HIP14 is the most highly conserved of 23 human palmitoyl acyltransferases (PATs) that catalyze the post-translational addition of palmitate to proteins, including huntingtin (HTT). HIP14 is dysfunctional in the presence of mutant HTT (mHTT), the causative gene for Huntington disease (HD), and we hypothesize that reduced palmitoylation of HTT and other HIP14 substrates contributes to the pathogenesis of the disease. Here we describe the yeast two-hybrid (Y2H) interactors of HIP14 in the first comprehensive study of interactors of a mammalian PAT. Unexpectedly, we discovered a highly significant overlap between HIP14 interactors and 370 published interactors of HTT, 4-fold greater than for control proteins (P = 8 × 10(-5)). Nearly half of the 36 shared interactors are already implicated in HD, supporting a direct link between HIP14 and the disease. The HIP14 Y2H interaction set is significantly enriched for palmitoylated proteins that are candidate substrates. We confirmed that three of them, GPM6A, and the Sprouty domain-containing proteins SPRED1 and SPRED3, are indeed palmitoylated by HIP14; the first enzyme known to palmitoylate these proteins. These novel substrates functions might be affected by reduced palmitoylation in HD. We also show that the vesicular cargo adapter optineurin, an established HTT-binding protein, co-immunoprecipitates with HIP14 but is not palmitoylated. mHTT leads to mislocalization of optineurin and aberrant cargo trafficking. Therefore, it is possible that optineurin regulates trafficking of HIP14 to its substrates. Taken together, our data raise the possibility that defective palmitoylation by HIP14 might be an important mechanism that contributes to the pathogenesis of HD.

Highlights d A map of 30,000 connections of 5,000 human proteins relevant to neurodegeneration d Network approach identifies modifiers of mutant TDP-43 and HTT toxicity in flies d ARF-GEP 100 controls misfolding and aggregation of diseasecausing proteins d MKL1 and ataxin-1 abnormally aggregate in AD patient brains

Microglia maintain homeostasis in the brain. However, with age, they become primed and respond more strongly to inflammatory stimuli. We show here that microglia from aged mice upregulated mammalian target of rapamycin (mTOR) complex 1 signaling regulating translation, as well as protein levels of inflammatory mediators. Genetic ablation of mTOR signaling showed a dual, yet contrasting effect on microglia priming: it caused an NF-kB-dependent upregulation of priming genes at mRNA level; however, mice displayed reduced cytokine protein levels, diminished microglia activation and milder sickness behavior. The effect on translation was dependent on reduced phosphorylation of 4EBP1, resulting in decreased binding of eIF4E to eIF4G. Similar changes were present in aged human microglia and in damage-associated microglia, indicating upregulation of mTOR-dependent translation is an essential step licensing microglia priming in aging and neurodegeneration.

Proteins with long, pathogenic polyglutamine (polyQ) sequences have an enhanced propensity to spontaneously misfold and self-assemble into insoluble protein aggregates. Here, we have identified 21 human proteins that influence polyQ-induced ataxin-1 misfolding and proteotoxicity in cell model systems. By analyzing the protein sequences of these modifiers, we discovered a recurrent presence of coiled-coil (CC) domains in ataxin-1 toxicity enhancers, while such domains were not present in suppressors. This suggests that CC domains contribute to the aggregation- and toxicity-promoting effects of modifiers in mammalian cells. We found that the ataxin-1–interacting protein MED15, computationally predicted to possess an N-terminal CC domain, enhances spontaneous ataxin-1 aggregation in cell-based assays, while no such effect was observed with the truncated protein MED15ΔCC, lacking such a domain. Studies with recombinant proteins confirmed these results and demonstrated that the N-terminal CC domain of MED15 (MED15CC) per se is sufficient to promote spontaneous ataxin-1 aggregation in vitro. Moreover, we observed that a hybrid Pum1 protein harboring the MED15CC domain promotes ataxin-1 aggregation in cell model systems. In strong contrast, wild-type Pum1 lacking a CC domain did not stimulate ataxin-1 polymerization. These results suggest that proteins with CC domains are potent enhancers of polyQ-mediated protein misfolding and aggregation in vitro and in vivo.

A 51 kDa fusion protein incorporating the N-methyltransferase domain of the multienzyme enniatin synthetase from Fusarium scirpi was expressed in Saccharomyces cerevisiae. The protein was purified and found to bind S-adenosyl methionine (AdoMet) as demonstrated by cross-linking experiments with (14)C-methyl-AdoMet under UV irradiation. Cofactor binding at equilibrium conditions was followed by saturation transfer difference (STD) NMR spectroscopy, and the native conformation of the methyltransferase was assigned. STD NMR spectroscopy yielded significant signals for H(2) and H(8) of the adenine moiety, H(1') of D-ribose, and S-CH(3) group of AdoMet. Methyl group transfer catalyzed by the enzyme was demonstrated by using aminoacyl-N-acetylcysteamine thioesters (aminoacyl-SNACs) of L-Val, L-Ile, and L-Leu, which mimic the natural substrate amino acids of enniatin synthetase presented by the enzyme bound 4'-phosphopantetheine arm. In these experiments the enzyme was incubated in the presence of the corresponding aminoacyl-SNAC and (14)C-methyl-AdoMet for various lengths of time, for up to 30 min. N-[(14)C-Methyl]-aminoacyl-SNAC products were extracted with EtOAc and separated by TLC. Acid hydrolysis of the isolated labeled compounds yielded the corresponding N-[(14)C-methyl] amino acids. Further proof for the formation of N-(14)C-methyl-aminoacyl-SNACs came from MALDI-TOF mass spectrometry which yielded 23 212 Da for N-methyl-valyl-SNAC, accompanied by the expected postsource decay (PSD) pattern. Interestingly, L-Phe, which is not a substrate amino acid of enniatin synthetase, also proved to be a methyl group acceptor. D-Val was not accepted as a substrate; this indicates selectivity for the L isomer.