Targeted cell ablation in zebrafish using optogenetic transcriptional control.

Cell ablation is a powerful method for elucidating the contributions of individual cell populations to embryonic development and tissue regeneration. Targeted cell loss in whole organisms has been typically achieved through expression of a cytotoxic or prodrug-activating gene product in the cell type of interest. This approach depends on the availability of tissue-specific promoters, and it does not allow further spatial selectivity within the promoter-defined region(s). To address this limitation, we have used the light-inducible GAVPO transactivator in combination with two genetically encoded cell-ablation technologies: the nitroreductase/nitrofuran system and a cytotoxic variant of the M2 ion channel. Our studies establish ablative methods that provide the tissue specificity afforded by cis-regulatory elements and the conditionality of optogenetics. Our studies also demonstrate differences between the nitroreductase and M2 systems that influence their efficacies for specific applications. Using this integrative approach, we have ablated cells in zebrafish embryos with both spatial and temporal control.

Ultrasensitive optical imaging with lanthanide lumiphores.

In principle, the millisecond emission lifetimes of lanthanide chelates should enable their ultrasensitive detection in biological systems by time-resolved optical microscopy. In practice, however, lanthanide imaging techniques have provided no better sensitivity than conventional fluorescence microscopy. Here, we identified three fundamental problems that have impeded lanthanide microscopy: low photon flux, inefficient excitation, and optics-derived background luminescence. We overcame these limitations with a new lanthanide imaging modality, transreflected illumination with luminescence resonance energy transfer (trLRET), which increases the time-integrated signal intensities of lanthanide lumiphores by 170-fold and the signal-to-background ratios by 75-fold. We demonstrate that trLRET provides at least an order-of-magnitude increase in detection sensitivity over that of conventional epifluorescence microscopy when used to visualize endogenous protein expression in zebrafish embryos. We also show that trLRET can be used to optically detect molecular interactions in vivo. trLRET promises to unlock the full potential of lanthanide lumiphores for ultrasensitive, autofluorescence-free biological imaging.

Topological analysis of Hedgehog acyltransferase, a multipalmitoylated transmembrane protein.

Hedgehog proteins are secreted morphogens that play critical roles in development and disease. During maturation of the proteins through the secretory pathway, they are modified by the addition of N-terminal palmitic acid and C-terminal cholesterol moieties, both of which are critical for their correct function and localization. Hedgehog acyltransferase (HHAT) is the enzyme in the endoplasmic reticulum that palmitoylates Hedgehog proteins, is a member of a small subfamily of membrane-bound O-acyltransferase proteins that acylate secreted proteins, and is an important drug target in cancer. However, little is known about HHAT structure and mode of function. We show that HHAT is comprised of ten transmembrane domains and two reentrant loops with the critical His and Asp residues on opposite sides of the endoplasmic reticulum membrane. We further show that HHAT is palmitoylated on multiple cytosolic cysteines that maintain protein structure within the membrane. Finally, we provide evidence that mutation of the conserved His residue in the hypothesized catalytic domain results in a complete loss of HHAT palmitoylation, providing novel insights into how the protein may function in vivo.

Cholesterylation: a tail of hedgehog.

Cholesterylation is a post-translational attachment of sterol to proteins. This modification has been a characteristic of a single family of hedgehog proteins (Hh). Hh is a well-established morphogenic molecule important in embryonic development. It was also found to be involved in the progression of many cancer types. Herein, we describe the mechanism of biosynthesis of cholesterylated Hh, the role of this unusual modification on protein functions and novel chemical probes, which could be used to specifically target this modification, both in vitro and in vivo.

Multifunctional reagents for quantitative proteome-wide analysis of protein modification in human cells and dynamic profiling of protein lipidation during vertebrate development.

Novel multifunctional reagents were applied in combination with a lipid probe for affinity enrichment of myristoylated proteins and direct detection of lipid-modified tryptic peptides by mass spectrometry. This method enables high-confidence identification of the myristoylated proteome on an unprecedented scale in cell culture, and allowed the first quantitative analysis of dynamic changes in protein lipidation during vertebrate embryonic development.

Proteome-wide analysis of protein lipidation using chemical probes: in-gel fluorescence visualization, identification and quantification of N-myristoylation, N- and S-acylation, O-cholesterylation, S-farnesylation and S-geranylgeranylation.

Protein lipidation is one of the most widespread post-translational modifications (PTMs) found in nature, regulating protein function, structure and subcellular localization. Lipid transferases and their substrate proteins are also attracting increasing interest as drug targets because of their dysregulation in many disease states. However, the inherent hydrophobicity and potential dynamic nature of lipid modifications makes them notoriously challenging to detect by many analytical methods. Chemical proteomics provides a powerful approach to identify and quantify these diverse protein modifications by combining bespoke chemical tools for lipidated protein enrichment with quantitative mass spectrometry-based proteomics. Here, we report a robust and proteome-wide approach for the exploration of five major classes of protein lipidation in living cells, through the use of specific chemical probes for each lipid PTM. In-cell labeling of lipidated proteins is achieved by the metabolic incorporation of a lipid probe that mimics the specific natural lipid, concomitantly wielding an alkyne as a bio-orthogonal labeling tag. After incorporation, the chemically tagged proteins can be coupled to multifunctional 'capture reagents' by using click chemistry, allowing in-gel fluorescence visualization or enrichment via affinity handles for quantitative chemical proteomics based on label-free quantification (LFQ) or tandem mass-tag (TMT) approaches. In this protocol, we describe the application of lipid probes for N-myristoylation, N- and S-acylation, O-cholesterylation, S-farnesylation and S-geranylgeranylation in multiple cell lines to illustrate both the workflow and data obtained in these experiments. We provide detailed workflows for method optimization, sample preparation for chemical proteomics and data processing. A properly trained researcher (e.g., technician, graduate student or postdoc) can complete all steps from optimizing metabolic labeling to data processing within 3 weeks. This protocol enables sensitive and quantitative analysis of lipidated proteins at a proteome-wide scale at native expression levels, which is critical to understanding the role of lipid PTMs in health and disease.

trLRET microscopy: Ultrasensitive imaging of lanthanide luminophores.

In principle, the long emission lifetimes of lanthanide chelates should enable their ultrasensitive detection in biological systems by time-resolved optical microscopy. However, most lanthanide-imaging systems cannot achieve sensitivities that exceed those of conventional fluorescence microscopes, since they are limited by inefficient lanthanide excitation, the low photon flux of excited lanthanide luminophores, and optics-derived background photoluminescence. We recently reported a new lanthanide-imaging modality, trans-reflected illumination with luminescence resonance energy transfer (trLRET), which overcomes each of these constraints. Here we provide a detailed procedure for visualizing endogenous protein expression in zebrafish embryos, using lanthanide-labeled antibodies, Q-switched laser illumination, and trLRET microscopy. These methods allow ultrasensitive molecular imaging in cells and organisms, establishing a new paradigm for biological exploration and discovery.

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.

Myristoylation profiling in human cells and zebrafish.

Human cells (HEK 293, HeLa, MCF-7) and zebrafish embryos were metabolically tagged with an alkynyl myristic acid probe, lysed with an SDS buffer and tagged proteomes ligated to multifunctional capture reagents via copper-catalyzed alkyne azide cycloaddition (CuAAC). This allowed for affinity enrichment and high-confidence identification, by delivering direct MS/MS evidence for the modification site, of 87 and 61 co-translationally myristoylated proteins in human cells and zebrafish, respectively. The data have been deposited to ProteomeXchange Consortium (Vizcaíno et al., 2014 Nat. Biotechnol., 32, 223-6) (PXD001863 and PXD001876) and are described in detail in Multifunctional reagents for quantitative proteome-wide analysis of protein modification in human cells and dynamic protein lipidation during vertebrate development׳ by Broncel et al., Angew. Chem. Int. Ed.

Attenuation of hedgehog acyltransferase-catalyzed sonic Hedgehog palmitoylation causes reduced signaling, proliferation and invasiveness of human carcinoma cells.

Overexpression of Hedgehog family proteins contributes to the aetiology of many cancers. To be highly active, Hedgehog proteins must be palmitoylated at their N-terminus by the MBOAT family multispanning membrane enzyme Hedgehog acyltransferase (Hhat). In a pancreatic ductal adenocarcinoma (PDAC) cell line PANC-1 and transfected HEK293a cells Hhat localized to the endoplasmic reticulum. siRNA knockdown showed that Hhat is required for Sonic hedgehog (Shh) palmitoylation, for its assembly into high molecular weight extracellular complexes and for functional activity. Hhat knockdown inhibited Hh autocrine and juxtacrine signaling, and inhibited PDAC cell growth and invasiveness in vitro. In addition, Hhat knockdown in a HEK293a cell line constitutively expressing Shh and A549 human non-small cell lung cancer cells inhibited their ability to signal in a juxtacrine/paracrine fashion to the reporter cell lines C3H10T1/2 and Shh-Light2. Our data identify Hhat as a key player in Hh-dependent signaling and tumour cell transformed behaviour.

New chemical probes targeting cholesterylation of Sonic Hedgehog in human cells and zebrafish.

Sonic Hedgehog protein (Shh) is a morphogen molecule important in embryonic development and in the progression of many cancer types in which it is aberrantly overexpressed. Fully mature Shh requires attachment of cholesterol and palmitic acid to its C- and N-termini, respectively. The study of lipidated Shh has been challenging due to the limited array of tools available, and the roles of these posttranslational modifications are poorly understood. Herein, we describe the development and validation of optimised alkynyl sterol probes that efficiently tag Shh cholesterylation and enable its visualisation and analysis through bioorthogonal ligation to reporters. An optimised probe was shown to be an excellent cholesterol biomimetic in the context of Shh, enabling appropriate release of tagged Shh from signalling cells, formation of multimeric transport complexes and signalling. We have used this probe to determine the size of transport complexes of lipidated Shh in culture medium and expression levels of endogenous lipidated Shh in pancreatic ductal adenocarcinoma cell lines through quantitative chemical proteomics, as well as direct visualisation of the probe by fluorescence microscopy and detection of cholesterylated Hedgehog protein in developing zebrafish embryos. These sterol probes provide a set of novel and well-validated tools that can be used to investigate the role of lipidation on activity of Shh, and potentially other members of the Hedgehog protein family.