NLRP3 Cys126 palmitoylation by ZDHHC7 promotes inflammasome activation.

Nucleotide oligomerization domain (NOD)-like receptor protein 3 (NLRP3) inflammasome hyperactivation contributes to many human chronic inflammatory diseases, and understanding how NLRP3 inflammasome is regulated can provide strategies to treat inflammatory diseases. Here, we demonstrate that NLRP3 Cys126 is palmitoylated by zinc finger DHHC-type palmitoyl transferase 7 (ZDHHC7), which is critical for NLRP3-mediated inflammasome activation. Perturbing NLRP3 Cys126 palmitoylation by ZDHHC7 knockout, pharmacological inhibition, or modification site mutation diminishes NLRP3 activation in macrophages. Furthermore, Cys126 palmitoylation is vital for inflammasome activation in vivo. Mechanistically, ZDHHC7-mediated NLRP3 Cys126 palmitoylation promotes resting NLRP3 localizing on the trans-Golgi network (TGN) and activated NLRP3 on the dispersed TGN, which is indispensable for recruitment and oligomerization of the adaptor ASC (apoptosis-associated speck-like protein containing a CARD). The activation of NLRP3 by ZDHHC7 is different from the termination effect mediated by ZDHHC12, highlighting versatile regulatory roles of S-palmitoylation. Our study identifies an important regulatory mechanism of NLRP3 activation that suggests targeting ZDHHC7 or the NLRP3 Cys126 residue as a potential therapeutic strategy to treat NLRP3-related human disorders.

GCP16 stabilizes the DHHC9 subfamily of protein acyltransferases through a conserved C-terminal cysteine motif.

Protein S-acylation is a reversible lipid post-translational modification that allows dynamic regulation of processes such as protein stability, membrane association, and localization. Palmitoyltransferase ZDHHC9 (DHHC9) is one of the 23 human DHHC acyltransferases that catalyze protein S-acylation. Dysregulation of DHHC9 is associated with X-linked intellectual disability and increased epilepsy risk. Interestingly, activation of DHHC9 requires an accessory protein-GCP16. However, the exact role of GCP16 and the prevalence of a requirement for accessory proteins among other DHHC proteins remain unclear. Here, we report that one role of GCP16 is to stabilize DHHC9 by preventing its aggregation through formation of a protein complex. Using a combination of size-exclusion chromatography and palmitoyl acyltransferase assays, we demonstrate that only properly folded DHHC9-GCP16 complex is enzymatically active in vitro. Additionally, the ZDHHC9 mutations linked to X-linked intellectual disability result in reduced protein stability and DHHC9-GCP16 complex formation. Notably, we discovered that the C-terminal cysteine motif (CCM) that is conserved among the DHHC9 subfamily (DHHC14, -18, -5, and -8) is required for DHHC9 and GCP16 complex formation and activity in vitro. Co-expression of GCP16 with DHHCs containing the CCM improves DHHC protein stability. Like DHHC9, DHHC14 and DHHC18 require GCP16 for their enzymatic activity. Furthermore, GOLGA7B, an accessory protein with 75% sequence identity to GCP16, improves protein stability of DHHC5 and DHHC8, but not the other members of the DHHC9 subfamily, suggesting selectivity in accessory protein interactions. Our study supports a broader role for GCP16 and GOLGA7B in the function of human DHHCs.

DHHC9 and GCP16 constitute a human protein fatty acyltransferase with specificity for H- and N-Ras.

Covalent lipid modifications mediate the membrane attachment and biological activity of Ras proteins. All Ras isoforms are farnesylated and carboxyl-methylated at the terminal cysteine; H-Ras and N-Ras are further modified by palmitoylation. Yeast Ras is palmitoylated by the DHHC cysteine-rich domain-containing protein Erf2 in a complex with Erf4. Here we report that H- and N-Ras are palmitoylated by a human protein palmitoyltransferase encoded by the ZDHHC9 and GCP16 genes. DHHC9 is an integral membrane protein that contains a DHHC cysteine-rich domain. GCP16 encodes a Golgi-localized membrane protein that has limited sequence similarity to yeast Erf4. DHHC9 and GCP16 co-distribute in the Golgi apparatus, a location consistent with the site of mammalian Ras palmitoylation in vivo. Like yeast Erf2.Erf4, DHHC9 and GCP16 form a protein complex, and DHHC9 requires GCP16 for protein fatty acyltransferase activity and protein stability. Purified DHHC9.GCP16 exhibits substrate specificity, palmitoylating H- and N-Ras but not myristoylated G (alphai1) or GAP-43, proteins with N-terminal palmitoylation motifs. Hence, DHHC9.GCP16 displays the properties of a functional human ortholog of the yeast Ras palmitoyltransferase.

Purification of recombinant G protein alpha subunits from Escherichia coli.

The purification of recombinant G protein a subunits expressed in Escherichia coli (E. coli) is a convenient and inexpensive method to obtain homogeneous preparations of protein for biochemical and biophysical analyses. Wild-type and mutant forms of G alpha are easily produced for analysis of their intrinsic biochemical properties, as well as for reconstitution with receptors, effectors, regulators, and G protein beta gamma subunits. Methods are described for the expression of Gi alpha and Gs alpha proteins in E. coli. Protocols are provided for the purification of untagged G protein a subunits using conventional chromatography and histidine (His)-tagged subunits using metal chelate chromatography. Modification of G alpha with myristate can be recapitulated in E. coli by expressing N-myristoyltransferase (NMT) with its G protein substrate. Protocols for the production and purification of myristoylated G alpha are presented.

Identification of a Ras palmitoyltransferase in Saccharomyces cerevisiae.

Most Ras proteins are posttranslationally modified by a palmitoyl lipid moiety through a thioester linkage. However, the mechanism by which this occurs is not known. Here, evidence is presented that the Ras2 protein of Saccharomyces cerevisiae is palmitoylated by a Ras protein acyltransferase (Ras PAT) encoded by the ERF2 and ERF4 genes. Erf2p is a 41-kDa protein localized to the membrane of the endoplasmic reticulum and contains a conserved DHHC cysteine-rich domain (DHHC-CRD). Erf2p co-purifies with Erf4p (26 kDa) when it is expressed in yeast or in Escherichia coli. The Erf2p/Erf4p complex is required for Ras PAT activity, and mutations within conserved residues (Cys(189), His(201), and Cys(203)) of the Erf2p DHHC-CRD domain abolish Ras PAT activity. Furthermore, a palmitoyl-Erf2p intermediate is detected suggesting that Erf2p is directly involved in palmitate transfer. ERF2 and ERF4 are the first genes identified that encode a palmitoyltransferase for a Ras GTPase.