Encyclopedia of Signaling Molecules

2018 Edition
| Editors: Sangdun Choi

DHHC Proteins

Reference work entry
DOI: https://doi.org/10.1007/978-3-319-67199-4_322


Historical Background

Protein palmitoylation is the first discovered and the most common lipid modification. This posttranslational change involves addition of the saturated 16-carbon palmitate to specific cysteine residues by a labile thioester linkage (Linder and Deschenes 2007). Although reversible palmitoylation was discovered over 30 years ago, the enzymes that add palmitate to proteins (palmitoyl acyl transferases, PATs) and those that cleave the thioester bond (palmitoyl protein thioesterases, PPTs) had been elusive. In 2002, genetic screening in yeast identified proteins that mediate PAT activity. Erf2/4 (Lobo et al. 2002) and Akr1 (Roth et al. 2002) were identified as PATs for yeast Ras2 and Yck2, respectively. Erf2 and Akr1 share a conserved DHHC (Asp-His-His-Cys) cysteine-rich domain (CRD) (Fig. 1) and have four or six transmembrane domains. The DHHC sequence and its surrounding CRD sequence are essential for their enzymatic activity. In 2004, 23 kinds of mammalian DHHC proteins were systematically isolated and some of them were characterized as PATs (Fukata et al. 2004). DHHC proteins have now emerged as evolutionally conserved PATs from plants, yeast (7 genes), Caenorhabditis elegans (15 genes), Drosophila melanogaster (22 genes) to mammals (24 genes).
DHHC Proteins, Fig. 1

The domain structure of DHHC3, a prototype of mammalian DHHC proteins. DHHC proteins contain four or six transmembrane domains and a conserved DHHC (Asp-His-His-Cys) motif in the cytoplasmic cysteine-rich domain (CRD). Some members of DHHC proteins have unique motif/domain, such as PDZ-binding motif (in DHHC3). The consensus sequence of DHHC-CRD is indicated (green and red). X, a variety of amino acids

Subfamily Classification of Mammalian DHHC Proteins

The large family of DHHC proteins can be classified into several subfamilies based on the homology of the DHHC-CRD core catalytic domains. DHHC2 and DHHC15 belong to one subfamily, while DHHC3 and DHHC7 form another subfamily. Importantly, DHHC proteins in the same subfamily often share their substrates (Fig. 2) (Fukata and Fukata 2010; Greaves and Chamberlain 2011). DHHC3/7 subfamily palmitoylates most of palmitoyl-proteins, such as PSD-95, GAP43, Gα, SNAP-25, NCAM-140, and GABAA receptor γ subunit. In contrast, DHHC2/15 subfamily more specifically palmitoylates PSD-95 and GAP43 (Fukata et al. 2004), but not Gα, NCAM-140 nor GABAA receptor γ subunit (Fig. 2). H/N-Ras is the only well-established substrate for DHHC9/18 subfamily. Because the number of identified substrate-enzyme pairs is limited, enzymatic activities of some DHHC proteins, such as DHHC1/10 subfamily, still remain undocumented.
DHHC Proteins, Fig. 2

Phylogenetic tree of mammalian DHHC protein family members. 23 DHHC proteins can be categorized into several subfamilies based on the homology of the catalytic DHHC domains. For example, DHHC2 and DHHC15 belong to one subfamily (in red), while DHHC3 and DHHC7 form another subfamily (in blue). The discovery of this mammalian DHHC protein family and the establishment of simple screening system using DHHC protein library have facilitated identification of palmitoyl substrate-enzyme pairs. Importantly, the same subfamily of DHHC proteins often shares substrates, for example, DHHC2/15 subfamily specifically palmitoylates PSD-95 and GAP-43. DHHC3/7 subfamily palmitoylates most of palmitoyl-proteins, such as PSD-95, GAP-43, Gα, GABAARγ2, and SNAP-25. DHHC9/18 subfamily (in purple) palmitoylates H-Ras, DHHC21 (in brown) palmitoylates Lck and eNOS, and DHHC17 (in yellow) palmitoylates SNAP-25 and huntingtin. Note that some DHHC clone numbers initially collected by Fukata et al. (2004) are different from a current standard nomenclature (ZDHHC clone numbers) (Fukata and Fukata 2010). At present, 24 zDHHC genes have been annotated in the mouse genome (ZDHHC22 is recently annotated)

Enzymatic and Regulatory Mechanisms for DHHC Proteins

DHHC-CRD region is the catalytic core site as mutation of the cysteine in the DHHC motif makes DHHC proteins catalytically inactive. Cysteines in this DHHC-CRD region are often autopalmitoylated (Roth et al. 2002; Fukata et al. 2004). Two mechanisms for palmitate transfer to a substrate have been proposed: (1) DHHC protein directly transfers palmitate from palmitoyl-CoA to the substrate and (2) DHHC protein first forms a thioester intermediate with palmitate (i.e., autopalmitoylation of DHHC proteins), followed by the transfer of the palmitate to the substrate. Recent mutational analysis in yeast revealed that palmitoylation reaction by DHHC proteins, at least yeast Erf2, is mediated by the latter two-step reaction (Mitchell et al. 2010). In addition to the catalytically critical DHHC-CRD domain, some DHHC proteins have regulatory regions such as SH3 domain and ankyrin repeats at the C-terminal or N-terminal cytoplasmic regions. These domains may recruit specific substrate proteins to the DHHC enzyme. In fact, ankyrin repeats of DHHC17/HIP14 contribute to determining the substrate specificity (Huang et al. 2009).

DHHC3/7, 17, and 2/15 subfamily members do not require additional proteins (cofactors) for their PAT activity. In contrast, yeast Erf2 and its mammalian ortholog DHHC9 are inactive PATs in the absence of accessory proteins, Erf4 (for Erf2) or GCP16 (for DHHC9; Golgi complex protein of 16 kDa) (Swarthout et al. 2005). So far, the regulatory mechanism that directly activates or inactivates DHHC PAT activity is not known.

Akin to protein phosphorylation, protein palmitoylation is a reversible reaction that can be regulated by specific extracellular signals, for example, β-adrenergic receptor activation accelerates depalmitoylation and palmitoylation levels of Gαs (Degtyarev et al. 1993; Wedegaertner and Bourne 1994). Palmitoylation of  LAT, linker for activation of T-cells, is reduced by T-cell receptor activation by ionomycin (Hundt et al. 2006). Also, glutamate-induced synaptic activity induces depalmitoylation of PSD-95 scaffolding protein (El-Husseini et al. 2002) and Cdc42 small GTPase (Kang et al. 2008). It is conceivable that unidentified PPTs, rather than DHHC-PATs, might play dominant roles in these processes. In contrast, blockade of neuronal activity rapidly increases PSD-95 palmitoylation. This dynamic palmitoylation is mediated by synaptic translocation of a PSD-95 PAT, DHHC2. This contrasts with the constitutive PSD-95 palmitoylation mediated by Golgi-resident DHHC3. Thus, the large family of DHHC palmitoylating enzymes are differentially localized and regulated in polarized cells (Noritake et al. 2009).

Inhibitors of DHHC Proteins

Several lipid-based compounds including 2-bromopalmitate (2BP), tunicamycin, and cerulenin have been used to inhibit protein palmitoylation (Ducker et al. 2006). Among them, the palmitate analog 2BP has been the most widely used. 2BP irreversibly inhibits DHHC-mediated palmitoylation directly acting on DHHC proteins in vitro (Fukata et al. 2004; Jennings et al. 2009), although 2BP also inhibits fatty acyl-CoA ligase and other enzymes involved in lipid metabolism in cells. In addition, 2-(2-hydroxy-5-nitro-benzylidene)-benzo[b]thiophen-3-one (Compound V) reversibly inhibits DHHC-mediated palmitoylation (Jennings et al. 2009). Because these inhibitors are not specific for individual DHHC-PATs, development of DHHC member-specific inhibitors is awaited.

Pathophysiological Significance of DHHC Proteins

DHHC protein members are linked to several human diseases (Table 1). DHHC8 is one of susceptibility genes for schizophrenia. Originally, microdeletions of human chromosome 22q11.2 locus, containing the DHHC8 gene, were reported to cause cognitive deficits and be associated with a high risk of developing schizophrenia. DHHC8 knockout mice showed behavioral phenotypes, a deficiency in prepulse inhibition and decreased exploratory activity in a new environment, and abnormalities in dendritic spines of neurons (Mukai et al. 2004, 2008). DHHC17/HIP14 is an associated protein with huntingtin protein, which is a causal gene for Huntington’s disease. DHHC17 palmitoylates huntingtin and regulates its trafficking and function (Yanai et al. 2006). Polyglutamine expansion of huntingtin gene reduces interaction of huntingtin protein with DHHC17, resulting in decreased huntingtin palmitoylation. Knockout mice of DHHC17/HIP14 display behavioral, biochemical, and neuropathological defects that are reminiscent of Huntington’s disease (Singaraja et al. 2011). Finally, mutations of DHHC9 and DHHC15 have been reported to be associated with X-linked mental retardation.
DHHC Proteins, Table 1

Human disorders associated with DHHC proteins

DHHC protein

Disorders associated with DHHC-PATs


Colorectal cancer




X-linked mental retardation

Colorectal cancer


Bladder cancer


X-linked mental retardation


Huntington’s disease


DHHC proteins are evolutionally conserved palmitoylating enzymes and add palmitates to proteins at specific cysteine residues by a reversible thioester linkage. DHHC proteins are integral membrane proteins with DHHC-CRD domain as a catalytic domain. Enzymatic activity and subcellular distribution of DHHC proteins can be dynamically regulated by extracellular stimulations. However, signal transduction pathways from receptors on the plasma membrane to DHHC proteins are poorly understood. Because mutations of DHHC proteins are closely associated with human diseases like schizophrenia and cancers, it would be beneficial to develop drugs acting on a specific DHHC protein for human health.


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© Springer International Publishing AG 2018

Authors and Affiliations

  1. 1.Division of Membrane Physiology, Department of Cell PhysiologyNational Institute for Physiological SciencesOkazakiJapan
  2. 2.Department of Physiological Sciences, School of Life ScienceThe Graduate University for Advanced Studies (SOKENDAI)OkazakiJapan