title: Altered ubiquitin causes perturbed calcium homeostasis, hyperactivation of calpain, dysregulated differentiation, and cataract.
abstract: Although the ocular lens shares many features with other tissues, it is unique in that it retains its cells throughout life, making it ideal for studies of differentiation/development. Precipitation of proteins results in lens opacification, or cataract, the major blinding disease. Lysines on ubiquitin (Ub) determine fates of Ub-protein substrates. Information regarding ubiquitin proteasome systems (UPSs), specifically of K6 in ubiquitin, is undeveloped. We expressed in the lens a mutant Ub containing a K6W substitution (K6W-Ub). Protein profiles of lenses that express wild-type ubiquitin (WT-Ub) or K6W-Ub differ by only ∼2%. Despite these quantitatively minor differences, in K6W-Ub lenses and multiple model systems we observed a fourfold Ca(2+) elevation and hyperactivation of calpain in the core of the lens, as well as calpain-associated fragmentation of critical lens proteins including Filensin, Fodrin, Vimentin, β-Crystallin, Caprin family member 2, and tudor domain containing 7. Truncations can be cataractogenic. Additionally, we observed accumulation of gap junction Connexin43, and diminished Connexin46 levels in vivo and in vitro. These findings suggest that mutation of Ub K6 alters UPS function, perturbs gap junction function, resulting in Ca(2+) elevation, hyperactivation of calpain, and associated cleavage of substrates, culminating in developmental defects and a cataractous lens. The data show previously unidentified connections between UPS and calpain-based degradative systems and advance our understanding of roles for Ub K6 in eye development. They also inform about new approaches to delay cataract and other protein precipitation diseases.
authors.affiliation: Laboratory for Nutrition and Vision Research, US Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, MA 02111; Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Science, Sichuan University, Chengdu, Sichuan 610064, People's Republic of China; allen.taylor@tufts.edu kliu@scu.edu.cn.
authors.forename: Ke
authors.initials: K
authors.lastname: Liu
authors.affiliation: Laboratory for Nutrition and Vision Research, US Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, MA 02111; Key Laboratory of Bio-Resources and Eco-Environment of Ministry of Education, College of Life Science, Sichuan University, Chengdu, Sichuan 610064, People's Republic of China;
authors.forename: Lei
authors.initials: L
authors.lastname: Lyu
authors.affiliation: Departments of Structural Biology and Developmental Neurobiology, St. Jude Proteomics Facility, St. Jude Children's Research Hospital, Memphis, TN 38105;
authors.forename: David
authors.initials: D
authors.lastname: Chin
authors.affiliation: Department of Physiology and Biophysics, State University of New York, Stony Brook, NY 11794;
authors.forename: Junyuan
authors.initials: J
authors.lastname: Gao
authors.affiliation: Department of Physiology and Biophysics, State University of New York, Stony Brook, NY 11794;
authors.forename: Xiurong
authors.initials: X
authors.lastname: Sun
authors.affiliation: Laboratory for Nutrition and Vision Research, US Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, MA 02111;
authors.forename: Fu
authors.initials: F
authors.lastname: Shang
authors.affiliation: Laboratory for Nutrition and Vision Research, US Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, MA 02111;
authors.forename: Andrea
authors.initials: A
authors.lastname: Caceres
authors.affiliation: Laboratory for Nutrition and Vision Research, US Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, MA 02111;
authors.forename: Min-Lee
authors.initials: ML
authors.lastname: Chang
authors.affiliation: Laboratory for Nutrition and Vision Research, US Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, MA 02111;
authors.forename: Sheldon
authors.initials: S
authors.lastname: Rowan
authors.affiliation: Departments of Structural Biology and Developmental Neurobiology, St. Jude Proteomics Facility, St. Jude Children's Research Hospital, Memphis, TN 38105;
authors.forename: Junmin
authors.initials: J
authors.lastname: Peng
authors.affiliation: Department of Physiology and Biophysics, State University of New York, Stony Brook, NY 11794;
authors.forename: Richard
authors.initials: R
authors.lastname: Mathias
authors.affiliation: Physiology and Functional Genomics, University of Florida College of Medicine, Gainesville, FL 32610-0274; and.
authors.forename: Hideko
authors.initials: H
authors.lastname: Kasahara
authors.affiliation: Laboratory for Nutrition and Vision Research, US Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, MA 02111;
authors.forename: Shuhong
authors.initials: S
authors.lastname: Jiang
authors.affiliation: Laboratory for Nutrition and Vision Research, US Department of Agriculture Human Nutrition Research Center on Aging, Tufts University, Boston, MA 02111; Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 7610001, Israel allen.taylor@tufts.edu kliu@scu.edu.cn.
authors.forename: Allen
authors.initials: A
authors.lastname: Taylor
mesh_terms: D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000235:genetics / Q000378:metabolism; D002386:Cataract / Q000235:genetics / Q000378:metabolism / Q000473:pathology; D004789:Enzyme Activation; D005136:Eye Proteins / Q000235:genetics / Q000378:metabolism; D017629:Gap Junctions / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007908:Lens, Crystalline / Q000378:metabolism / Q000473:pathology; D051379:Mice; D008822:Mice, Transgenic; D020125:Mutation, Missense; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism; D059748:Proteolysis; D025801:Ubiquitin / Q000235:genetics / Q000378:metabolism
chemical_list: D005136:Eye Proteins; D025801:Ubiquitin; D002154:Calpain; D046988:Proteasome Endopeptidase Complex; D002118:Calcium
keywords: calpain; cataract; connexin; development; ubiqutin