Xinxin Gao

Single-cell imaging of Wnt palmitoylation by the acyltransferase porcupine

Wnts are secreted palmitoylated glycoproteins that are important in embryonic development and human cancers. Here we report a method for imaging the palmitoylated form of Wnt proteins with subcellular resolution using clickable bioorthogonal fatty acids and in situ proximity ligation. Palmitoylated Wnt3a is visualized throughout the secretory pathway and trafficks to multivesicular bodies that act as export sites in secretory cells. We establish that glycosylation is not required for Wnt3a palmitoylation, which is necessary but not sufficient for Wnt3a secretion. Wnt3a is palmitoylated by fatty acids 13-16 carbons in length at Ser209 but not at Cys77, consistent with a slow turnover rate. We find that porcupine (PORCN) itself is palmitoylated, demonstrating what is to our knowledge the first example of palmitoylation of an MBOAT protein, and this modification partially regulates Wnt palmitoylation and signaling. Our data reveal the role of O-palmitoylation in Wnt signaling and suggest another layer of cellular control over PORCN function and Wnt secretion.

Engineering and Structural Characterization of a Linear Polyubiquitin-Specific Antibody

Polyubiquitination is an essential posttranslational modification that plays critical roles in cellular signaling. PolyUb (polyubiquitin) chains are formed by linking the carboxyl-terminus of one Ub (ubiquitin) subunit to either a lysine residue or the amino-terminus of an adjacent Ub. Linkage through the amino-terminus results in linear polyubiquitination that has recently been demonstrated to be a key step in nuclear factor κB activation; however, tools to study linear chains have been lacking. We therefore engineered a linear-linkage-specific antibody that is functional in Western blot, immunoprecipitation, and immunofluorescence applications. A crystal structure of the linear-linkage-specific antibody Fab fragment in complex with linear diubiquitin provides molecular insight into the nature of linear chain specificity. We use the antibody to demonstrate that linear polyUb is up-regulated upon tumor necrosis factor α stimulation of cells, consistent with a critical role in nuclear factor κB signaling. This antibody provides an essential tool for further investigation of the function of linear chains.

Method for cellular imaging of palmitoylated proteins with clickable probes and proximity ligation applied to Hedgehog, tubulin, and Ras.

Hedgehog protein undergoes post-translational palmitoylation, which is critical for its signaling activity during embryonic development and in adult tissues. Due to a lack of suitable imaging methods, the trafficking route of palmitoylated Hedgehog has remained unclear in secretory cells. Here, we report a novel method for imaging the subcellular distribution of palmitoylated forms of cellular proteins with high resolution. The method utilizes clickable chemical reporters to label the entire palmitoylated proteome, followed by proximity ligation on antibodies to the click-conjugated dye and the protein of interest to reveal the spatial localization of specific palmitoylated proteins, as exemplified by sonic Hedgehog, tubulin, and Ras. Palmitoylated sonic Hedgehog is found in the endoplasmic reticulum, the Golgi apparatus, and at the plasma membrane but not the endosomal system in Hedgehog-secreting cells. Palmitoylated tubulin is found along microtubule tracks and also partially associated with the plasma membrane, while palmitoylated H-Ras is visualized at various cellular locations including the plasma membrane, Golgi apparatus and endoplasmic reticulum. Our method is broadly applicable to imaging the palmitoylation of cellular proteins as well as other protein post-translational modifications that are detectable by clickable chemical reporters.

Membrane targeting of palmitoylated Wnt and Hedgehog revealed by chemical probes

Palmitoylation of the Wnt and Hedgehog proteins is critical for maintaining their physiological functions. To date, there are no reported studies that characterize the cellular distribution of the palmitoylated forms of these proteins. Here, we describe the subcellular localization of palmitoylated Wnt and Sonic Hedgehog by using a highly sensitive and non‐radioactive labeling method that utilizes alkynyl palmitic acid. We show that palmitoylated Wnt and Sonic Hedgehog localize to cellular membrane fractions only, highlighting a role for palmitoylation in the membrane association of these proteins. The method described herein has the utility to validate inhibitors of Wnt and Hedgehog acyltransferases in drug discovery, and enables further investigations of the role of palmitoylation in the secretion and signaling of these proteins.

Single-cell in situ imaging of palmitoylation in fatty-acylated proteins

Dissecting the subcellular distribution of a fatty-acylated protein is key to the understanding of the molecular mechanisms regulating protein movement and function in a cell. This protocol describes how to perform single-cell imaging of palmitoylation in a fatty-acylated protein of interest with high sensitivity using click chemistry, proximity ligation and fluorescence microscopy. The initial steps in this protocol involve optimization of conditions for (i) metabolic incorporation of an alkynyl analog of palmitic acid into cellular proteins coupled with click chemistry and (ii) detecting a specific protein of interest with primary antibodies using automated fluorescence microscopy, followed by (iii) imaging palmitoylation of the target fatty-acylated protein of interest, such as Wnt, Sonic Hedgehog or H-Ras. Furthermore, we outline strategies for imaging specific fatty-acylated proteins with subcellular organelles and/or total proteome palmitoylation, and we discuss special considerations that need to be given depending on the experimental design. The use of clickable fatty acids with proximity ligation may have promising applications to the investigation of fatty acylation cell biology. The entire protocol takes ∼3 weeks to complete.

Identification of the WNT1 residues required for palmitoylation by Porcupine

The post‐translational palmitoylation of WNT morphogens is critical for proper signaling during embryogenesis and adult homeostasis. The addition of palmitoyl groups to WNT proteins is catalyzed by Porcupine (PORCN). However, the Wnt amino acid residues required for recognition and palmitoylation by PORCN have not been fully characterized. We show that WNT1 residues 214–234 are sufficient for PORCN‐dependent palmitoylation of Ser224. Substitution of Ser224 with Thr, but not Cys, is tolerated in palmitoylation and biological assays. Our data highlight the importance of palmitoylation for WNT1 activity and establish PORCN as an O‐acyl transferase for WNT1.

A Decade of Click Chemistry in Protein Palmitoylation: Impact on Discovery and New Biology

Protein palmitoylation plays diverse roles in regulating the trafficking, stability, and activity of cellular proteins. The advent of click chemistry has propelled the field of protein palmitoylation forward by providing specific, sensitive, rapid, and easy-to-handle methods for studying protein palmitoylation. This year marks the 10th anniversary since the first click chemistry-based fatty acid probes for detecting protein lipid modifications were reported. The goal of this review is to highlight key biological advancements in the field of protein palmitoylation during the past 10 years. In particular, we discuss the impact of click chemistry on enabling protein palmitoylation proteomics methods, uncovering novel lipid modifications on proteins and elucidating their functions, as well as the development of non-radioactive biochemical and enzymatic assays. In addition, this review provides context for building and exploring new research avenues in protein palmitoylation through the use of clickable fatty acid probes.

Cellular uptake of a cystine-knot peptide and modulation of its intracellular trafficking

Cyclotides or cyclic cystine-knot peptides have emerged as a promising class of pharmacological ligands that modulate protein function. Interestingly, very few cyclotides have been shown to enter into cells. Yet, it remains unknown whether backbone cyclization is required for their cellular internalization. In this report, we studied the cellular behavior of EETI-II, a model acyclic cystine-knot peptide. Even though synthetic methods have been used to generate EETI-II, recombinant methods that allow efficient large scale biosynthesis of EETI-II have been lagging. Here, we describe a novel protocol for recombinant generation of folded EETI-II in high yields and to near homogeneity. We also uncover that EETI-II is efficiently uptaken via an active endocytic pathway to early endosomes in mammalian cells, eventually accumulating in late endosomes and lysosomes. Notably, co-incubation with a cell-penetrating peptide enhanced the cellular uptake and altered the trafficking of EETI-II, leading to its evasion of lysosomes. Our results demonstrate the feasibility of modulating the subcellular distribution and intracellular targeting of cystine-knot peptides, and hence enable future exploration of their utility in drug discovery and delivery.

Visualizing Wnt Palmitoylation in Single Cells

Wnt palmitoylation regulates its secretion and signaling activity in cells. Methods to monitor cellular Wnt palmitoylation are instrumental in investigating Wnt activity, secretion, and its interaction with cellular membrane compartments. This protocol describes a method we have recently developed to detect cellular Wnt palmitoylation. The method, combining click chemistry, bio-orthogonal fatty acid probes, and proximity ligation assay (PLA), provides high sensitivity and subcellular resolution for detection of Wnt palmitoylation. It is also compatible with multiple imaging platforms, and is applicable to detecting palmitoylated forms of other fatty acylated proteins.