Increased levels of circulating saturated free fatty acids, such as palmitate, have been implicated in the etiology of type II diabetes and cancer. In addition to being a constituent of glycerolipids and a source of energy, palmitate also covalently attaches to numerous cellular proteins via a process named palmitoylation. Recognized for its roles in membrane tethering, cellular signaling, and protein trafficking, palmitoylation is also emerging as a potential regulator of metabolism. Indeed, we showed previously that the acylation of two mitochondrial proteins at their active site cysteine residues result in their inhibition. Herein, we sought to identify other palmitoylated proteins in mitochondria using a nonradioactive bio-orthogonal azido-palmitate analog that can be selectively derivatized with various tagged triarylphosphines. Our results show that, like palmitate, incorporation of azido-palmitate occurred on mitochondrial proteins via thioester bonds at sites that could be competed out by palmitoyl-CoA. Using this method, we identified 21 putative palmitoylated proteins in the rat liver mitochondrial matrix, a compartment not recognized for its content in palmitoylated proteins, and confirmed the palmitoylation of newly identified mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase. We postulate that covalent modification and perhaps inhibition of various mitochondrial enzymes by palmitoyl-CoA could lead to the metabolic impairments found in obesity-related diseases.

For lipid synthesis, energy production via ␤ -oxidation, or for protein fatty acylation to occur, long-chain fatty acids (LCFAs) must be activated by conversion to their CoA derivatives (LCFA-CoAs) by fatty acyl-CoA synthetase (FAS ). Protein fatty acylation is one of many types of posttranslational modifi cations of proteins by lipids, which also includes isoprenoids, glycosylphosphatidylinositols, and cholesterol. Typically, lipids covalently attached to proteins serve as hydrophobic membrane anchors ( 1-6 ).Protein fatty acylation is mainly divided into two categories: N-myristoylation and S-acylation. The corresponding reactions are catalyzed by N-myristoyl transferases (NMT1 and NMT2) and two families of protein acyltransferases (PATs) referred to as zinc fi nger, Asp-His-His-Cys PATs Abstract Progress in understanding the biology of protein fatty acylation has been impeded by the lack of rapid direct detection and identifi cation methods. We fi rst report that a synthetic -alkynyl-palmitate analog can be readily and specifi cally incorporated into GAPDH or mitochondrial 3-hydroxyl-3-methylglutaryl-CoA synthase in vitro and reacted with an azido-biotin probe or the fl uorogenic probe 3-azido-7-hydroxycoumarin using click chemistry for rapid detection by Western blotting or fl at bed fl uorescence scanning. The acylated cysteine residues were confi rmed by MS. Second, -alkynyl-palmitate is preferentially incorporated into transiently expressed H-or N-Ras proteins (but not nonpalmitoylated K-Ras), compared with -alkynyl-myristate or -alkynyl-stearate, via an alkali sensitive thioester bond. Third, -alkynyl-myristate is specifi cally incorporated into endogenous co-and posttranslationally myristoylated proteins. The competitive inhibitors 2-bromopalmitate and 2-hydroxymyristate prevented incorporation of -alkynylpalmitate and -alkynyl-myristate into palmitoylated and myristoylated proteins, respectively. Labeling cells with -alkynyl-palmitate does not affect membrane association of N-Ras. Furthermore, the palmitoylation of endogenous proteins including H-and N-Ras could be easily detected using -alkynyl-palmitate as label in cultured HeLa, Jurkat, and COS-7 cells, and, promisingly, in mice. The -alkynylmyristate and -palmitate analogs used with click chemistry

Excessive liver production of ketone bodies is one of many metabolic complications that can arise from diabetes, and in severe untreated cases, it can result in ketoacidosis, coma, and death. Mitochondrial HMG-CoA synthase (HMGCS2), the rate-limiting enzyme in ketogenesis, has been shown to interact with PPARalpha and act as a coactivator to up-regulate transcription from the PPRE of its own gene. Although protein palmitoylation is typically a cytosolic process that promotes membrane association, we recently identified 21 palmitoylated proteins in rat liver mitochondria, including HMGCS2. Herein, our data support a mechanism whereby palmitate is first added onto HMGCS2 active site Cys166 and then transacylated to Cys305. Palmitoylation promotes the HMGCS2/PPARalpha interaction, resulting in transcriptional activation from the Hmgcs2 PPRE. These results, together with the fact that 8 of the 21 palmitoylated mitochondrial proteins that we previously identified have nuclear receptor interacting motifs, demonstrate a novel--and perhaps ubiquitous--role for palmitoylation as a modulator of transcription.

BACKGROUND:The incidence of oropharyngeal squamous cell carcinoma caused by oncogenic HPV (HPV-OPSCC) is rising worldwide. HPV-OPSCC is commonly diagnosed by RT-qPCR of HPV-16 E6 and E7 oncoproteins or by cyclin-dependent kinase inhibitor 2A, multiple tumor suppressor 1 (p16) immunohistochemistry (IHC). Droplet digital PCR (ddPCR) has been recently reported as ultrasensitive and highly precise method of nucleic acid quantification for biomarker analysis. We aimed to validate this method for the detection of HPV-16 E6 and E7 in HPV-OPSCC. METHODS: Participants were recruited from January 2015-November 2015 at initial presentation to the University of Alberta Head and Neck Oncology Clinic. RNA was extracted, purified and quantified from prospectively collected participant tissues, and ddPCR was performed with fluorescent probes detecting HPV-16 E6 and E7. Results from ddPCR were compared with p16 IHC performed by clinical pathology as standard of care. RESULTS: Head and neck tissues were prospectively obtained from 68 participants including 29 patients with OPSCC, 29 patients with non-OPSCC and 10 patients without carcinoma. 79.2% of patients with OPSCC were p16 positive. The sensitivity and specificity of ddPCR HPV E6/E7 compared with p16 IHC in OPSCC was 91.3 and 100%, respectively. The amount of target RNA used was 1 ng, 20-50 times lower than reported by other for RT-qPCR HPV E6/E7. CONCLUSIONS: The ddPCR of HPV E6/E7 is a novel and highly specific method of detecting HPV-16 in OPSCC.

BackgroundThe incidence of oropharyngeal squamous cell carcinoma (OPSCC) caused by oncogenic human papillomavirus (HPV) is rising worldwide. HPV-OPSCC is commonly diagnosed by RT-qPCR of HPV E6 and E7 oncoproteins or by p16 immunohistochemistry (IHC). Droplet digital PCR (ddPCR) has been recently reported as an ultra-sensitive and highly precise method of nucleic acid quantification for biomarker analysis. To validate the use of a minimally invasive assay for detection of oncogenic HPV based on oropharyngeal swabs using ddPCR. Secondary objectives were to compare the accuracy of ddPCR swabs to fresh tissue p16 IHC and RT-qPCR, and to compare the cost of ddPCR with p16 IHC.MethodsWe prospectively included patients with p16+ oral cavity/oropharyngeal cancer (OC/OPSCC), and two control groups: p16− OC/OPSCC patients, and healthy controls undergoing tonsillectomy. All underwent an oropharyngeal swab with ddPCR for quantitative detection of E6 and E7 mRNA. Surgical specimens had p16 IHC performed. Agreement between ddPCR and p16 IHC was determined for patients with p16 positive and negative OC/OPSCC as well as for healthy control patients. The sensitivity and specificity of ddPCR of oropharyngeal swabs were calculated against p16 IHC for OPSCC.Results122 patients were included: 36 patients with p16+OPSCC, 16 patients with p16−OPSCC, 4 patients with p16+OCSCC, 41 patients with p16−OCSCC, and 25 healthy controls. The sensitivity and specificity of ddPCR of oropharyngeal swabs against p16 IHC were 92 and 98% respectively, using 20–50 times less RNA than that required for conventional RT-qPCR. Overall agreement between ddPCR of tissue swabs and p16 of tumor tissue was high at ĸ = 0.826 [0.662-0.989].ConclusionOropharyngeal swabs analyzed by ddPCR is a quantitative, rapid, and effective method for minimally invasive oncogenic HPV detection. This assay represents the most sensitive and accurate mode of HPV detection in OPSCC without a tissue biopsy in the available literature.Electronic supplementary materialThe online version of this article (doi:10.1186/s40463-016-0177-8) contains supplementary material, which is available to authorized users.