Palmitoylation is the post-translational addition of a palmitate moiety to a cysteine residue through a covalent thioester bond. The addition and removal of this modification is controlled by both palmitoyl acyl-transferases and thioesterases. Using bioinformatic analysis, we identified 22 DHHC family palmitoyl acyl-transferase homologs in the Drosophila genome. We used in situ hybridization, RT-PCR, and published FlyAtlas microarray data to characterize the expression patterns of all 22 fly homologs. Our results indicate that all are expressed genes, but several, including CG1407, CG4676, CG5620, CG6017/dHIP14, CG6618, CG6627, and CG17257 appear to be enriched in neural tissues suggesting that they are important for neural function. Furthermore, we have found that several may be expressed in a sex-specific manner with adult male-specific expression of CG4483 and CG17195. Using tagged versions of the DHHC genes, we demonstrate that fly DHHC proteins are primarily located in either the Golgi Apparatus or Endoplasmic Reticulum in S2 cells, except for CG1407, which was found on the plasma membrane. We also characterized the subcellular localization and expression of the three known thioesterases: Palmitoyl-protein Thioesterase 1 (Ppt1), Palmitoyl-protein Thioesterase 2 (Ppt2), and Acyl-protein Thioesterase 1 (APT1). Our results indicate that Ppt1 and Ppt2 are the major lysosomal thioesterases while APT1 is the likely cytoplasmic thioesterase. Finally, in vivo rescue experiments show that Ppt2 expression cannot rescue the neural inclusion phenotypes associated with loss of Ppt1, further supporting distinct functions and substrates for these two thioesterases. These results will serve as the basis for a more complete understanding of the protein palmitoylome's normal cellular functions in the fly and will lead to further insights into the molecular etiology of diseases associated with the mis-regulation of palmitoylation.

Infantile neuronal ceroid lipofuscinosis (INCL) is a pediatric neurodegenerative disease caused by mutations in the human CLN1 gene. CLN1 encodes palmitoyl-protein thioesterase 1 (PPT1), suggesting an important role for the regulation of palmitoylation in normal neuronal function. To further elucidate Ppt1 function, we performed a gain-of-function modifier screen in Drosophila using a collection of enhancerpromoter transgenic lines to suppress or enhance the degeneration produced by overexpression of Ppt1 in the adult visual system. Modifier genes identified in our screen connect Ppt1 function to synaptic vesicle cycling, endo-lysosomal trafficking, synaptic development, and activity-dependent remodeling of the synapse. Furthermore, several homologs of the modifying genes are known to be regulated by palmitoylation in other systems and may be in vivo substrates for Ppt1. Our results complement recent work on mouse Ppt1À/À cells that shows a reduction in synaptic vesicle pools in primary neuronal cultures and defects in endosomal trafficking in human fibroblasts. The pathways and processes implicated by our modifier loci shed light on the normal cellular function of Ppt1. A greater understanding of Ppt1 function in these cellular processes will provide valuable insight into the molecular etiology of the neuronal dysfunction underlying the disease.

Infantile-onset Neuronal Ceroid Lipofuscinosis (INCL) is a severe pediatric neurodegenerative disorder produced by mutations in the gene encoding palmitoyl-protein thioesterase 1 (Ppt1). This enzyme is responsible for the removal of a palmitate post-translational modification from an unknown set of substrate proteins. To better understand the function of Ppt1 in neurons, we performed an unbiased dominant loss-of-function genetic modifier screen in Drosophila using a previously characterized Ppt1 gain-of-function system. The enhancers and suppressors identified in our screen make novel connections between Ppt1 and genes involved in cellular trafficking and the modulation of synaptic growth. We further support the relevance of our screen by demonstrating that Garland cells from Ppt1 loss-of-function mutants have defects in endocytic trafficking. Endocytic tracer uptake and ultrastructural analysis of these non-neuronal cells points to Ppt1 playing a role in modulating the early stages of vesicle formation. This work lays the groundwork for further experimental exploration of these processes to better understand their contributions to the INCL disease process.

Background: Poly-N-acetyl glucosamine nanofibers derived from a marine diatom have been used to increase cutaneous wound healing. These nanofibers exert their activity by specifically activating integrins, which makes them a useful tool for dissecting integrin-mediated pathways. We have shown that short-fiber poly-N-acetyl glucosamine nanofiber (sNAG) treatment of endothelial cells results in increased cell motility and metabolic rate in the absence of increased cell proliferation. Results: Using a Seahorse Bioanalyzer to measure oxygen consumption in real time, we show that sNAG treatment increases oxygen consumption rates, correlated with an integrin-dependent activation of Akt1. Akt1 activation leads to an increase in the expression of the transcriptional coactivator, peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α). This is not due to increased mitochondrial biogenesis, but is associated with an increase in the expression of pyruvate dehydrogenase kinase 4 (PDK4), suggesting regulation of fatty acid oxidation. Blockade of fatty acid oxidation with etomoxir, an O-carnitine palmitoyltransferase-1 inhibitor, blocks the sNAG-dependent increased oxygen consumption. ³H-palmitate uptake experiments indicate a PDK4-dependent increase in fatty acid oxidation, which is required for nanofiber-induced cell motility. Conclusions: Our findings imply a linear pathway whereby an integrin-dependent activation of Akt1 leads to increased PGC-1α and PDK4 expression resulting in increased energy production by fatty acid oxidation.