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Cholesterylation: a tail of hedgehog Paulina Ciepla*†, Anthony I. Magee†‡1 and Edward W. Tate*†1 *Department of Chemistry, Imperial College London, Exhibition Road, London SW7 2AZ, U.K. †Institute of Chemical Biology, Imperial College London, Exhibition Road, London SW7 2AZ, U.K. ‡National Heart and Lung Institute, Imperial College London, Exhibition Road, London SW7 2AZ, U.K.

Abstract 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.

Introduction

Mechanism of Hh cholesterylation

Cholesterylation is a post-translational modification (PTM) characterized by the covalent attachment of the sterol to proteins [1] and to date appears to be a unique feature of the hedgehog (Hh) protein family, although the incorporation of cholesterylation probes to other as yet unidentified proteins has previously been described [1,2]. Hh proteins belong to a group of morphogenic molecules involved in the patterning of many organs during embryonic development, including limbs, skin, neuronal progenitor cells, inner ear, eyes, taste buds and hair follicles. In postnatal and adult organisms, its role is extended to the maintenance of stem cells and tissue repair and regeneration [3,4]. Hh pathway activation is also associated with generation and progression of many cancers [5]. The expression of Hh is conserved between invertebrate and vertebrate organisms, with higher species usually expressing a larger number of Hh homologues. Humans express three Hh proteins from different genes: Sonic (Shh), Indian (Ihh) and Desert (Dhh) [6]. The attachment of cholesterol and palmitate (the second, post-translationally attached lipid) are widely conserved between these homologues but they differ in their sites of expression. Recently-reported probes for cholesterylation permit analysis and imaging of lipidated Hh proteins both in vitro and in vivo and open up new approaches to understanding the role of this unusual class of PTM [2,7].

The post-translational attachment of cholesterol to the signalling Hh N-domain (HhN, 19 kDa) of the Hh precursor (∼45 kDa) is a product of the autocatalytic cleavage reaction and removal of the Hh C-domain (HhC; homologous to self-splicing proteins), through an intein-like mechanism (Figure 1A). In this process, cholesterol acts as a reaction cofactor, highlighting a secondary role of HhC as a cholesterol transferase [1]. Mechanistically, the cleavage is a two-step process initiated by nucleophilic attack at the carbonyl group of the C-terminal glycine of HhN (Gly257 ) by the N-terminal cysteine thiol of HhC (Cys258 , numbering based on the Drosophila Hh), resulting in the formation of a thioester intermediate. The activation of the thiol occurs through the action of disulfide isomerases protein disulfide isomerase (PDI) and protein disulfide isomerase, pancreatic (PDIp), which reduce the disulfide-bond formed between two HhC cysteines: Cys258 and Cys400 [8]. The second step of the autocatalytic cleavage is attack on the thioester by the cholesterol 3β-hydroxy group and formation of processed and cholesterol-modified HhNp (p = processed) and free HhC. Kinetic studies of Drosophila Hh processing assigned an apparent Michaelis–Menten (K m app ) value of 14 μM for cholesterol [9]. Studies using cholesterol analogues performed by Beachy and co-workers [10] identified important cholesterol features required for successful cleavage and sterol attachment. The 3β-position of the hydroxy group is essential and its replacement with other functionalities, such as a ketone, ester or thiol, resulted in the abolition of auto-processing. This study also identified non-essential functionalities, such as an aliphatic side chain, whose absence or branching does not result in observable changes in processing. A decrease in efficiency is however observed by the addition of hydroxy groups to the side chain or ring structures (positions 4 and 19). Another non-essential but important functionality is the C5(6) double bond, whose absence

Key words: cholesterylation, hedgehog protein, bioorthogonal lipid probes, post-translational modification, lipidation, alkynyl cholesterol probe. Abbreviations: AzChol, azide cholesterol; Disp, Dispatched; Hh, hedgehog; HhC, Hh C-domain; HhN, Hh N-domain; HPE, holoprosencephaly; hpf, hours post fertilization; Ihh, Indian hedgehog; Ptc, Patched; PTM, post-translational modification; Shh, Sonic hedgehog; SSD, sterol-sensing domain; YnChol, alkynyl cholesterol. 1

Correspondence may be addressed to either [email protected] or [email protected]).

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Biochem. Soc. Trans. (2015) 43, 262–267; doi:10.1042/BST20150032

Protein acylation: from mechanism to drug discovery

Figure 1 Hh cholesterylation (A) Mechanism of Hh cholesterylation. (B) Hh signalling pathway with red boxes highlighting processes where cholesterylation or the presence of cholesterylation are thought to be involved. Hhat (Hh acyltransferase) catalyses the attachment of palmitate. (C) Bio-orthogonal cholesterol probes: AzChol [2] and YnChol [7]. Versatility of analytic cholesterylation assays performed using chemical-tagging approach with YnChol probe.

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reduces (although does not completely abolish) autoprocessing. Interestingly, nucleophiles such as dithiothreitol, glutathione, hydroxylamine, 2-mercaptoethanol and free cysteine can also participate in the cleavage reaction in vitro [1,8–11]. The product of such reactions is a non-cholesterylated protein: HhN. There is currently no robust evidence for such reactions occurring in vivo. The mechanistic details of Hh cholesterylation can also explain the specific structure of HhC. The conserved amino acids found in different homologues of Hh are mainly involved in the formation of the active site and thus are responsible for the generation of the thioester or the binding and transfer of cholesterol. Amino acids important for thioester formation (apart from Cys258 ) are His329 , Thr326 and Cys400 . They are responsible for formation of hydrogen bonds with the α-amino group of Cys258 or activation of the free thiol [8,12]. The amino acids required for cholesterol attachment are Asp303 (replaced by histidine in some Hh homologues) crucial for activating the cholesterol hydroxyl and 63 C-terminal amino acids which form a hydrophobic pocket that is hypothesized to mediate cholesterol binding. An in vitro assay of Drosophila Hh auto-processing performed by Jiang and Paulus [9] also identified two putative auto-processing inhibitors: zafirlukast, a reversible inhibitor with an IC50 value of 10 μM and honokiol, an apparently irreversible inhibitor. These compounds inhibited cholesterol-dependent processing but were inactive towards processing induced by small thiols, suggesting that they target cholesterol binding rather than the catalytic site of HhC. These compounds are not yet proven to be active in vivo; given the high hydrophobicity of honokiol and the known receptor antagonist pharmacology of zafirlukast, these compounds are likely to have important off-target effects in cells.

Role of Hh cholesterylation The absence of HhN cholesterol modification was found to have a pronounced effect on embryonic development. Mice unable to incorporate cholesterol, although producing the processed ShhN (obtained by excision of the ShhC domain, ShhN, mutants), showed significant developmental defects characteristic of holoprosencephaly (HPE)-like phenotypes, a disease characterized by genetic mutations in Hh [13]. The absence of cholesterol also resulted in decreased ShhN concentration near the zone of polarizing activity affecting digit development in ShhN- mice limbs, whereas increasing the anterior spreading of ShhN, resulting in an increase in digit counts in ShhN + limbs [14]. The effect of cholesterol deficiency is however less severe than the absence of Shh expression, suggesting that cholesterol is responsible for the formation of an appropriate Shh signalling gradient rather than signal activity itself. Patched (Ptc) is a transmembrane protein, responsible for constitutive inhibition of Hh signalling [15]. Binding of Hh to Ptc represses this inhibitory effect and sequesters Hh spreading by endocytotic internalization and the activation  C The

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of HhNp degradation [15,16]. Although Ptc contains the sterol-sensing domain (SSD; similar to proteins involved in cholesterol biogenesis, homoeostasis or lipid storage) [17], ShhNp and ShhN have comparable binding affinities to Ptc [18,19]. This observation suggests that the change in the morphogenic gradient in developing embryos is caused by changes in extracellular transport rather than changes in the binding affinity of Shh to its receptor. The absence of cholesterol in invertebrates also results in an increase in HhN spread, usually accompanied by increased activation of low-level Hh response genes in cells distant form the Hh source and reduced activation of high-level targets [11,20–24]. Sterol-deficient mutants were thus unable to form an appropriate extracellular gradient, further demonstrated by enlarged notum containing extra-macrochaetae and altered wing pattern in adult phenotypes, similar to ectopic activation of the Hh pathway or hypomorphic Ptc [21]. Figure 1(B) summarizes the role of Hh cholesterylation on Hh signalling based on in vitro studies. Firstly, cholesterol is crucial for HhNp membrane tethering, specifically to membrane microdomains in Hh signalling cells [25]. The sterol influence on this process varies between cell lines; however, it was quantified as ∼60 % in vertebrate C17 cells [26]. Similarly, in invertebrate CL8 cells, HhNp was found to form punctate structures at plasma membranes, whereas a cholesterol-deficient mutant showed no membrane accumulation [22]. The introduction of the cholesterylation site to soluble proteins (e.g. Halo–tagged) was sufficient to target them to membranes and reduce their cellular secretion [27]. The influence of palmitate on membrane binding is also significant; only dually-lipidated molecules achieve an almost quantitative membrane association and chaperone protein activity appears to be required to handle these lipid anchors during Hh secretion Dispatched (Disp) is a multipass transmembrane protein involved in HhNp release from signalling cells [15]. In vitro assays of vertebrate cells showed that HhNp can bind to Disp and that cholesterylation is important for the Disp–HhNp interaction [27]. Similar results were observed in vivo in Drosophila Disp − mutants, where only cholesterol-deficient HhN was secreted, whereas the wild-type protein was fully cell-associated [19]. Like Ptc, Disp contains a SSD, which in this case is responsible for the specific interaction between Disp and HhNp. The release of HhNp is also dependent on the Scube2 secreted glycoprotein either expressed in the signalling cells or supplied to the cell culture medium. In a similar fashion to Disp, Scube2 increases the release of HhNp, but not the non-cholesterylated mutant [27,28]. Further, Disp and Scube2 were also proposed to recognize different functional groups of cholesterol: the aliphatic chain (Disp) and ring structures (Scube2), suggesting a hand-off of HhNp between these proteins during cellular release [27]. Interestingly, the introduction of the cholesterylation site to a soluble protein (e.g. maltose-binding protein) allows its successful secretion from signalling cells in a Scube2dependent manner [27], suggesting that the presence of cholesterol and not protein–protein interactions between

Protein acylation: from mechanism to drug discovery

HhNp–Disp–Scube2 is the driving force of HhNp secretion. Interestingly, Scube2 has only been identified in vertebrates to date and further experiments will be required to confirm these mechanistic hypotheses. Further, the secreted HhNp forms various transport species, which permits its travel through extracellular space. In one model, multimeric complexes of HhNp are formed due to presence of terminal lipids of HhNp, which hydrophobic interactions create a template for the formation of such species [29]. Thus, the presence of cholesterol is essential for the formation of such complexes; however the synergistic role of both HhNp lipids is distinguishable since the amount of monomeric HhN in cell culture medium significantly increases after the deletion of HhC (resulting in noncholesterylated but palmitoylated HhN) or mutation of the palmitoylation site (resulting in cholesterylated nonpalmitoylated HhNp) [26]. Similarly, in invertebrates, the multimeric complexes are only formed by dually-lipidated proteins and only these can associate with the receiving cell membrane [21,22]. Interestingly, multimers are also thought to be more successful at Hh signalling activation than monomers [30]. A recent theory by Grobe and co-workers [31,32] proposes a role for proteases (e.g. sheddases) in the cleavage of both lipidation sites, which consequently solubilizes the protein and allows its transport; in this model, protease recruitment and activation can be induced by Scube 2 or mechanically, by treatment with cyclodextrin. This theory is however not in agreement with other studies presenting multimeric, sterylated HhNp in cell culture medium [7,29] and its predictions require further supporting evidence and studies. Studies of Drosophila larvae imaginal discs identified extracellular HhNp associated with lipoprotein particles [33], whereas proteomic analysis of human plasma found Ihh (an Hh family member) associated with very low density lipoproteins (VLDLs), which promote the survival of primary endothelial cells [34]. Drosophila mutants expressing reduced levels of apolipoprotein L1 (ApoL1) were characterized by the accumulation of HhNp close to its source, with a limited number of particles found further from signalling cells [33]. This resulted in a decreased range of Hh gradient and a phenotypically-reduced area of imaginal disks [33]. Both invertebrate and vertebrate sterol-deficient proteins lose their ability to bind to lipoproteins [35]. Thus, lipoproteins may also play a significant role as transporters of sterol-modified molecules. There are a number of conflicting reports concerning the role of cholesterol on the activation of Hh-receiving cells. For example, in vitro assays studying the expression of Shh target genes in rat E1 telencephalic explants showed a reduced differentiation obtained by the addition of ShhNp compared with ShhN, suggesting a role of cholesterylation in reduction in the activity of palmitoylated Shh [26]. Overexpression studies found a significant increase in Shh-responsive gene expression in the forebrain of zebrafish embryos microinjected with either ShhNp or ShhN expression constructs [26], suggesting that cholesterylation

has no additive or restrictive effect on the activation of these genes. Studies of invertebrate cells further showed that cholesterol attachment and its involvement on the ability to form multimeric HhNp complexes caused a 3-fold increase in the induction of the Hh pathway [22]. Taken together, these observations imply that the effect of Hh cholesterylation on pathway activation is complex and may be species and tissuedependent and further studies are required to resolve these discrepancies. A problematic oversight of mutation studies that remove cholesterylation altogether is their inability to determine the direct effect of cholesterol on Hh processing and the formation of HhN. The depletion of cholesterol with cyclodextrin or point mutations in the sterol-binding domain of the ShhC in HPE resulted in the inhibition of Shh processing and accumulation of the precursor in signalling cells [10,27,36,37]. This phenotype was not rescued by endogenous, cellular nucleophiles (contradictory to in vitro assays). Further, sterol depletion allowed determination of Hh precursor and HhNp cellular stability, suggesting half-lives of 23 and 37 min respectively [36]. Thus, processing of Hh precursor is responsible for deciding the fate of Hh, targeting it either for secretion (after cleavage and lipid attachment) or for degradation (unprocessed protein). Contradicting reports however suggest that the unprocessed Hh molecule can still be secreted and induce Hh signalling [36,38], but the presence of such species is not yet confirmed in vivo. Understanding of Hh cholesterylation is significantly hindered by the limited number of tools available for its study. With the exception of genetic mutations in, or deletion of, the HhC domain, which prevents attachment of cholesterol, only two techniques for the visualization of the sterolmodified HhN have been reported to date: radiolabelling with isotopic [3 H]-cholesterol [1,8,29,31] or labelling with sterol analogues containing bio-orthogonal alkynyl (YnChol) [7] or azide (AzChol) [2] functionalities. These can be imaged by radiography or by reactions with complementary bioorthogonal functionalities containing fluorophores, biotin or other tags [39], giving the chemical-tagging approach remarkable versatility (Figure 1C). This technique allows the presence of lipidated proteins to be monitored in different tissue and cell samples and more recently in specific cellular compartments [40]. Recently, our laboratory has established the first technology for labelling sterylated Hh homologues in vivo in developing zebrafish embryos, which endogenously express elevated levels of Hh proteins [7]. Sterol probes containing terminal alkynes were introduced into the solution of zebrafish water containing dechorionated embryos at 4 hours post fertilization (hpf). Within 48 hpf, probes were absorbed by the embryos, transported into Hh producing cells and translocated into specific Hh processing sites. After whole embryo lysis, visualization of the probe-modified HhN was achieved by ligation to an azide-containing fluorophore. Interestingly, the discrimination of zebrafish HhC protein between the tested probes was the same as that observed in vitro in mammalian cells [human embryonic kidney  C The

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(HEK)293a cells stably expressing human Shh]. The same probe achieved the highest level of incorporation into HhN both in vivo and in vitro, whereas structures suffering from low incorporation levels in cell-based experiments were undetectable in zebrafish embryo assays. These observations suggested that the HhC is a versatile domain in zebrafish, mammalian and fruit fly systems and can accommodate various sterol molecules in the processing reaction, highlighting the similarities in their cholesterol-binding sites. We also believe that this approach can be extended beyond analysis in embryonic lysates and with appropriate and optimized labelling, fixation and permeabilization protocols this technique can be used further to study the spatial resolution of sterylated HhN inside developing embryos or adult organisms, especially in the context of in vivo disease models driven by Hh overexpression.

Conclusion The role of Hh cholesterylation is complex and appears to be highly context-dependent. Significant differences have been highlighted by experiments performed in different organisms, tissue samples or even in vitro cell models. However, these cholesterylation studies raise an interesting hypothesis whereby the unique nature of Hh processing and cholesterylation, rather than protein structure itself, is responsible for targeting HhN to specific cellular and extracellular transport routes. The presence of cholesterol is thus a decisive factor in precursor cleavage, association of the protein with the plasma membrane, binding to specific membrane receptors, triggering secretion and transport (Disp, Scube2) and association into defined transport molecules, which further form a tightly-regulated protein gradient and enable activation of specific target genes. These findings highlight the importance of Hh cholesterylation and further suggest that Hh processing may be considered an interesting drug target for diseases that require modulation of the Hh signalling pathway such as certain genetic disorders or cancers. However, it also prompted discovery of new tools and techniques for visualization of sterylation, which may allow the localization of sterylated HhNp to be studied in real time to obtain better understanding of the full signalling pathway of sterol-modified proteins and thus the biological role of this unusual PTM.

Funding This work was supported by the Imperial College London Institute of Chemical Biology EPSRC Centre for Doctoral Training [grant number EP/F500416/1] to P.C.

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Received 28 January 2015 doi:10.1042/BST20150032

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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 ...
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