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.— Huang, K., Sanders, S., Singaraja, R., Orban, P., Cij‐souw, T., Arstikaitis, P., Yanai, A., Hayden, M. R., El‐Husseini, A. Neuronal palmitoyl acyl transferases exhibit distinct substrate specificity. FASEB J. 23, 2605–2615 (2009)

DNA double-strand breaks (DSBs) can efficiently kill cancer cells, but they can also produce unwanted chromosome rearrangements (CRs) when DNA ends from different DSBs are erroneously joined. Movement of DSB-containing chromatin domains might facilitate these DSB interactions and promote formation of CRs. We, therefore, analyzed the mobility of DSB-containing chromatin domains in living mammalian cells and compared it to the mobility of non-damaged chromatin on a time-scale relevant for DSB repair. We found that DSB-containing chromatin domains are significantly more mobile than intact chromatin and capable of roaming a more than 2-fold larger area of the cell nucleus. Moreover, this increased DSB mobility, but not mobility of undamaged chromatin, can be reduced by agents that affect higher-order chromatin organization.

Munc18-1 is a soluble protein essential for synaptic transmission. To investigate the dynamics of endogenous Munc18-1 in neurons, we created a mouse model expressing fluorescently tagged Munc18-1 from the endogenous munc18-1 locus. We show using fluorescence recovery after photobleaching in hippocampal neurons that the majority of Munc18-1 trafficked through axons and targeted to synapses via lateral diffusion together with syntaxin-1. Munc18-1 was strongly expressed at presynaptic terminals, with individual synapses showing a large variation in expression. Axon–synapse exchange rates of Munc18-1 were high: during stimulation, Munc18-1 rapidly dispersed from synapses and reclustered within minutes. Munc18-1 reclustering was independent of syntaxin-1, but required calcium influx and protein kinase C (PKC) activity. Importantly, a PKC-insensitive Munc18-1 mutant did not recluster. We show that synaptic Munc18-1 levels correlate with synaptic strength, and that synapses that recruit more Munc18-1 after stimulation have a larger releasable vesicle pool. Hence, PKC-dependent dynamic control of Munc18-1 levels enables individual synapses to tune their output during periods of activity.

Synapses are specialized neuronal cell-cell contacts that underlie network communication in the mammalian brain. Across neuronal populations and circuits, a diverse set of synapses is utilized, and they differ in their molecular composition to enable heterogenous connectivity patterns and functions. In addition to pre- and post-synaptic specializations, the synaptic cleft is now understood to be an integral compartment of synapses that contributes to their structural and functional organization. Aiming to map the cleft proteome, this study applied a peroxidase-mediated proximity labeling approach and used the excitatory synaptic cell adhesion protein SynCAM 1 fused to horseradish peroxidase (HRP) as a reporter in cultured cortical neurons. This reporter marked excitatory synapses as measured by confocal microcopy and was targeted to the edge zone of the synaptic cleft as determined using 3D dSTORM super-resolution imaging. Proximity labeling with a membrane-impermeant biotin-phenol compound restricted labeling to the cell surface, and Label-Free Quantitation (LFQ) mass spectrometry combined with ratiometric HRP tagging of membrane vs. synaptic surface proteins was used to identify the proteomic content of excitatory clefts. Novel cleft candidates were identified, and Receptor-type tyrosine-protein phosphatase zeta was selected and successfully validated. This study supports the robust applicability of peroxidase-mediated proximity labeling for synaptic cleft proteomics and its potential for understanding synapse heterogeneity in health and changes in diseases such as psychiatric disorders and addiction.