Expression and purification of fused kinase from insect cells for in vitro kinase assay.

The Fused (Fu) kinase is a key transducer of Hedgehog signaling, but its relevant substrates have remained obscured due to the difficulty of obtaining active Fu for in vitro kinase assay. Based on the mechanism of Fu activation in vivo, we engineered a constitutively active Fu and expressed it in Sf9 cells using the baculovirus system. The kinase was affinity purified and applied for in vitro kinase assay using recombinant GST-fusion proteins as substrates to identify Fu-specific phosphorylation sites. For complete details on the use and execution of this protocol, please refer to Han et al. (2019).

Inhibition of tetrameric Patched1 by Sonic Hedgehog through an asymmetric paradigm.

The Hedgehog (Hh) pathway controls embryonic development and postnatal tissue maintenance and regeneration. Inhibition of Hh receptor Patched (Ptch) by the Hh ligands relieves suppression of signaling cascades. Here, we report the cryo-EM structure of tetrameric Ptch1 in complex with the palmitoylated N-terminal signaling domain of human Sonic hedgehog (ShhNp) at a 4:2 stoichiometric ratio. The structure shows that four Ptch1 protomers are organized as a loose dimer of dimers. Each dimer binds to one ShhNp through two distinct inhibitory interfaces, one mainly through the N-terminal peptide and the palmitoyl moiety of ShhNp and the other through the Ca2+-mediated interface on ShhNp. Map comparison reveals that the cholesteryl moiety of native ShhN occupies a recently identified extracellular steroid binding pocket in Ptch1. Our structure elucidates the tetrameric assembly of Ptch1 and suggests an asymmetric mode of action of the Hh ligands for inhibiting the potential cholesterol transport activity of Ptch1.

Management of high-risk and advanced basal cell carcinoma

Despite that basal cell carcinoma (BCC) is curative in the vast majority of cases, some patients are at high risk of recurrence and, in a few patients, lesions can progress to a point unsuitable for local therapy and prognosis is quite poor. The aim of the present work is to review clinical and pathologic characteristics as well as classical and new treatment options for high-risk, metastatic and locally advanced BCC. Surgery and radiotherapy remain the selected treatments for the majority of high-risk lesions. However, some patients are located on a blurry clinical boundary between high-risk and locally advanced BCC. Treatment of these patients is challenging and need an individualized and highly specialized approach. The treatment of locally advanced BCC, in which surgery or radiotherapy is unfeasible, inappropriate or contraindicated, and metastatic BCC has changed with new Hedgehog pathway inhibitors of which vismodegib is the first drug approved by FDA and EMA (AU)

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Identification of a family of fatty-acid-speciated sonic hedgehog proteins, whose members display differential biological properties.

Hedgehog (HH) proteins are proteolytically processed into a biologically active form that is covalently modified by cholesterol and palmitate. However, most studies of HH biogenesis have characterized protein from cells in which HH is overexpressed. We purified Sonic Hedgehog (SHH) from cells expressing physiologically relevant levels and showed that it was more potent than SHH isolated from overexpressing cells. Furthermore, the SHH in our preparations was modified with a diverse spectrum of fatty acids on its amino termini, and this spectrum of fatty acids varied dramatically depending on the growth conditions of the cells. The fatty acid composition of SHH affected its trafficking to lipid rafts as well as its potency. Our results suggest that HH proteins exist as a family of diverse lipid-speciated proteins that might be altered in different physiological and pathological contexts in order to regulate distinct properties of HH proteins.

Automation of large scale transient protein expression in mammalian cells.

Traditional mammalian expression systems rely on the time-consuming generation of stable cell lines; this is difficult to accommodate within a modern structural biology pipeline. Transient transfections are a fast, cost-effective solution, but require skilled cell culture scientists, making man-power a limiting factor in a setting where numerous samples are processed in parallel. Here we report a strategy employing a customised CompacT SelecT cell culture robot allowing the large-scale expression of multiple protein constructs in a transient format. Successful protocols have been designed for automated transient transfection of human embryonic kidney (HEK) 293T and 293S GnTI⁻ cells in various flask formats. Protein yields obtained by this method were similar to those produced manually, with the added benefit of reproducibility, regardless of user. Automation of cell maintenance and transient transfection allows the expression of high quality recombinant protein in a completely sterile environment with limited support from a cell culture scientist. The reduction in human input has the added benefit of enabling continuous cell maintenance and protein production, features of particular importance to structural biology laboratories, which typically use large quantities of pure recombinant proteins, and often require rapid characterisation of a series of modified constructs. This automated method for large scale transient transfection is now offered as a Europe-wide service via the P-cube initiative.

Purification and bioassay of hedgehog ligands for the study of cell death and survival.

The Hedgehog (Hh) family of secreted ligands-composed of Sonic Hedgehog (Shh), Indian Hedgehog (Ihh), and Desert Hedgehog (Dhh)-possesses many roles during embryonic development, adult homeostasis, and cancer. The specific functions of the Hh proteins are intertwined with their requirement as survival factors in Hh-responsive cells. However, studies designed to dissect the anti-apoptotic role of Hhs have been hindered by the lack of simple approaches to purify large quantities of recombinant ligands in the average laboratory setting because of the natural modifications of these proteins with palmitic acid and cholesterol. In this chapter, we provide a comprehensive protocol for the expression of Shh, Ihh, and Dhh in Escherichia coli as fusion proteins with calmodulin-binding peptide to allow easy and rapid purification. The ligands are engineered with a new N-terminus containing two isoleucine residues to provide an essential hydrophobic interphase for achieving high biologic activity. The protocol includes a detailed description of a method for determination of the specific activity of the generated proteins by use of a cell culture-based luciferase approach.

Purifying the hedgehog protein and its variants.

The purification of recombinant versions of the N-terminal signaling fragment of Sonic hedgehog (ShhN) from E. coli, Hi-5 insect cells, yeast, and mammalian cell sources reveals diverse post-translational modifications that affect the potency of the purified protein. Modifications to the N-terminal cysteine with fatty acyl groups results in significant increases in potency, up to 100-fold, when compared with the unmodified protein. Proteolytic clipping at sites near the N-terminus results in inactivation of signaling activity. The ShhN protein is particularly sensitive to metal ion-induced oxidation, and the methods described here were developed to minimize this oxidation. The purification methods developed for ShhN were applicable to human Indian and Desert hedgehog N-terminal signaling proteins, and therefore should be useful for hedgehog proteins from other species.