title: Enhanced potency of human Sonic hedgehog by hydrophobic modification.
abstract: Post-translational modifications of the developmental signaling protein Sonic hedgehog (Shh) by a long-chain fatty acid at the N-terminus and cholesterol at the C-terminus greatly activate the protein in a cell-based signaling assay. To investigate the structural determinants of this activation phenomenon, hydrophobic and hydrophilic moieties have been introduced by chemical and mutagenic methods to the soluble N-terminal signaling domain of Shh and tested in both in vitro and in vivo assays. A wide variety of hydrophobic modifications increased the potency of Shh when added at the N-terminus of the protein, ranging from long-chain fatty acids to hydrophobic amino acids, with EC(50) values from 99 nM for the unmodified protein to 0.6 nM for the myristoylated form. The N-myristoylated Shh was as active as the natural form having both N- and C-terminal modifications. The degree of activation appears to correlate with the hydrophobicity of the modification rather than any specific chemical feature of the adduct; moreover, substitution with hydrophilic moieties decreased activity. Hydrophobic modifications at the C-terminus of Shh resulted in only a 2-3-fold increase in activity, and no activation was found with hydrophobic modification at other surface positions. The N-terminal modifications did not appear to alter the binding affinity of the Shh protein for the transfected receptor protein, Patched, and had no apparent effect on structure as measured by circular dichroism, thermal denaturation, and size determination. Activation of Desert Hh through modification of its N-terminus was also observed, suggesting that this is a common feature of Hh proteins.
authors: Biogen, Inc., 14 Cambridge Center, Cambridge, Massachusetts 02142, USA. Fred_Taylor@Biogen.com
authors: F R
authors: FR
authors: Taylor
authors: D
authors: D
authors: Wen
authors: E A
authors: EA
authors: Garber
authors: A N
authors: AN
authors: Carmillo
authors: D P
authors: DP
authors: Baker
authors: R M
authors: RM
authors: Arduini
authors: K P
authors: KP
authors: Williams
authors: P H
authors: PH
authors: Weinreb
authors: P
authors: P
authors: Rayhorn
authors: X
authors: X
authors: Hronowski
authors: A
authors: A
authors: Whitty
authors: E S
authors: ES
authors: Day
authors: A
authors: A
authors: Boriack-Sjodin
authors: R I
authors: RI
authors: Shapiro
authors: A
authors: A
authors: Galdes
authors: R B
authors: RB
authors: Pepinsky
mesh_terms: D000214:Acyl Coenzyme A / Q000737:chemistry; D000577:Amides; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D002460:Cell Line; D002942:Circular Dichroism; D003545:Cysteine / Q000737:chemistry / Q000235:genetics; D005033:Ethylmaleimide / Q000737:chemistry; D005227:Fatty Acids / Q000737:chemistry; D005557:Formaldehyde / Q000737:chemistry; D053823:Hedgehog Proteins; D006801:Humans; D007202:Indicators and Reagents; D047908:Intracellular Signaling Peptides and Proteins; D007460:Iodoacetamide / Q000031:analogs & derivatives / Q000737:chemistry; D008565:Membrane Proteins / Q000096:biosynthesis / Q000378:metabolism; D051379:Mice; D008809:Mice, Inbred C3H; D000072079:Patched Receptors; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011499:Protein Processing, Post-Translational / Q000235:genetics; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D011956:Receptors, Cell Surface; D015398:Signal Transduction / Q000235:genetics; D021241:Spectrometry, Mass, Electrospray Ionization; D013438:Sulfhydryl Compounds / Q000737:chemistry; D013844:Thiazoles / Q000737:chemistry / Q000378:metabolism; D053778:Thiazolidines; D015534:Trans-Activators; D015854:Up-Regulation / Q000235:genetics
chemical_list: D000214:Acyl Coenzyme A; D000577:Amides; D005227:Fatty Acids; D053823:Hedgehog Proteins; D007202:Indicators and Reagents; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D000072079:Patched Receptors; D010446:Peptide Fragments; D011506:Proteins; D011956:Receptors, Cell Surface; C509239:SHH protein, human; D013438:Sulfhydryl Compounds; D013844:Thiazoles; D053778:Thiazolidines; D015534:Trans-Activators; D005557:Formaldehyde; C080491:N-isopropyliodoacetamide; C003438:thiazolidine-4-carboxylic acid; D003545:Cysteine; D005033:Ethylmaleimide; D007460:Iodoacetamide