title: UBE3B Is a Calmodulin-regulated, Mitochondrion-associated E3 Ubiquitin Ligase.
abstract: Recent genome-wide studies found that patients with hypotonia, developmental delay, intellectual disability, congenital anomalies, characteristic facial dysmorphic features, and low cholesterol levels suffer fromKaufmanoculocerebrofacialsyndrome (KOS, also reported as blepharophimosis-ptosis-intellectual disability syndrome). The primary cause of KOS is autosomal recessive mutations in the geneUBE3BHowever, to date, there are no studies that have determined the cellular or enzymatic function of UBE3B. Here, we report that UBE3B is a mitochondrion-associated protein withhomologous to theE6-APCterminus (HECT) E3 ubiquitin ligase activity. Mutating the catalytic cysteine (C1036A) or deleting the entire HECT domain (amino acids 758-1068) results in loss of UBE3B's ubiquitylation activity. Knockdown of UBE3B in human cells induces changes in mitochondrial morphology and physiology, a decrease in mitochondrial volume, and a severe suppression of cellular proliferation. We also discovered that UBE3B interacts with calmodulin via its N-terminal isoleucine-glutamine (IQ) motif. Deletion of the IQ motif (amino acids 29-58) results in loss of calmodulin binding and a significant increase in thein vitroubiquitylation activity of UBE3B. In addition, we found that changes in calcium levelsin vitrodisrupt the calmodulin-UBE3B interaction. These studies demonstrate that UBE3B is an E3 ubiquitin ligase and reveal that the enzyme is regulated by calmodulin. Furthermore, the modulation of UBE3B via calmodulin and calcium implicates a role for calcium signaling in mitochondrial protein ubiquitylation, protein turnover, and disease.
authors.affiliation: From the Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261.|the Hillman Cancer Center, University of Pittsburgh Cancer Institute.
authors.forename: Andrea
authors.initials: A
authors.lastname: Braganza
authors.affiliation: the Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama 36604.
authors.forename: Jianfeng
authors.initials: J
authors.lastname: Li
authors.affiliation: Biomedical Mass Spectrometry Center, School of the Health Sciences, and.
authors.forename: Xuemei
authors.initials: X
authors.lastname: Zeng
authors.affiliation: the Hillman Cancer Center, University of Pittsburgh Cancer Institute.|Biomedical Mass Spectrometry Center, School of the Health Sciences, and.|the Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261, and.
authors.forename: Nathan A
authors.initials: NA
authors.lastname: Yates
authors.affiliation: the Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama 36604.
authors.forename: Nupur B
authors.initials: NB
authors.lastname: Dey
authors.affiliation: the Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama 36604.
authors.forename: Joel
authors.initials: J
authors.lastname: Andrews
authors.affiliation: the Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama 36604.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Clark
authors.affiliation: the Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama 36604.
authors.forename: Leila
authors.initials: L
authors.lastname: Zamani
authors.affiliation: the Hillman Cancer Center, University of Pittsburgh Cancer Institute.
authors.forename: Xiao-Hong
authors.initials: XH
authors.lastname: Wang
authors.affiliation: Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, Pennsylvania 15213.
authors.forename: Claudette
authors.initials: C
authors.lastname: St Croix
authors.affiliation: From the Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261.|the Hillman Cancer Center, University of Pittsburgh Cancer Institute.
authors.forename: Roderick
authors.initials: R
authors.lastname: O'Sullivan
authors.affiliation: From the Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261.|the Hillman Cancer Center, University of Pittsburgh Cancer Institute.
authors.forename: Laura
authors.initials: L
authors.lastname: Garcia-Exposito
authors.affiliation: the Department of Biological Sciences, University of Pittsburgh, Pittsburgh, Pennsylvania 15260.
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Brodsky
authors.affiliation: From the Department of Pharmacology and Chemical Biology, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, rwsobol@health.southalabama.edu.|the Hillman Cancer Center, University of Pittsburgh Cancer Institute.|the Department of Oncologic Sciences, Mitchell Cancer Institute, University of South Alabama, Mobile, Alabama 36604.
authors.forename: Robert W
authors.identifier: 0000-0001-7385-3563
authors.initials: RW
authors.lastname: Sobol
mesh_terms: D000595:Amino Acid Sequence; D002147:Calmodulin / Q000378:metabolism*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D055785:Gene Knockdown Techniques; D006801:Humans; D008928:Mitochondria / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D002147:Calmodulin; C482647:UBE3B protein, human; D044767:Ubiquitin-Protein Ligases
keywords: HECT; Kaufman oculocerebrofacial syndrome; calcium; mitochondria; oxidative stress; protein degradation; reactive oxygen species (ROS); super-resolution microscopy; ubiquitin/proteosome system; ubiquitylation (ubiquitination)