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Neuronal palmitoyl acyl transferases exhibit distinct substrate specificity.

ABSTRACT: Palmitoylation, a post-translational modification of cysteine residues with the lipid palmitate, has recently emerged as an important mechanism for regulating protein trafficking and function. With the identification of 23 DHHC mammalian palmitoyl acyl transferases (PATs), a key question was the nature of substrate-enzyme specificity for these PATs. Using the acyl-biotin exchange palmitoylation assay, we compared the substrate specificity of four neuronal PATs, namely DHHC-3, DHHC-8, HIP14L (DHHC-13), and HIP14 (DHHC-17). Exogenous expression of enzymes and substrates in COS cells reveals that HIP14L and HIP14 modulate huntingtin palmitoylation, DHHC-8 modulates paralemmin-1 palmitoylation, and DHHC-3 shows the least substrate specificity. These in vitro data were validated by lentiviral siRNA-mediated knockdown of endogenous HIP14 and DHHC-3 in cultured rat cortical neurons. PATs require the presence of palmitoylated cysteines in order to interact with their substrates. To understand the elements that influence enzyme/substrate specificity further, we fused the HIP14 ankryin repeat domain to the N terminus of DHHC-3, which is not a PAT for huntingtin. This modification enabled DHHC-3 to behave similarly to HIP14 by modulating palmitoylation and trafficking of huntingtin. Taken together, this study indicates that individual PATs have specific substrate preference, determined by regulatory domains outside the DHHC domain of the enzymes.

SUBMITTER: Huang K

PROVIDER: S-EPMC2717768 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Neuronal palmitoyl acyl transferases exhibit distinct substrate specificity.

Huang Kun K Sanders Shaun S Singaraja Roshni R Orban Paul P Cijsouw Tony T Arstikaitis Pamela P Yanai Anat A Hayden Michael R MR El-Husseini Alaa A

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20090319 8

Palmitoylation, a post-translational modification of cysteine residues with the lipid palmitate, has recently emerged as an important mechanism for regulating protein trafficking and function. With the identification of 23 DHHC mammalian palmitoyl acyl transferases (PATs), a key question was the nature of substrate-enzyme specificity for these PATs. Using the acyl-biotin exchange palmitoylation assay, we compared the substrate specificity of four neuronal PATs, namely DHHC-3, DHHC-8, HIP14L (DHH ...[more]

PMID: 19299482

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Project description:BackgroundPancreatic adenocarcinoma (PAAD) is one of the most leading causes of cancer-related death across the world with the limited efficiency and response rate of immunotherapy. Protein S-palmitoylation, a powerful post-translational lipid modification, is well-known to regulate the stability and cellular distribution of cancer-related proteins, which is mediated by a family of 23 palmitoyl transferases, namely zinc finger Asp-His-His-Cys-type (ZDHHC). However, whether palmitoyl transferases can determine tumor progression and the efficacy of immunotherapy in PAAD remains unknown.MethodsBioinformatics methods were used to identify differential ZDHHCs expression in PAAD. A systematic pan-cancer analysis was conducted to assess the immunological role of ZDHHC3 using RNA sequencing data from The Cancer Genome Atlas database. In vivo Panc 02 subcutaneous tumor model validated the anti-tumor effect of knockdown of ZDHHC3 or intraperitoneal injection of 2-bromopalmitate (2-BP), a typical broad-spectrum palmitoyl transferases inhibitor. Furthermore, we explored therapeutic strategies with combinations of 2-BP with PD-1/PD-L1-targeted immunotherapy in C57BL/6 mice bearing syngeneic Panc 02 pancreatic tumors.ResultsZDHHC enzymes were associated with distinct prognostic values of pancreatic cancer. We identified that ZDHHC3 expression promotes an immunosuppressive tumor microenvironment in PAAD. 2-BP suppressed pancreatic-tumor cell viability and tumor sphere-forming activities, as well as increased cell apoptosis in vitro, without affecting normal human pancreatic ductal epithelial cells. Furthermore, genetic inactivation of ZDHHC3 or intraperitoneal injection of 2-BP impeded tumor progression in Panc 02 pancreatic tumors with enhanced anti-tumor immunity. 2-BP treatment significantly enhanced the therapeutic efficacy of PD-1/PD-L1 inhibitors in Panc 02 pancreatic tumors.ConclusionThis study revealed some ZDHHC enzyme genes for predicting the prognosis of pancreatic cancer, and demonstrated that ZDHHC3 plays a critical oncogenic role in pancreatic cancer progression, highlighting its potential as an immunotherapeutic target of pancreatic cancer. In addition, combination therapy of 2-BP and PD-1/PD-L1 achieved synergic therapy effects in a mouse model of pancreatic cancer.

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Neuronal palmitoyl acyl transferases exhibit distinct substrate specificity.

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Neuronal palmitoyl acyl transferases exhibit distinct substrate specificity.

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