UBE3B is a mitochondria-associated E3 ubiquitin ligase whose activity is modulated by its interaction with Calmodulin to respond to oxidative stress

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Andrea Braganza
thesis / dissertation description
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 (also reported as blepharophimosis-ptosis-intellectual disability syndrome). The primary cause of Kaufman oculocerebrofacial syndrome (KOS) is autosomal recessive mutations in the gene UBE3B. However, to date, there are no studies that determine the cellular or enzymatic function of UBE3B. Here we report that UBE3B is a mitochondria-associated protein with HECT E3 ubiquitin ligase activity. Mutating the catalytic cysteine (C1036A) or deleting the entire HECT domain (aa758-1068) results in loss of UBE3B’s ubiquitylation activity. Knockdown of UBE3B in human cells results in changes in mitochondrial morphology, a decrease in mitochondria volume and an increase in mitochondrial oxidative stress. We also discovered that UBE3B strongly interacts with calmodulin (CaM) via the N-terminal IQ domain of UBE3B. Deletion of the IQ domain (aa29-58) results in loss of calmodulin binding. We also found that in vitro changes in the concentration of calcium resulted in decreased the interaction between UBE3B and CaM. In both these cases we saw an increase in the ubiquitylation activity of UBE3B. These studies are the first to demonstrate that UBE3B is an E3 ubiquitin ligase. Further, the changes in the interaction between UBE3B and calmodulin implicate a role for calcium signaling in mitochondrial protein ubiquitylation and turnover in the cell.