Stimulating Deubiquitination

Ubiquitination is associated with targeting of proteins for degradation (polyubiquitination) or regulation (monoubiquitination). Chen et al. stimulated synaptosomes (pinched off nerve terminals enriched in presynaptic components from brain) with high K + to depolarize the membranes, which in the presence of external calcium led to a global decrease in protein ubiquitination (detected by immunoblotting with antibodies that recognize ubiquitin). The decrease in ubiquitination was blocked by treatment of the synaptosomes with inhibitors of the calcium-dependent phosphatase calcineurin but was not blocked by inhibition of the proteasome. Analysis of the monoubiquitinated proteins, epsin 1 and Eps15, showed that depolarization also stimulated a calcium-dependent decrease in monoubiquitination. Notably, however, ubiquitinated epsin 1 represented only a small pool of the total epsin 1. Epsin 1 is a substrate for the deubiquitinating enzyme fat facets in Drosophila , and the mammalian homolog FAM was found to be localized to the same synapses in rat brain as epsin 1 and coprecipitates with rat epsin 1. In transfected cells, deubiquitination of epsin 1 did not occur in response to calcium ionophore if the cells were treated with small interfering RNA for FAM. However, siRNA to decrease FAM did not affect the calcium-triggered global decrease in general protein ubiquitination. Using a polyubiquitinated form of epsin 1, the authors showed that ubiquitinated epsin 1 did not interact with liposomes containing phosphatidylinositol 4,5-bisphosphate or endocytic coat proteins, which are both known to interact with nonubiquitinated epsin 1. Ubiquitinated epsin 1 did interact with a fragment of Eps15 containing the Eps15 homology domain. The role of this rapid change in protein ubiquitination in neuronal regulation remains to be determined, but these results suggest that acute stimulation profoundly alters protein ubiquitination, which likely affects various processes controlling synaptic activity. H. Chen, S. Polo, P. P. Di Fiore, P. V. De Camilli, Rapid Ca 2+ -dependent decrease of protein ubiquitination at synapses. Proc. Natl. Acad. Sci. U.S.A. 100 , 14908-14913 (2003). [Abstract] [Full Text]