The Aceylation of Hedgehog

The Drosophila Hedgehog protein and its vertebrate counterpart Sonic hedgehog are required for a wide variety of patterning events throughout development. Hedgehog proteins are secreted from cells and undergo autocatalytic cleavage and cholesterol modification to produce a mature signaling domain. This domain of Sonic hedgehog has recently been shown to acquire an N-terminal acyl group in cell culture. The in vivo role that such acylation might play in appendage patterning in mouse and Drosophila has been investigated; in both species Hedgehog proteins define a posterior domain of the limb or wing. A mutant form of Sonic hedgehog that cannot undergo acylation retains significant ability to repattern the mouse limb. However, the corresponding mutation in Drosophila Hedgehog renders it inactive in vivo, although it is normally processed. Furthermore, overexpression of the mutant form has dominant negative effects on Hedgehog signaling. These data suggest that the importance of the N-terminal cysteine of mature Hedgehog in patterning appendages differs between species (Lee, 2001a).

A mutant form of human Shh, which cannot be acylated because cysteine-24 is changed to serine (C24S-Shh-N), is at least 800-fold less active in inducing alkaline phosphatase activity in C3H10T1/2 cells than wild-type acylated Shh-N and can antagonize the effect of Shh-N on these cells. In addition, C24S-Shh has much less ability to ventralize the embryonic mouse forebrain than does acylated Shh. To further evaluate the impact of acylation on other aspects of Shh function during mouse development, retroviral vectors were used to misexpress full-length wild-type and C24S-Shh in developing limb buds. Shh and C24S-Shh sequences were inserted immediately upstream of an internal ribosome entry site (IRES) followed by a PLAP gene to allow easy detection of infected cells. Retroviral-mediated misexpression of wild-type Shh in the early mouse limb bud results in polydactyly and/or proximal deviation of the anterior digits, resembling the 'thumbing' hand signal of a hitchhiker. A strong correlation of such phenotypes with retroviral infection in the distal and anterior regions of the limb bud was observed. These results were consistent with previous studies demonstrating digit duplication and altered anterior-posterior patterning when ectopic Shh is delivered to the anterior limb mesenchyme, and were consistent with loss of anterior-posterior expansion of the handplate in the absence of Shh. In contrast, Shh overexpression by cells in the posterior limb where Shh is expressed endogenously or ectopic expression by cells in more proximal limb domains has no apparent impact on early limb patterning. Thus, as expected, the ability of Shh to influence limb development is dependent on its location along the anterior-posterior axis. The phenotypes, however, do not appear to require mesenchymal expression of Shh. Indeed, since retroviral injection after 9.0 dpc infects almost exclusively the surface ectoderm, the ectopic anterior outgrowths are most frequently induced by Shh-expressing cells located in close proximity to or within the apical ectodermal ridge (AER), an ectoderm-derived structure. Mutant C24S-Shh produces similar results in the limb bud. Ectopic anterior expression of C24S-Shh in the ectoderm induces polydactyly and deviation of the anterior digits. Also, similar to wild-type Shh, ectopic proximal or posterior C24S-Shh has no overt effects on limb patterning. Morphological analysis of 327 infected limbs indicates that both acylated and unacylated forms of Shh induce a similar spectrum of phenotypes, although C24S-Shh appears less active than wild type (Lee, 2001a).

In order to test whether acylation of the corresponding cysteine of Drosophila Hh could affect the activity of the protein, a mutant hh cDNA was constructed encoding serine instead of cysteine at position 84 (referred to hereafter as C84S-Hh). Cys-84 is the N-terminal residue of the mature Hh protein, corresponding to Cys-24 in Shh, and is followed by a conserved sequence of amino acids. The GAL4-UAS system was used to express wild-type and C84S-Hh in transgenic flies. To test whether C84S-Hh could replace wild-type Hh, C84S-Hh was expressed in the normal Hh pattern, under the control of engrailed-GAL4, in flies transheterozyogous for null and temperature-sensitive alleles of hh. At the restrictive temperature, embryos with temperature sensitive hh display a severe segment polarity phenotype in which the naked cuticle is lost. When a wild-type Hh transgene was expressed using en-GAL4 in hhts embryos, most of the cuticles examined were wild type or showed a mild segment polarity phenotype. Thus this en-GAL4 line can direct the production of sufficient protein to rescue Hh signaling. In contrast, when C84S-Hh is similarly expressed, the mutant embryos fail to hatch and show a segment polarity phenotype that is indistinguishable from the hhts phenotype in the absence of any rescuing construct (Lee, 2001a).

The inability of C84S-Hh to rescue the hh mutant phenotype could be caused by an effect of the C84S mutation on protein stability or processing. To test this, a Western blot analysis was perfomed of protein extracts from embryos overexpressing the C84S-Hh or wild-type Hh constructs under the control of the ubiquitous daughterless-GAL4 driver. Using a polyclonal antiserum against Hh protein, it was found that the abundance of the 19-kDa N-terminal fragment of Hh is greatly increased in protein extracts from embryos expressing either wild-type or C84S Hh. Thus the C84S mutation abolishes the function of Hh without affecting its processing (Lee, 2001a).

To test whether any residual function of C84S-Hh could be detected in a gain of function assay, a series of GAL4 lines was used to express either C84S-Hh or wild-type Hh in a variety of embryonic and larval tissues. While misexpression of wild-type Hh causes clear patterning defects, often leading to lethality, C84S-Hh displayed no Hh gain of function activity in any of the tissues tested. C84S-Hh misexpression caused a visible phenotype only in the developing wing and the ocelli (Lee, 2001a).

In the wing imaginal disc, Hh is produced by cells of the posterior compartment and signals to cells at the anterior-posterior (AP) border. Hh directly patterns the wing in the vicinity of the AP border, corresponding to the L3-L4 intervein region in the adult; Hh also indirectly patterns the remainder of the wing through the induction of dpp expression at the AP border. Overexpression of wild-type Hh in the posterior compartment of the wing disc using en-GAL4 results in an expansion of the anterior compartment. However, when C84S-Hh was overexpressed in the posterior compartment a loss of tissue was observed in the region surrounding the compartment boundary, between veins L3 and L4, and the partial fusion of these veins. This phenotype resembles loss of function mutations in the genes fused and cubitus interruptus, which are required for Hh signal transduction in the wing. Hh and C84S-Hh are also misexpressed with optomotor blind-GAL4, which is expressed in a broad domain of the wing disc centered on the AP compartment border. Expression of wild-type Hh using omb-GAL4 causes an expansion of the L3-L4 region; in contrast, C84S-Hh expression using omb-GAL4 results in an even stronger loss of L3-L4 tissue and partial or complete fusion of veins 3 and 4. These results suggest that, rather than mimicking wild-type Hh, C84S-Hh may interfere with the endogenous Hh signal. Likewise, when C84S-Hh is misexpressed in the ocellar region of the eye-antennal imaginal disc using eyeless-GAL4, formation of the ocelli is blocked; ocellar development requires Hh signaling (Lee, 2001a).

C84S-Hh may directly compete with wild-type Hh for binding to the Ptc receptor; alternatively, it could simply interfere with the production of wild-type Hh. To address this question, C84S-Hh was misexpressed in anterior wing disc cells that do not normally produce Hh protein, using ptc-GAL4. ptc is up-regulated in anterior cells responding to Hh; however, ptc is not expressed in the posterior cells that produce Hh. C84S-Hh expression under the control of ptc-GAL4 also reduces the amount of L3-L4 tissue produced. This result suggests that C84S-Hh interferes with Hh signaling in the extracellular environment and not at the level of intracellular processing (Lee, 2001a).

If C84S-Hh acts by competing with wild-type Hh, the severity of the phenotype should depend on the relative amounts of C84S-Hh and wild-type Hh present. This was tested by altering the ratio of wild type to mutant protein being expressed. When C84S-Hh is expressed in flies heterozygous for hh, the reduction of the L3-L4 region is enhanced, thus decreasing the dosage of wild-type Hh relative to C84S-Hh exacerbates the dominant negative phenotype (Lee, 2001a).

What role is played by the acylation? Fatty acid chains are frequently added to intracellular proteins in order to localize them to the plasma membrane. Acylation of extracellular proteins has very rarely been reported, but it would be expected to reduce their solubility and restrict their diffusion. Hh has already been shown to carry a C-terminal cholesterol modification that restricts its action to nearby cells; misexpression of the mature N-terminal signaling domain without cholesterol increases its range of activity, resulting in patterning defects. A single lipid modification is usually insufficient for membrane localization; thus acylation and cholesterol modification could both be required to tether Hh molecules. However, there is no evidence that C24S-Shh and C84S-Hh are less efficiently localized than wild-type Shh or Hh, since antibody staining shows no difference in the distribution of the two forms when misexpressed. Alternatively, the importance of acylation in addition to cholesterol modification could be to promote Hh association with raft membrane domains; this may be important for its cellular trafficking or signaling. Another possibility is that acylation might be required for Hh release from cells, perhaps localizing it to intracellular transport vesicles containing the Dispatched protein. However, the finding that C84S-Hh can inhibit the function of wild-type Hh even when expressed only in the responding cells using ptc-GAL4 suggests that it is able to exit the cell, rather than just interfering intracellularly with the release of wild-type Hh. Similarly, the ability of C24S-Shh expressed in the limb ectoderm to activate Hoxd13 expression in the underlying mesenchyme argues either that it can move freely between germ layers or that it activates a second signal with this ability. Thus it is likely that acylation is important for Hh signaling through Ptc, either because it causes more productive binding to Ptc itself or because it affects Hh interaction with other proteins (Lee, 2001a).

A critical step in maturation of the Hh protein is autoproteolytic cleavage at an internal site, during which the bioactive NH2-terminal product of cleavage acquires a COOH-terminally attached cholesterol. This modification can be bypassed by the expression of truncated or chimeric Hh proteins, such as HhN, that are produced in bioactive form without cleavage or cholesterol addition. Neither of these proteins is active in the absence of ski function, indicating that Ski must control a different property of the Hh signal, one that is shared by HhNp (N-palmitoylated Hh), HhN, and HhCD2, a Hh/Cd2 fusion protein (Chamoun, 2001).

It has recently been demonstrated that in addition to COOH-terminal addition of cholesterol, the human Sonic hedgehog (Shh) protein is further modified by NH2-terminal attachment of a palmitoyl adduct in a manner dependent upon the NH2-terminal cysteine residue (Pepinski, 1998). The finding that ski encodes a putative acyl transferase raises the possibility that it could function as an enzyme for the palmitoylation of Hh protein. To determine whether Drosophila Hh is NH2-terminally acylated, the protein was purified from Drosophila tissue culture cells and subjected to mass spectrometric analysis. The experimentally determined mass of HhNp (20,235 daltons) exceeds by 238 daltons the average mass expected for the amino-terminal signaling domain linked to cholesterol alone (19,996.8 daltons). The difference between these values corresponds closely to the expected mass of a palmitoyl adduct in ester or amide linkage (238.4 daltons). Furthermore, substitution of a serine in place of the NH2-terminal cysteine results in a protein modified by cholesterol alone. These results support the conclusion that Drosophila HhNp undergoes NH2-terminal palmitoylation (Chamoun, 2001).

If Ski functions as the Hh palmitoyl-transferase, then the absence of Ski should result in Hh protein that is not NH2-terminally modified, with a consequent loss or reduction of Hh signaling activity. Genetic evidence for this possibility derives from the functional equivalence between mutations that abolish the enzymatic activity of Ski and alterations in Hh proteins that prevent their NH2-terminal acylation. If the wild-type ski alleles are replaced by a ski transgene in which two presumptive active-site residues of Ski are mutated, Hh signaling activity is greatly reduced. The same phenotypes are observed when posterior wing cells express the NH2-terminally mutant form of Hh (HhC85S) instead of wild-type Hh protein. Interestingly, although mouse Shh protein also depends on ski function for maximal activity, it retains some signaling activity if secreted from ski mutant cells. The same partial reduction in Shh activity is observed with a mutant form of Shh in which the NH2-terminal cysteine residue; mutated Shh is not further reduced when secreted from ski mutant cells. These results suggest that Ski is specifically required for the NH2-terminal addition of palmitate to Hh (Chamoun, 2001).

Biochemical evidence for Ski's role in Hh palmitoylation was obtained by examination of Hh protein produced from cells with reduced ski function. Hh proteins were analyzed by reversed-phase high pressure liquid chromatography (RP-HPLC), which resolves doubly-modified HhNp from proteins lacking the NH2-terminal palmitate adduct. A small proportion of HhNp in normal cells and tissues displays the earlier elution profile characteristic of the HhNpC85Sprotein, suggesting that autoprocessing and cholesterol modification precede palmitoylation during Hh biogenesis. Drosophila cultured cells expressing HhNp were treated with double-stranded RNA corresponding to two regions of the ski coding sequence. A significantly increased proportion of the protein from these cells eluted at a position indicative of absence of the NH2-terminal palmitate. A similar shift in elution was observed for HhNp from tissues of mutant third instar larvae, except that all of the HhNp eluted earlier, as would be expected for a complete loss of palmitate transfer. Reduction or loss of Ski function thus reduces the hydrophobicity of HhNp to that of HhNpC85S, consistent with a loss of NH2-terminal palmitoylation and with a role for Ski function in palmitate transfer (Chamoun, 2001).

It has been proposed that the linkage of palmitate to the NH2-terminus of ShhNp is via an amide bond (Pepinsky, 1998), but the mechanism of amide formation is not clear. The membership of Ski in a family of enzymes catalyzing O-linked acyltransfers strongly argues for a mechanism in which a thioester intermediate is formed with the side chain of the NH2-terminal cysteine, followed by a rearrangement through a five-membered cyclic intermediate to form the amide. It is curious that addition of cholesterol, the other lipid modification of the mature Hh signal, also involves a cyclic intermediate and thioester chemistry but proceeds in reverse, from amide to ester (Chamoun, 2001).

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