Summary Palmitoylation regulates diverse aspects of neuronal protein trafficking and function. Here, a global characterization of the neuronal palmitoyl-proteome identifies most of the known neuronal palmitoyl-proteins (PPs), 68 in total, plus over 200 new PP candidates, with additional testing confirming palmitoylation for 21 of these candidates. New PPs include neurotransmitter receptors, transporters, adhesion molecules, scaffolding proteins, as well as SNAREs and other vesicular trafficking proteins. Of particular interest is a finding of palmitoylation for a brain-specific Cdc42 splice variant. The palmitoylated Cdc42 isoform (Cdc42-palm) differs from the canonical, prenylated form (Cdc42-prenyl) both with regard to localization and function: Cdc42-palm, concentrates in dendritic spines and plays a special role in inducing these post-synaptic structures. Finally, assessing palmitoylation dynamics in drug-induced activity paradigms finds rapidly induced changes both for Cdc42 as well as for other synaptic PPs, suggesting that palmitoylation may participate broadly in the activity-driven changes that shape synapse morphology and function.
In neurons, posttranslational modification by palmitate regulates the trafficking and function of signaling molecules, neurotransmitter receptors, and associated synaptic scaffolding proteins. However, the enzymatic machinery involved in protein palmitoylation has remained elusive. Here, using biochemical assays, we show that huntingtin (htt) interacting protein, HIP14, is a neuronal palmitoyl transferase (PAT). HIP14 shows remarkable substrate specificity for neuronal proteins, including SNAP-25, PSD-95, GAD65, synaptotagmin I, and htt. Conversely, HIP14 is catalytically invariant toward paralemmin and synaptotagmin VII. Exogenous HIP14 enhances palmitoylation-dependent vesicular trafficking of several acylated proteins in both heterologous cells and neurons. Moreover, interference with endogenous expression of HIP14 reduces clustering of PSD-95 and GAD65 in neurons. These findings define HIP14 as a mammalian palmitoyl transferase involved in the palmitoylation and trafficking of multiple neuronal proteins.
SUMMARY Neurotrophin receptor tyrosine kinases (Trks) have well-defined trophic roles in nervous system development through kinase activation by neurotrophins. Yet Trks have typical cell-adhesion domains and express non-catalytic isoforms suggesting additional functions. Here we discovered non-catalytic TrkC in an unbiased hippocampal neuron-fibroblast coculture screen for proteins that trigger differentiation of neurotransmitter release sites in axons. All TrkC isoforms, but not TrkA or TrkB, function directly in excitatory glutamatergic synaptic adhesion by neurotrophin-independent high-affinity trans-binding to axonal PTPσ tyrosine phosphatase receptor. PTPσ triggers and TrkC mediates clustering of postsynaptic molecules in dendrites, indicating bidirectional synaptic organizing functions. Effects of a TrkC neutralizing antibody that blocks TrkC-PTPσ interaction and TrkC knockdown in culture and in vivo reveal essential roles of TrkC-PTPσ in glutamatergic synapse formation. Thus, postsynaptic TrkC trans-interaction with presynaptic PTPσ generates bidirectional adhesion and recruitment essential for excitatory synapse development and positions these signaling molecules at the center of synaptic pathways.
Post-translational modification by the lipid palmitate is crucial for the correct targeting and function of many proteins. Here we show that huntingtin (htt) is normally palmitoylated at cysteine 214, which is essential for its trafficking and function. The palmitoylation and distribution of htt are regulated by the palmitoyl transferase huntingtin interacting protein 14 (HIP14). Expansion of the polyglutamine tract of htt, which causes Huntington disease, results in reduced interaction between mutant htt and HIP14 and consequently in a marked reduction in palmitoylation. Mutation of the palmitoylation site of htt, making it palmitoylation resistant, accelerates inclusion formation and increases neuronal toxicity. Downregulation of HIP14 in mouse neurons expressing wild-type and mutant htt increases inclusion formation, whereas overexpression of HIP14 substantially reduces inclusions. These results suggest that the expansion of the polyglutamine tract in htt results in decreased palmitoylation, which contributes to the formation of inclusion bodies and enhanced neuronal toxicity.
Palmitoylation, a post-translational modification of cysteine residues with the lipid palmitate, has recently emerged as an important mechanism for regulating protein trafficking and function. With the identification of 23 DHHC mammalian palmitoyl acyl transferases (PATs), a key question was the nature of substrate-enzyme specificity for these PATs. Using the acyl-biotin exchange palmitoylation assay, we compared the substrate specificity of four neuronal PATs, namely DHHC-3, DHHC-8, HIP14L (DHHC-13), and HIP14 (DHHC-17). Exogenous expression of enzymes and substrates in COS cells reveals that HIP14L and HIP14 modulate huntingtin palmitoylation, DHHC-8 modulates paralemmin-1 palmitoylation, and DHHC-3 shows the least substrate specificity. These in vitro data were validated by lentiviral siRNA-mediated knockdown of endogenous HIP14 and DHHC-3 in cultured rat cortical neurons. PATs require the presence of palmitoylated cysteines in order to interact with their substrates. To understand the elements that influence enzyme/substrate specificity further, we fused the HIP14 ankryin repeat domain to the N terminus of DHHC-3, which is not a PAT for huntingtin. This modification enabled DHHC-3 to behave similarly to HIP14 by modulating palmitoylation and trafficking of huntingtin. Taken together, this study indicates that individual PATs have specific substrate preference, determined by regulatory domains outside the DHHC domain of the enzymes.
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