations, and contributes to the observed phenotypes. In this review we summarize recent studies describing the molecular mechanisms by which OPTN controls immunity and autophagy, and discuss these findings in the context of several diseases that have been associated with OPTN (mal)function.

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^☆: Sequence data from this article have been deposited with the GenBank Data Library under Accession Nos. AY340623-AY340635AY340623AY340624AY340625AY340626AY340627AY340628AY340629AY340630AY340631AY340632AY340633AY340634AY340635.

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Molecular cloning, genomic structure, and protein characterization of mouse optineurin☆

Abstract

Section snippets

Dummy introduction

Isolation and characterization of the mouse optineurin gene

Discussion

Cell culture

cDNA cloning and sequence analysis of mouse optineurin

Acknowledgments

Genomics

Am. J. Ophthalmol.

Am. J. Hum. Genet.

Genomics

The global impact of glaucoma

Bull. World Health Organ.

The epidemiology of open-angle glaucoma: a review

Am. J. Epidemiol.

Department of Health and Social Security. Reports on Public Health and Medical Subjects. No. 128. The incidence and causes of blindness in England and Wales 1963–68, with an appendix on services available for incipient blindness

Reports on Public Health & Medical Subjects

Low-tension glaucoma

Int. Ophthalmol. Clin.

Low tension glaucoma—its place in modern glaucoma practice

Br. J. Ophthalmol.

Clinically detectable nerve fiber atrophy precedes the onset of glaucomatous field loss

Arch. Ophthalmol.

Genetic linkage of familial open angle glaucoma to chromosome 1q21–q31

Nat. Genet.

Mapping a gene for adult-onset primary open-angle glaucoma to chromosome 3q

Am. J. Hum. Genet.