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 from Kaufman oculocerebrofacial syndrome (KOS, also reported as blepharophimosis-ptosis-intellectual disability syndrome). The primary cause of KOS is autosomal recessive mutations in the gene UBE3B. However, 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 with homologous to the E6-AP C terminus (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 the in vitro ubiquitylation activity of UBE3B. In addition, we found that changes in calcium levels in vitro disrupt 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.Ubiquitylation is a reversible post-translational modification that impacts most cellular processes. However, it is best known for targeting proteins for degradation by the 26S proteasome, and the ubiquitin/proteasome system (UPS) 2 is a major part of the cellular protein quality control network (1-5). Substrate modification can occur either by the attachment of a single ubiquitin molecule or a polyubiquitin chain, where one ubiquitin is conjugated to the next via an isopeptide bond (6). Conjugation of ubiquitin to one or more lysine residues on the substrate protein is mediated by the sequential action of a ubiquitin-activating enzyme (E1), a ubiquitin-conjugating enzyme (E2), and a ubiquitin ligase (E3). Although there is limited diversity in E1 and E2 enzymes, numerous cellular E3 ligases specify the timing and substrate selection of ubiquitylation reactions.E3 ligases are mainly composed of three major families, the really interesting new gene (RING) family, the homologous to the E6-AP C terminus (HECT) family, and the RING-between-RING (RBR) family. The RING ligases catalyze the direct transfer of ubiquitin from the E2 to the substrate (7,8). Conversely, the HECT and RBR E3 ligases covalently transfer the ubiquitin from the E2 to a resident catalytic cysteine. This catalytic cysteine in the E3 ligase active site can then serve as the site of attachment for a thioester-linked polyubiqu...

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