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1.
STAR Protoc ; 2(1): 100376, 2021 03 19.
Article in English | MEDLINE | ID: mdl-33681825

ABSTRACT

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).


Subject(s)
Cloning, Molecular/methods , Hedgehog Proteins/isolation & purification , Proteins/isolation & purification , Animals , Baculoviridae/genetics , Cell Line , Hedgehog Proteins/metabolism , Phosphorylation , Protein Serine-Threonine Kinases , Recombinant Fusion Proteins/biosynthesis , Sf9 Cells , Signal Transduction
2.
Nat Commun ; 10(1): 2320, 2019 05 24.
Article in English | MEDLINE | ID: mdl-31127104

ABSTRACT

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.


Subject(s)
Hedgehog Proteins/ultrastructure , Patched-1 Receptor/ultrastructure , Protein Domains , Cholesterol/metabolism , Cryoelectron Microscopy , HEK293 Cells , Hedgehog Proteins/chemistry , Hedgehog Proteins/isolation & purification , Hedgehog Proteins/metabolism , Humans , Ligands , Lipoylation , Models, Molecular , Patched-1 Receptor/isolation & purification , Patched-1 Receptor/metabolism , Protein Binding , Protein Multimerization , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure
3.
Clin. transl. oncol. (Print) ; 17(7): 497-503, jul. 2015. tab, ilus
Article in English | IBECS | ID: ibc-138445

ABSTRACT

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)


No disponible


Subject(s)
Female , Humans , Male , Neoplasms, Basal Cell/diagnosis , Neoplasms, Basal Cell/surgery , Neoplasm Metastasis/diagnosis , Neoplasm Metastasis/pathology , Microsurgery/methods , Mohs Surgery/methods , Hedgehog Proteins/isolation & purification , Neoplasms, Basal Cell/complications , Neoplasms, Basal Cell/physiopathology , Mohs Surgery/instrumentation , Mohs Surgery/trends , Mohs Surgery
4.
Cell Rep ; 10(8): 1280-1287, 2015 Mar 03.
Article in English | MEDLINE | ID: mdl-25732819

ABSTRACT

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.


Subject(s)
Fatty Acids/metabolism , Hedgehog Proteins/metabolism , Animals , Cell Line , Chick Embryo , Chickens/metabolism , Chromatography, High Pressure Liquid , Fatty Acids/chemistry , Hedgehog Proteins/chemistry , Hedgehog Proteins/isolation & purification , Humans , Peptides/analysis , Peptides/chemistry , Tandem Mass Spectrometry
5.
J Struct Biol ; 175(2): 209-15, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21571074

ABSTRACT

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.


Subject(s)
Automation, Laboratory/instrumentation , Recombinant Proteins/biosynthesis , Automation, Laboratory/methods , Cell Culture Techniques , DNA, Circular/isolation & purification , HEK293 Cells , Hedgehog Proteins/biosynthesis , Hedgehog Proteins/isolation & purification , Humans , Plasmids/isolation & purification , Recombinant Proteins/isolation & purification , Transfection/methods
6.
Methods Enzymol ; 446: 189-204, 2008.
Article in English | MEDLINE | ID: mdl-18603123

ABSTRACT

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.


Subject(s)
Cell Death/physiology , Cell Survival/physiology , Hedgehog Proteins/analysis , Hedgehog Proteins/isolation & purification , Animals , Biological Assay/methods , Chick Embryo , Hedgehog Proteins/physiology , Nervous System/cytology , Protein Precursors/metabolism , Recombinant Proteins/biosynthesis , Signal Transduction/physiology
7.
Methods Mol Biol ; 397: 1-22, 2007.
Article in English | MEDLINE | ID: mdl-18025709

ABSTRACT

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.


Subject(s)
Chemistry Techniques, Analytical/methods , Hedgehog Proteins/isolation & purification , Mutant Proteins/isolation & purification , Acylation , Animals , Cell Line , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Hedgehog Proteins/chemistry , Humans , Insecta , Mice , Molecular Weight , Mutant Proteins/chemistry , Pichia , Protein Engineering , Protein Processing, Post-Translational , Rats , Solubility
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