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 Cterminus (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.
Highlights
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, reported as blepharophimosis-ptosis-intellectual disability syndrome)
UBE3B’s Predicted Structure Resembles homologous to the E6-AP C terminus (HECT) E3 Ligases— From a previous report, it was proposed that UBE3B belongs to the HECT subfamily of E3 ubiquitin ligases [31]
The C terminus of UBE3B is the HECT domain, which is composed of two lobes as follows: the N-lobe that is predicted to bind to the ubiquitin-conjugating enzyme (E2), and the C-lobe that is predicted to contain the catalytic cysteine, which forms a covalent thioester bond with ubiquitin
Summary
UBE3B’s Predicted Structure Resembles HECT E3 Ligases— From a previous report, it was proposed that UBE3B belongs to the HECT subfamily of E3 ubiquitin ligases [31]. The N terminus of UBE3B is predicted to contain the substrate binding domain and a calmodulin-binding (IQ) motif [35]. To assess the non-mitochondrial component of the GFP signal, cells were fractionated into mitochondrial, ER, and cytoplasmic fractions and probed with an antibody against copGFP (Fig. 2B). Full-length copGFP fusions were observed in the mitochondrial fraction, but not in the ER fraction This suggests that full-length UBE3B is associated primarily, if not exclusively, with the mitochondria. The non-mitochondrial component of the observed GFP signal does not represent full-length UBE3B and is likely partly constituted of fragmented copGFP (25 kDa) species in the ER and cytoplasm. B, alignment of UBE3B with calmodulin binding domains as predicted by Phyre and using ClustalW2. C, alignment of UBE3B with HECT E3 ligase domains as predicted by Phyre and using ClustalW2. For extremely high accuracy models, the % ID should be above 30 – 40%; if the confidence is high, even very low % ID (Ͻ15%) can be very useful
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