Journal: Journal of developmental biology
Article Title: A Residual N-Terminal Peptide Enhances Signaling of Depalmitoylated Hedgehog to the Patched Receptor.
doi: 10.3390/jdb12020011
Figure Lengend Snippet: Figure 4. A minimal N-terminal amino acid sequence contributes to Shh signaling in vitro. (A) The N-terminal Shh peptide, including the Cardin–Weintraub (CW) motif (green) is shown. A G-to-R exchange (shown in blue) just upstream of the CW site renders G32RShh susceptible to furin cleavage (inset, right lane, red arrowhead). PCSK7 cleaves the wild-type Shh peptide at the same site (inset, and red arrowhead). (B) qPCR confirmed that ShhC and C25AShhC increased the transcription of Ptch1, Gli1, and Alp1 in C3H10T1/2 cells to a similar extent. Target gene transcription was much less induced by furin- or PCSK7-cleaved proteins, indicating that processing rendered them inactive. (C) Cryo-EM derived structures (pdb: 6e1h) reveal interactions between the palmitoylated Shh N-peptide (white backbone, nitrogens stained blue, oxygens stained red, palmitate stained green) and Ptch residues (stained yellow) [22]. (D) Shh and mutant proteins lacking the N-terminal C25 to prevent palmitoylation, and their consecutively N-truncated counterparts were expressed, pulled down with heparin, and immunoblotted. All proteins were expressed at similar levels, as indicated by polyclonal α-Shh reactivity. Bottom: C3H10T1/2 osteoblast progenitor cells were incubated with similar amounts of ShhC, C25SShhC, and their truncated variants, and relative increases in Alp1 activity were determined as biological readouts. Media obtained from mock-transfected Bosc23 cells was used as a negative control, and ShhC-conditioned media as a positive control. Inhibition of ShhC
Article Snippet: Goat-α-Shh antibodies (R&D Systems, AF464) were used for all blots to detect full-length unprocessed Shh (decreased el ctrophoretic mobility and, top) and N-truncated pro ins that were solubilized from the cellular precurso (increased electrophoretic mobility band, bottom).
Techniques: Sequencing, In Vitro, Cryo-EM Sample Prep, Derivative Assay, Staining, Mutagenesis, Incubation, Activity Assay, Transfection, Negative Control, Positive Control, Inhibition
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![Figure 2. Reverse-phase HPLC reveals the decreased hydrophobicity of ShhC released in the presence of serum or of pharmacological cholesterol <t>acceptors.</t> <t>Goat-α-Shh</t> antibodies (R&D Systems, <t>AF464)</t> were used for all blots to detect full-length unprocessed Shh (decreased electrophoretic mobility band, top) and N-truncated proteins that were solubilized from the cellular precursor (increased electrophoretic mobility band, bottom). Lower fraction numbers (fr#) indicate more hydrophilic (delipidated) proteins and higher fraction numbers indicate more lipophilic (lipidated) proteins. (A–D) RP-HPLC calibration. Consistent with its dual lipidation, R&D 8908-SH positive control proteins elute predominantly in the late fraction #37 (black arrowhead). Artificial monolipidated cellular ShhN elutes in fractions #27–29 (red arrowhead) and monolipidated cellular C25SShh (this artificial variant has the cysteine palmitate acceptor replaced with a non-accepting serine) elutes predominantly in fraction #32 (white arrowhead). Overexpressed soluble C25SShhN, another engi- neered control protein lacking both lipids, elutes in fractions #26–28 from the C4 column (green arrowhead). N-terminal Shh peptides in the schematics are labeled in orange. (E) Overexpressed cellular Shh elutes predominantly in fraction #37 (black arrowhead); a small fraction that probably did not undergo Hhat-dependent N-terminal palmitoylation elutes in fraction #33 (white arrowhead). (F) ShhC, solubilized by Disp and Scube2 from its dually lipidated cellular precursor (E), also eluted in fractions #32–34, showing that it retained the C-cholesterol moiety but not the N-palmitate after its release (white arrowhead). (G) Consistent with this, the artificially produced soluble control C25SShhC, blocked in its ability to undergo N-palmitoylation during biosynthesis, also eluted in fractions #32–34 (white arrowhead). The increased electrophoretic mobility of the protein indicated that its N-terminus was also processed. (H,I) Similar hydrophobicity of ShhC and C25SShhC was expressed in the presence of 600 µg/mL of the pharmacological cholesterol acceptor CD. Note that the electrophoretic mobility of the most soluble ShhC is again increased (lower band), consistent with proteolytic processing of the palmitoylated N-terminal peptide during Disp- and Scube2-mediated ShhC release [32,36].](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_1456/pm38651456/pm38651456__page7_image1.jpg)
![Figure 4. A minimal N-terminal amino acid sequence contributes to Shh signaling in vitro. (A) The N-terminal Shh peptide, including the Cardin–Weintraub (CW) motif (green) is shown. A G-to-R exchange (shown in blue) just upstream of the CW site renders G32RShh susceptible to furin cleavage (inset, right lane, red arrowhead). PCSK7 cleaves the wild-type Shh peptide at the same site (inset, and red arrowhead). (B) qPCR confirmed that ShhC and C25AShhC increased the transcription of Ptch1, Gli1, and Alp1 in C3H10T1/2 cells to a similar extent. Target gene transcription was much less induced by furin- or PCSK7-cleaved proteins, indicating that processing rendered them inactive. (C) Cryo-EM derived structures (pdb: 6e1h) reveal interactions between the palmitoylated Shh N-peptide (white backbone, nitrogens stained blue, oxygens stained red, palmitate stained green) and Ptch residues (stained yellow) [22]. (D) Shh and mutant proteins lacking the N-terminal C25 to prevent palmitoylation, and their consecutively N-truncated counterparts were expressed, pulled down with heparin, and immunoblotted. All proteins were expressed at similar levels, as indicated by polyclonal α-Shh reactivity. Bottom: C3H10T1/2 osteoblast progenitor cells were incubated with similar amounts of ShhC, C25SShhC, and their truncated variants, and relative increases in Alp1 activity were determined as biological readouts. Media obtained from mock-transfected Bosc23 cells was used as a negative control, and ShhC-conditioned media as a positive control. Inhibition of ShhC](https://pub-med-unpaywalled-images-cdn.bioz.com/pub_med_ids_ending_with_1456/pm38651456/pm38651456__page11_image1.jpg)