Summary
The microRNA pathway has been implicated in the regulation of synaptic protein synthesis and ultimately dendritic spine morphogenesis, a phenomenon associated with long-lasting forms of memory. However, the particular microRNAs (miRNAs) involved are largely unknown. We performed a functional screen to identify specific miRNAs that function at synapses to control dendritic spine structure. One of the identified miRNAs, miR-138, is highly enriched in the brain, localized within dendrites and negatively regulates the size of dendritic spines in rat hippocampal neurons. miR-138 controls the expression of Acyl protein thioesterase 1 (APT1), an enzyme regulating the palmitoylation status of proteins that are known to function at the synapse, including G protein alpha subunits (Gα). RNAi-mediated knockdown of APT1 and expression of membrane-localized Gα both suppress spine enlargement caused by miR-138 inhibition, suggesting that APT1-regulated depalmitoylation of Gα might be an important downstream event of miR-138 function. Our results uncover a novel miRNA-dependent mechanism in neurons and demonstrate a previously unrecognized complexity of miRNA-dependent control of dendritic spine morphogenesis.
Cycles of depalmitoylation and repalmitoylation critically control the steady-state localization and function of various peripheral membrane proteins, such as Ras proto-oncogene products. Interference with acylation using small molecules is a strategy to modulate cellular localization--and thereby unregulated signaling--caused by palmitoylated Ras proteins. We present the knowledge-based development and characterization of a potent inhibitor of acyl protein thioesterase 1 (APT1), a bona fide depalmitoylating enzyme that is, so far, poorly characterized in cells. The inhibitor, palmostatin B, perturbs the cellular acylation cycle at the level of depalmitoylation and thereby causes a loss of the precise steady-state localization of palmitoylated Ras. As a consequence, palmostatin B induces partial phenotypic reversion in oncogenic HRasG12V-transformed fibroblasts. We identify APT1 as one of the thioesterases in the acylation cycle and show that this protein is a cellular target of the inhibitor.
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