Geranylgeranyl generosity: a new prenyl-transferase gives a fat to a SNARE protein.

Protein prenylation, a well-defined protein consensus motifs direct modification by one of three prenyl-transferases, has been an area of fairly settled science for 20 or 30 years. Protein prenylation, the specific prenyl modification (farnesyl or geranylgeranyl), as well as the prenyl-transferases involved can be inferred by protein sequence. Two new papers now upset this settled wisdom with the discovery of a fourth prenyl-transferase, namely geranylgeranyl-transferase-III (GGTase-III) (Kuchay et al, 2019; Shirakawa et al, 2020).

Targeting the Ras palmitoylation/depalmitoylation cycle in cancer.

The Ras proteins are well-known drivers of many cancers and thus represent attractive targets for the development of anticancer therapeutics. Inhibitors that disrupt the association of the Ras proteins with membranes by blocking the addition of the farnesyl lipid moiety to the Ras C-terminus failed in clinical trials. Here, we explore the possibility of targeting a second lipid modification, S-acylation, commonly referred to as palmitoylation, as a strategy to disrupt the membrane interaction of specific Ras isoforms. We review the enzymes involved in adding and removing palmitate from Ras and discuss their potential roles in regulating Ras tumorigenesis. In addition, we examine other proteins that affect Ras protein localization and may serve as future drug targets.

Neuronal ceroid lipofuscinosis with DNAJC5/CSPα mutation has PPT1 pathology and exhibit aberrant protein palmitoylation.

Neuronal ceroid lipofuscinoses (NCL) are a group of inherited neurodegenerative disorders with lysosomal pathology (CLN1-14). Recently, mutations in the DNAJC5/CLN4 gene, which encodes the presynaptic co-chaperone CSPα were shown to cause autosomal-dominant NCL. Although 14 NCL genes have been identified, it is unknown if they act in common disease pathways. Here we show that two disease-associated proteins, CSPα and the depalmitoylating enzyme palmitoyl-protein thioesterase 1 (PPT1/CLN1) are biochemically linked. We find that in DNAJC5/CLN4 patient brains, PPT1 is massively increased and mis-localized. Surprisingly, the specific enzymatic activity of PPT1 is dramatically reduced. Notably, we demonstrate that CSPα is depalmitoylated by PPT1 and hence its substrate. To determine the consequences of PPT1 accumulation, we compared the palmitomes from control and DNAJC5/CLN4 patient brains by quantitative proteomics. We discovered global changes in protein palmitoylation, mainly involving lysosomal and synaptic proteins. Our findings establish a functional link between two forms of NCL and serve as a springboard for investigations of NCL disease pathways.

Neural palmitoyl-proteomics reveals dynamic synaptic palmitoylation.

Palmitoylation regulates diverse aspects of neuronal protein trafficking and function. Here a global characterization of rat neural palmitoyl-proteomes identifies most of the known neural palmitoyl proteins-68 in total, plus more than 200 new palmitoyl-protein candidates, with further testing confirming palmitoylation for 21 of these candidates. The new palmitoyl proteins include neurotransmitter receptors, transporters, adhesion molecules, scaffolding proteins, as well as SNAREs and other vesicular trafficking proteins. Of particular interest is the 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 has a special role in inducing these post-synaptic structures. Furthermore, assessing palmitoylation dynamics in drug-induced activity models identifies rapidly induced changes for Cdc42 as well as for other synaptic palmitoyl proteins, suggesting that palmitoylation may participate broadly in the activity-driven changes that shape synapse morphology and function.

Wild-type HTT modulates the enzymatic activity of the neuronal palmitoyl transferase HIP14.

Huntington disease (HD) is caused by polyglutamine expansion in the huntingtin (HTT) protein. Huntingtin-interacting protein 14 (HIP14), one of 23 DHHC domain-containing palmitoyl acyl transferases (PATs), binds to HTT and robustly palmitoylates HTT at cysteine 214. Mutant HTT exhibits reduced palmitoylation and interaction with HIP14, contributing to the neuronal dysfunction associated with HD. In this study, we confirmed that, among 23 DHHC PATs, HIP14 and its homolog DHHC-13 (HIP14L) are the two major PATs that palmitoylate HTT. Wild-type HTT, in addition to serving as a palmitoylation substrate, also modulates the palmitoylation of HIP14 itself. In vivo, HIP14 palmitoylation is decreased in the brains of mice lacking one HTT allele (hdh+/-) and is further reduced in mouse cortical neurons treated with HTT antisense oligos (HTT-ASO) that knockdown HTT expression by ∼95%. Previously, it has been shown that palmitoylation of DHHC proteins may affect their enzymatic activity. Indeed, palmitoylation of SNAP25 by HIP14 is potentiated in vitro in the presence of wild-type HTT. This influence of HTT on HIP14 activity is lost in the presence of CAG expansion. Furthermore, in both brains of hdh+/- mice and neurons treated with HTT-ASO, we observe a significant reduction in palmitoylation of endogenous SNAP25 and GluR1, synaptic proteins that are substrates of HIP14, suggesting wild-type HTT also influences HIP14 enzymatic activity in vivo. This study describes an important biochemical function for wild-type HTT modulation of HIP14 palmitoylation and its enzymatic activity.

Altered palmitoylation and neuropathological deficits in mice lacking HIP14.

Huntingtin interacting protein 14 (HIP14, ZDHHC17) is a huntingtin (HTT) interacting protein with palmitoyl transferase activity. In order to interrogate the function of Hip14, we generated mice with disruption in their Hip14 gene. Hip14-/- mice displayed behavioral, biochemical and neuropathological defects that are reminiscent of Huntington disease (HD). Palmitoylation of other HIP14 substrates, but not Htt, was reduced in the Hip14-/- mice. Hip14 is dysfunctional in the presence of mutant htt in the YAC128 mouse model of HD, suggesting that altered palmitoylation mediated by HIP14 may contribute to HD.

Palmitoylation by the DHHC protein Pfa4 regulates the ER exit of Chs3.

The yeast chitin synthase Chs3 provides a well-studied paradigm for polytopic membrane protein trafficking. In this study, high-throughput analysis of the yeast deletion collection identifies a requirement for Pfa4, which is an uncharacterized protein with protein acyl transferase (PAT) homology, in Chs3 transport. PATs, which are the enzymatic mediators of protein palmitoylation, have only recently been discovered, and few substrates have been identified. We find that Chs3 is palmitoylated and that this modification is Pfa4-dependent, indicating that Pfa4 is indeed a PAT. Chs3 palmitoylation is required for ER export, but not for interaction with its dedicated ER chaperone, Chs7. Nonetheless, both palmitoylation and chaperone association are required to prevent the accumulation of Chs3 in high-molecular mass aggregates at the ER. Our data indicate that palmitoylation is necessary for Chs3 to attain an export-competent conformation, and suggest the possibility of a more general role for palmitoylation in the ER quality control of polytopic membrane proteins.

Palmitoylation and function of glial glutamate transporter-1 is reduced in the YAC128 mouse model of Huntington disease.

Excitotoxicity plays a key role in the selective vulnerability of striatal neurons in Huntington disease (HD). Decreased glutamate uptake by glial cells could account for the excess glutamate at the synapse in patients as well as animal models of HD. The major molecule responsible for clearing glutamate at the synapses is glial glutamate transporter GLT-1. In this study, we show that GLT-1 is palmitoylated at cysteine38 (C38) and further, that this palmitoylation is drastically reduced in HD models both in vitro and in vivo. Palmitoylation is required for normal GLT-1 function. Blocking palmitoylation either with the general palmitoylation inhibitor, 2-bromopalmitate, or with a GLT-1 C38S mutation, severely impairs glutamate uptake activity. In addition, GLT-1-mediated glutamate uptake is indeed impaired in the YAC128 HD mouse brain, with the defect in the striatum evident as early as 3 months prior to obvious neuropathological findings, and in both striatum and cortex at 12 months. These phenotypes are not a result of changes in GLT1 protein expression, suggesting a crucial role of palmitoylation in GLT-1 function. Thus, it appears that impaired GLT-1 palmitoylation is present early in the pathogenesis of HD, and may influence decreased glutamate uptake, excitotoxicity, and ultimately, neuronal cell death in HD.

Protein aggregation induced during glass bead lysis of yeast.

Yeast cell lysates produced by mechanical glass bead disruption are widely used in a variety of applications, including for the analysis of native function, e.g. protein-protein interaction, enzyme assays and membrane fractionations. Below, we report a striking case of protein denaturation and aggregation that is induced by this lysis protocol. Most of this analysis focuses on the type 1 casein kinase Yck2, which normally tethers to the plasma membrane through C-terminal palmitoylation. Surprisingly, when cells are subjected to glass bead disruption, non-palmitoylated, cytosolic forms of the kinase denature and aggregate, while membrane-associated forms, whether attached through their native palmitoyl tethers or through a variety of artificial membrane-tethering sequences, are wholly protected from denaturation and aggregation. A wider look at the yeast proteome finds that, while the majority of proteins resist glass bead-induced aggregation, a significant subset does, in fact, succumb to such denaturation. Thus, yeast researchers should be aware of this potential artifact when embarking on biochemical analyses that employ glass bead lysates to look at native protein function. Finally, we demonstrate an experimental utility for glass bead-induced aggregation, using its fine discrimination of membrane-associated from non-associated Yck2 forms to discern fractional palmitoylation states of Yck2 mutants that are partially defective for palmitoylation.

The yeast DHHC cysteine-rich domain protein Akr1p is a palmitoyl transferase.

Protein palmitoylation has been long appreciated for its role in tethering proteins to membranes, yet the enzymes responsible for this modification have eluded identification. Here, experiments in vivo and in vitro demonstrate that Akr1p, a polytopic membrane protein containing a DHHC cysteine-rich domain (CRD), is a palmitoyl transferase (PTase). In vivo, we find that the casein kinase Yck2p is palmitoylated and that Akr1p function is required for this modification. Akr1p, purified to near homogeneity from yeast membranes, catalyzes Yck2p palmitoylation in vitro, indicating that Akr1p is itself a PTase. Palmitoylation is stimulated by added ATP. Furthermore, during the reaction, Akr1p is itself palmitoylated, suggesting a role for a palmitoyl-Akr1p intermediate in the overall reaction mechanism. Mutations introduced into the Akr1p DHHC-CRD eliminate both the trans- and autopalmitoylation activities, indicating a central participation of this conserved sequence in the enzymatic reaction. Finally, our results indicate that palmitoylation within the yeast cell is controlled by multiple PTase specificities. The conserved DHHC-CRD sequence, we propose, is the signature feature of an evolutionarily widespread PTase family.

The yeast casein kinase Yck3p is palmitoylated, then sorted to the vacuolar membrane with AP-3-dependent recognition of a YXXPhi adaptin sorting signal.

Our previous work found the two yeast plasma membrane-localized casein kinases Yck1p and Yck2p to be palmitoylated on C-terminal Cys-Cys sequences by the palmitoyl transferase Akr1p. The present work examines a third casein kinase, Yck3p, which ends with the C-terminal sequence Cys-Cys-Cys-Cys-Phe-Cys-Cys-Cys. Yck3p is palmitoylated and localized to the vacuolar membrane. While the C-terminal cysteines are required for this palmitoylation, Akr1p is not. Palmitoylation requires the C-terminal Yck3p residues 463-524, whereas information for vacuolar sorting maps to the 409-462 interval. Vacuolar sorting is disrupted in cis through deletion of the 409-462 sequences and in trans through mutation of the AP-3 adaptin complex; both cis- and trans-mutations result in Yck3p missorting to the plasma membrane. This missorted Yck3p restores 37 degrees C viability to yck1Delta yck2-ts cells. yck1Delta yck2-ts suppressor mutations isolated within the YCK3 gene identify the Yck3p vacuolar sorting signal-the tetrapeptide YDSI, a perfect fit to the YXXPhi adaptin-binding consensus. Although YXXPhi signals have a well-appreciated role in the adaptin-mediated sorting of mammalian cells, this is the first signal of this class to be identified in yeast.

Transcriptome-facilitated development of SNPs for the Sonoran Desert rock fig, Ficus petiolaris (Moraceae).

PREMISE OF THE STUDY: Single-nucleotide polymorphism (SNP) primers were developed for a native North American desert fig, Ficus petiolaris (Moraceae), to provide markers for population genetic studies designed to quantify patterns of gene flow across a complex landscape. METHODS AND RESULTS: Transcriptome sequencing and bioinformatic protocols were implemented to discover SNPs in single-copy protein-coding genes. Multiplexes of 30 nuclear and 24 organellar (chloroplast and mitochondrial) SNPs were selected for primer development and genotyping on the Sequenom MASSArray System. Of these 54 loci, 49 reliably amplified across a panel of 96 F. petiolaris individuals. CONCLUSIONS: This study has provided SNP primers that can be applied in future studies investigating population genetics of F. petiolaris and its coevolution with associated pollinating and nonpollinating fig wasps.

Tracking brain palmitoylation change: predominance of glial change in a mouse model of Huntington's disease.

Protein palmitoylation, a reversible lipid modification of proteins, is widely used in the nervous system, with dysregulated palmitoylation being implicated in a variety of neurological disorders. Described below is ABE/SILAM, a proteomic strategy that couples acyl-biotinyl exchange (ABE) purification of palmitoyl-proteins to whole animal stable isotope labeling (SILAM) to provide an accurate tracking of palmitoylation change within rodent disease models. As a first application, we have used ABE/SILAM to look at Huntington's disease (HD), profiling palmitoylation change in two HD-relevant mouse mutants: the transgenic HD model mouse YAC128 and the hypomorphic Hip14-gt mouse, which has sharply reduced expression for HIP14 (Zdhhc17), a palmitoyl-transferase implicated in the HD disease process. Rather than mapping to the degenerating neurons themselves, the biggest disease changes instead map to astrocytes and oligodendrocytes (i.e., the supporting glial cells).

The yeast kinase Yck2 has a tripartite palmitoylation signal.

The yeast kinase Yck2 tethers to the cytoplasmic surface of the plasma membrane through dual palmitoylation of its C-terminal Cys-Cys dipeptide, mediated by the Golgi-localized palmitoyl-transferase Akr1. Here, the Yck2 palmitoylation signal is found to consist of three parts: 1) a 10-residue-long, conserved C-terminal peptide (CCTP) that includes the C-terminal Cys-Cys dipeptide; 2) the kinase catalytic domain (KD); and mapping between these two elements; and 3) a 176-residue-long, poorly conserved, glutamine-rich sequence. The CCTP, which contains the C-terminal cysteines as well as an important Phe-Phe dipeptide, likely serves as an Akr1 recognition element, because CCTP mutations disrupt palmitoylation within a purified in vitro palmitoylation system. The KD contribution appears to be complex with roles for both KD activity (e.g., Yck2-mediated phosphorylation) and structure (e.g., Akr1 recognition elements). KD and CCTP mutations are strongly synergistic, suggesting that, like the CCTP, the KD may also participate at the Yck2-Akr1 recognition step. The long, glutamine-rich domain, which is located between the KD and CCTP, is predicted to be intrinsically disordered and may function as a flexible, interdomain linker, allowing a coupled interaction of the KD and CCTP with Akr1. Multipart palmitoylation signals may prove to be a general feature of this large class of palmitoylation substrates. These soluble proteins have no clear means of accessing membranes and thus may require active capture out of the cytoplasm for palmitoylation by their membrane-localized transferases.

Palmitoylated proteins: purification and identification.

This proteomic protocol purifies and identifies palmitoylated proteins (i.e., S-acylated proteins) from complex protein extracts. The method relies on an acyl-biotinyl exchange chemistry in which biotin moieties are substituted for the thioester-linked protein acyl-modifications through a sequence of three in vitro chemical steps: (i) blockade of free thiols with N-ethylmaleimide; (ii) cleavage of the Cys-palmitoyl thioester linkages with hydroxylamine; and (iii) labeling of thiols, newly exposed by the hydroxylamine, with biotin-HPDP (Biotin-HPDP-N-[6-(Biotinamido)hexyl]-3'-(2'-pyridyldithio)propionamide. The biotinylated proteins are then affinity-purified using streptavidin-agarose and identified by multi-dimensional protein identification technology (MuDPIT), a high-throughput, tandem mass spectrometry (MS/MS)-based proteomic technology. MuDPIT also affords a semi-quantitative analysis that may be used to assess the gross changes induced to the global palmitoylation profile by mutation or drugs. Typically, 2-3 weeks are required for this analysis.

Proteomic identification of palmitoylated proteins.

A proteomic method that purifies and identifies palmitoylated proteins from complex protein extracts is described. Using the fatty acid exchange labeling chemistry (described in the preceding report), palmitoyl modifications are exchanged for biotinylated compounds, allowing the subset of palmitoyl-proteins to be affinity-purified and then identified by mass spectroscopic protein identification technologies. The advantages and pitfalls of this new technology are discussed within the context of the recent application of this method in the yeast Saccharomyces cerevisiae.

Global analysis of protein palmitoylation in yeast.

Protein palmitoylation is a reversible lipid modification that regulates membrane tethering for key proteins in cell signaling, cancer, neuronal transmission, and membrane trafficking. Palmitoylation has proven to be a difficult study: Specifying consensuses for predicting palmitoylation remain unavailable, and first-example palmitoylation enzymes--i.e., protein acyltransferases (PATs)--were identified only recently. Here, we use a new proteomic methodology that purifies and identifies palmitoylated proteins to characterize the palmitoyl proteome of the yeast Saccharomyces cerevisiae. Thirty-five new palmitoyl proteins are identified, including many SNARE proteins and amino acid permeases as well as many other participants in cellular signaling and membrane trafficking. Analysis of mutant yeast strains defective for members of the DHHC protein family, a putative PAT family, allows a matching of substrate palmitoyl proteins to modifying PATs and reveals the DHHC family to be a family of diverse PAT specificities responsible for most of the palmitoylation within the cell.

Transmembrane topology of the protein palmitoyl transferase Akr1.

The two recently identified protein acyl transferases (PATs), Akr1p and Erf2p/Erf4p, point toward the DHHC protein family as a likely PAT family. The DHHC protein family, defined by the novel, zinc finger-like DHHC cysteine-rich domain (DHHC-CRD), is a diverse collection of polytopic membrane proteins extending through all eukaryotes. To define the PAT domains that are oriented to the cytoplasm and are thus available to effect the cytoplasmically limited palmitoyl modification, we have determined the transmembrane topology of the yeast PAT Akr1p. Portions of the yeast protein invertase (Suc2p) were inserted in-frame at 10 different hydrophilic sites within the Akr1 polypeptide. Three of the Akr1-Suc2-Akr1 insertion proteins were found to be extensively glycosylated, indicating that the invertase segment inserted at these Akr1p sites is luminally oriented. The remaining seven insertion proteins were not glycosylated, consistent with a cytoplasmic orientation for these sites. The results support a model in which the Akr1 polypeptide crosses the bilayer six times with the bulk of its hydrophilic domains disposed toward the cytoplasm. Cytoplasmic domains include both the relatively large, ankyrin repeat-containing N-terminal domain and the DHHC-CRD, which maps to a cytosolic loop segment. Functionality of the different Akr1-Suc2-Akr1 proteins also was examined. Insertions at only 4 of the 10 sites were found to disrupt Akr1p function. Interestingly, these four sites all map cytoplasmically, suggesting key roles for these cytoplasmic domains in Akr1 PAT function. Finally, extrapolating from the Akr1p topology, topology models are proposed for other DHHC protein family members.

Ubiquitin-independent entry into the yeast recycling pathway.

The yeast a-factor receptor (Ste3p) is subject to two mechanistically distinct modes of endocytosis: a constitutive, ligand-independent pathway links to vacuolar degradation of the receptor, while a ligand-dependent uptake pathway links primarily to recycling and thus, receptor reutilization. Ste3p ubiquitination triggers its uptake into the constitutive pathway. The present work considers the role of the receptor ubiquitination associated with the Ste3p ligand-dependent endocytosis mechanism. The doa4delta mutation which reduces the cellular availability of ubiquitin blocks the Ste3p constitutive uptake. Uptake into the Ste3p ligand-dependent recycling pathway, however, continues unimpaired. The ubiquitin independence of Ste3p ligand-dependent uptake was further indicated by analysis of receptor mutants having Lys-to-Arg substitutions at all possible ubiquitin acceptor sites. Again, the ligand-induced internalization was unimpaired. Furthermore, no discernible effect was seen on either recycling or on the slow PEP4-dependent turnover of the receptor (for receptor internalized via the ligand-dependent mechanism, trafficking to the vacuole/lysosome is the minor, alternate fate to recycling). However, one striking effect of the Lys-to-Arg mutations was noted. Following a prolonged exposure of the cells to the a-factor ligand, rather than being delivered to the vacuolar lumen, the Lys-to-Arg receptor was found to localize instead to the limiting membrane of the vacuole. Thus, while receptor ubiquitination clearly is not required for either the a-factor-dependent uptake into recycling pathway or for the recycling itself, it does affect the routing of receptor to the vacuole, likely by affecting the routing through the late endosomal, multivesicular body: ubiquitinated receptor may be selected into the internal, lumenal vesicles, while unmodified receptor may be left to reside at the limiting external membrane.