Identification of conserved regions and residues within hedgehog acyltransferase critical for palmitoylation of Sonic hedgehog Journal Article
| Authors: | Buglino, J. A.; Resh, M. D. |
| Article Title: | Identification of conserved regions and residues within hedgehog acyltransferase critical for palmitoylation of Sonic hedgehog |
| Abstract: | Background: Sonic hedgehog (Shh) is a palmitoylated protein that plays key roles in mammalian development and human cancers. Palmitoylation of Shh is required for effective long and short range Shh-mediated signaling. Attachment of palmitate to Shh is catalyzed by Hedgehog acyltransferase (Hhat), a member of the membrane bound O-acyl transferase (MBOAT) family of multipass membrane proteins. The extremely hydrophobic composition of MBOAT proteins has limited their biochemical characterization. Except for mutagenesis of two conserved residues, there has been no structure-function analysis of Hhat, and the regions of the protein required for Shh palmitoylation are unknown. Methodology/Principal Findings: Here we undertake a systematic approach to identify residues within Hhat that are required for protein stability and/or enzymatic activity. We also identify a second, novel MBOAT homology region (residues 196-234) that is required for Hhat activity. In total, ten deletionmutants and eleven point mutants were generated and analyzed. Truncations at the N- and C-termini of Hhat yielded inactive proteins with reduced stability. Four Hhat mutants with deletions within predicted loop regions and five pointmutants retained stability but lost palmitoylation activity.We purified two pointmutants, W378A and H379A, with defective Hhat activity. Kinetic analyses revealed alterations in apparent Km and Vmax for Shh and/or palmitoyl CoA, changes that likely explain the catalytic defects observed for these mutants. Conclusions/Significance: This study has pinpointed specific regions and multiple residues that regulate Hhat stability and catalysis. Our findings should be applicable to other MBOAT proteins that mediate lipid modification of Wnt proteins and ghrelin, and should serve as a model for understanding how secreted morphogens are modified by palmitoyl acyltransferases. © 2010 Buglino, Resh. |
| Keywords: | controlled study; unclassified drug; human cell; genetics; nonhuman; animal cell; animal; metabolism; mammalia; animals; carboxy terminal sequence; cell line; sonic hedgehog protein; hedgehog proteins; enzyme activity; phosphorylation; chemistry; amino terminal sequence; nucleotide sequence; base sequence; armyworm; spodoptera; catalysis; dna primers; primer dna; enzyme kinetics; enzyme structure; erinaceidae; mutagenesis; acyltransferase; isoenzymes; mutant; cyclic amp dependent protein kinase; cyclic amp-dependent protein kinases; microscopy, electron, transmission; palmitoylation; enzyme stability; hedgehog acyltransferase; palmitoyl coenzyme a; cholesterol esterase; isoenzyme; cold; lipoylation; transmission electron microscopy; cold temperature; sterol esterase |
| Journal Title: | PLoS ONE |
| Volume: | 5 |
| Issue: | 6 |
| ISSN: | 1932-6203 |
| Publisher: | Public Library of Science |
| Date Published: | 2010-06-23 |
| Start Page: | e11195 |
| Language: | English |
| DOI: | 10.1371/journal.pone.0011195 |
| PUBMED: | 20585641 |
| PROVIDER: | scopus |
| PMCID: | PMC2890405 |
| DOI/URL: | |
| Notes: | --- - "Export Date: 20 April 2011" - "Art. No.: e11195" - "Source: Scopus" |
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