Identification of palmitoylated mitochondrial proteins using a bio‐orthogonal azido‐palmitate analogue

Abstract

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.

Protein myristoylation in health and disease

“…Together with the previous studies on the identification of palmitoylated mitochondrial proteins [144] and post-translationally myristoylated proteins associated with apoptosis [106] (both discussed above), this work demonstrates that this chemical proteomics approach can be used to profile fatty-acylated proteomes. The study also employed fluorescent probes to detect alkynyl-tagged proteins, using YnC16, and coppercatalysed click reaction as the bioorthogonal ligation.…”

“…Work by Kostiuk and co-workers looked at the detection of palmitoylated mitochondrial proteins using in vitro incorporation of an azido-palmitate analogue (AzC14) and Staudinger Ligation with a variety of labelled phosphines (including biotinylated and fluorescein-conjugated reagents) [144]. Proteins were separated first by charge and then on the basis of size by SDS-PAGE and identified by Tandem-MS. Incorporation via thioester bonds was demonstrated by hydroxylamine and alkali sensitivity.…”

“…Proteins were separated first by charge and then on the basis of size by SDS-PAGE and identified by Tandem-MS. Incorporation via thioester bonds was demonstrated by hydroxylamine and alkali sensitivity. In comparison with in vitro radiolabelling, the technique was found to be rapid, sensitive and specific [144]. A similar method was applied to the detection of proteins posttranslationally myristoylated in apoptotic Jurkat T cells [106].…”

Rapid and selective detection of fatty acylated proteins using ω-alkynyl-fatty acids and click chemistry

“…The alkynyl moiety has been used as a tag to generate bio-orthogonal analogs that could readily be reacted with azidetagged probes using the Cu(I)-catalyzed azide-alkyne [3+2] cycloaddition, a type of reaction also known as "click chemistry" (29)(30)(31)(32)(33). To complement the -azido-fatty acid series of chemical reporters ( 16,20,21 ), we sought to investigate the potential of -alkynyl-fatty acids and click chemistry as a means to detect S-acylation and N-myristoylation. In addition to increasing the reaction rate, the copper (I) catalyst also sensitizes alkynes toward dipolar reagents such as azides in a specifi c manner ( 33 ).…”

The Protein Lipidation and Its Analysis

“…This was proven by labeling of a myristoylated and palmitoylated Lck, a src family member protein kinase essential for T-cell activation. Later Berthiaume´s group also exploited it for the identification of palmitoylated mitochondrial proteins [122].…”