With isolation of the Drosophila hedgehog gene. Isolation of hedgehog genes in higher organisms, including mammals. Our biochemical studies revealed the derivation of the mature, active Hedgehog signal from its protein precursor by a novel autocatalytic processing reaction. This reaction proceeds via a thioester intermediate that replaces a main-chain peptide bond. Cholesterol attack of this thioester then results in cleavage of the precursor and covalent linkage of cholesterol to the carboxyl terminus of the amino-terminal fragment. This amino-terminal fragment is further modified by addition of palmitate at its amino terminus and is then responsible for all signaling activities. The carboxyl-terminal domain initiates processing of the precursor and is required for production of the active signal.
Crystallographic structural studies, in collaboration with Daniel Leahy (Johns Hopkins University), revealed distinct and surprising evolutionary origins for the signaling and processing domains of the Hedgehog protein. The amino-terminal signaling domain displays a striking resemblance to an enzyme involved in shaping the bacterial cell wall. Catalytic activity, however, is no longer required for Hedgehog signaling; instead, this conserved domain appears to function as a structural scaffold for residues that are important in Hedgehog function as a protein ligand. In addition, a portion of the Hedgehog carboxyl-terminal processing domain displays unmistakable structural similarity to the inteins of self-splicing proteins. The inteins constitute a widely disseminated group of genetic parasites whose excision from host proteins also proceeds by an initial step involving a thioester intermediate; the Hedgehog processing domain in addition has acquired a carboxyl-terminal appendage that mediates participation of cholesterol in the second step of the processing reaction. These structural findings provide a remarkable view of the Hedgehog protein as an embryonic patterning signal that evolved by assembly and adaptation of ancient domains for novel functions.
Normal and Pathological Functions of Hedgehog Signaling
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Identification of the active Hedgehog signal facilitated studies of its distribution and activities in embryonic tissues, and in turn led to the finding that Hedgehog signaling elicits concentration-dependent responses in a range of developing organs. Expression of the vertebrate Hedgehog family member Sonic hedgehog (Shh), for example, was found to be localized in structures with the ability to organize development of surrounding tissues. Furthermore, embryonic tissue explants respond to the Hedgehog signal in vitro in a concentration-dependent manner. In embryos, localized production of the Hedgehog signal elicits distinct responses from surrounding cells as a function of the length of exposure to or distance from the source. Such graded responses are critical for formation of the normal spatial pattern of structures as diverse as the digits of the limb or neuronal types in the spinal cord.
In mice lacking function of Shh or other pathway components, the organs affected include the brain, spinal cord, axial skeleton and musculature, the limbs, and endodermally derived organs such as the gastrointestinal tract and the lungs. A striking aspect of the Shh mutant phenotype is the occurrence of cyclopia and ventral forebrain-patterning defects, and these malformations suggested a potential connection between embryonic disruption of Hedgehog signaling and human cyclopia. Although rare, human cyclopia is the extreme manifestation of holoprosencephaly, a term that includes a spectrum of birth defects. Genetic or environmental perturbations of Hedgehog signaling are now well recognized as causally linked to human holoprosencephaly.
Cyclopia in Shh mutant mice also led to the discovery that the plant-derived teratogen, cyclopamine, specifically antagonizes Hedgehog signaling through binding and inhibition of the pathway component Smoothened. With the use of cyclopamine as a potent and specific Hedgehog pathway antagonist, our studies have shifted from patterning activities in embryos to postembryonic activities in maintenance of tissue pattern and neoplastic growth. We have thus noted a critical role for continuous Hedgehog pathway activity in driving the growth of a broad group of deadly human cancers that include esophageal, pancreatic, biliary tract, gastric, prostate, and small-cell lung cancers. These cancers arise in the epithelia of endodermally derived organs and in aggregate account for a significant proportion of human cancer deaths. Our studies show that treatment with the Hedgehog pathway antagonist cyclopamine can block cell proliferative effects associated with pathway activation and can cause complete regression of aggressive human and rodent cancers growing in mice. The use of cyclopamine or other pathway antagonists thus may represent a novel, nontoxic approach to therapies for lethal human cancers.
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Why is Hedgehog pathway activity a factor in the growth of so many types of cancer? Recent studies point to a requirement for Hedgehog pathway activity in the renewal and maintenance of postembryonic tissues, probably through effects on division and activity of tissue stem cells. These findings are relevant to cancer because of the possible derivation of cancer stem cells-the minority of cells within a cancer that are capable of its propagation-from adult tissue stem cells. We have proposed that the association between Hedgehog pathway activation and cancer growth is related to the normal function of this pathway in regulating stem cell activity. Given the increased cancer incidence associated with chronic tissue injury, we have further proposed that Hh pathway activation and consequent expansion of tissue stem cells may be induced by tissue injury and that inappropriate continuation of pathway activity may initiate cancer growth by transforming these cells into cancer stem cells. Much of our current work is aimed at investigating the normal mechanism and biological role of Hedgehog pathway activation in response to injury, and the pathological mechanism by which pathway activity inappropriately persists and leads to oncogenic transformation.
The Hedgehog (Hh) proteins are evolutionarily conserved signaling molecules that control the normal growth and patterning of diverse animals including Drosophila and humans. In flies Hh is required for multiple developmental processes such as embryonic segment patterning, eye and appendage development (for reviews see [1,2]) In vertebrates, three Hh homologues are expressed in a tissue specific manner and are responsible for the morphogenesis of various organs such as the neural tube and the limbs, and for cartilage and male germinal cell differentiation [3,4]. In mammals, deregulation of the Hh pathway is responsible for cancers, especially basal cell carcinoma and medulloblastoma [5-7]. In all cases described so far, Hh initiates and/or maintains the transcription of target genes in responsive cells. Among the targets are patched (ptc), which encodes a Hh receptor protein, and genes encoding signaling molecules. In Drosophila, decapentaplegic (dpp), a signal of the TGFβ class, and wingless (wg), a member of the Wnt family, are transcribed in response to Hh. Genes encoding related signaling molecules such as TGFβ, FGF, and Wnt are transcriptionally induced by Hh signals in vertebrates, as are genes for a variety of transcription factors.
In Drosophila, Cubitus interruptus (Ci), a zinc finger transcription factor of the vertebrate Gli family, plays a central and complex role in the transcriptional regulation of Hh target genes. Ci acts as either transcriptional activator or repressor in a Hh-dependent manner (for reviews see [1,8,9]). In the absence of Hh signal, most of Ci is cleaved to generate a 75 kD nuclear protein (Ci75-R) consisting of the N-terminus and the zinc finger DNA binding domain of the protein. Ci75-R acts as a repressor on hh and dpp transcription [10-12]. Ci cleavage is proteasome dependent and requires Ci phosphorylation by the protein kinase A (PKA), the activity of the kinesin-related protein Costal2 (Cos2) and of Slimb (Slb), a F-box WD-40 protein (other members of this family are known to direct ubiquitin-mediated proteolysis of specific phospho-proteins) [13-21]. In the absence of Hh, Ci75-R is mainly localized into the nucleus while the uncleaved fraction of Ci (155kD, Ci-155) is retained in the cytoplasm by Cos2 and Suppressor of Fused (Su(fu)), a putative PEST motif-containing protein [14,19,22-25]. The exclusion of full-length Ci155 from the nucleus is also ensured by its constitutive export [14,24]. Hh signaling inhibits Ci proteolysis, probably by reducing Ci phosphorylation level via the action of a phosphatase [10,14,18]. This results in the accumulation of full-length Ci-155. Hedgehog reception also relieves Ci-155 cytoplasmic retention and allows Ci-155 to be translocated into the nucleus via a basic nuclear localization sequence [14,18,19,25]. Nevertheless, the persistence of its export leads to the accumulation of the vast majority of Ci-155 in the cytoplasm. Last, proper induction of Hh target genes also requires Hedgehog signaling to produce, by an unknown mechanism, an activated form of Ci called Ci-A [11,23-26]. In the absence of Hh, Su (fu) appears to prevent Ci activation. Upon Hh reception, both the ser-thr protein kinase Fused (Fu) and Cos2 counteract Su(fu) to produce Ci-A [19,23-27].
The molecular mechanism by which Hh signaling controls Ci remains poorly understood. Both full-length and truncated forms of Ci are detected in the cytoplasm as part of one or more large molecular weight protein complexes [10,28-30]. The cytoplasmic complex that has been studied also includes Fu and Cos2 and Su(fu) [28-31]. In the absence of Hh signal, Fu-Cos2-Ci ternary complex binds microtubules, probably through Cos2 [28-30]. Cultured cell experiments showed that Hh signal triggers the release of the Fu-Cos2-Ci complex from the microtubules [28,29]. Concomitantly Hh signal increases the level of phosphorylation of both Fu and Cos2 [28,32].
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Our working hypothesis is that Hh signal could control Ci fate (i.e. cleavage, subcellular localization, and/or activation) by inducing changes in the activity, composition, and/or subcellular localization of the transducing cytoplasmic complex. Since Cos2 is a putative motor protein with microtubule-binding activity, it could play a central role in this process by regulating the association of the complex with the microtubules and perhaps by directing its movement to specific locations within the cell.
In order to better understand how Hh transducing complex may function, we focused on the precise relationships among its different members. The physical association of Cos2 with Fu and Ci has been previously demonstrated using gel filtration chromatography and co-immunoprecipitation from embryo and cultured cell extracts [28-30]. Here we have undertaken the identification and the mapping of the molecular interactions taking place between Cos2, Fu, Su(fu), and Ci using the yeast two-hybrid method and an in vitro biochemical assay. Our results show that (i) Cos2, as Su(fu), interacts with both the catalytic and regulatory domains of Fu and with the N-terminal part of Ci; (ii) Ci and Fu associate with Cos2 in the neck domain, located C-terminally to the motor domain. The precise identification of the interaction region of each protein in vitro provide new insights into the structure and possible mechanism of action of the complex during Hh signal transduction.