Membrane Topology of Hedgehog Acyltransferase

Matevossian, Armine; Resh, Marilyn D.

doi:10.1074/jbc.m114.625764

Cited by 48 publications

(57 citation statements)

References 31 publications

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“…A Clustal Omega alignment of the hGOAT and mGOAT sequences reveals a number of cysteine residues in the human enzyme that are not conserved in mGOAT (Figure 6d, yellow highlights), 71 with the majority of these non-conserved cysteines lying outside the conserved MBOAT domain in the C-terminal sequence of GOAT. 62, 72–74 …”

Section: Resultsmentioning

confidence: 99%

“…hGOAT contains a total of 16 cysteine residues (Figure 5a), with several of these cysteines lying in the conserved C-terminal “MBOAT” domain within hGOAT. 62, 72, 74 Mutational analyses of the three protein-modifying members of the MBOAT family (Hhat, PORCN, and GOAT) have revealed functionally required residues but none have implicated cysteine residues as functionally essential. 30, 62, 65, 73, 75–77 While Hhat and PORCN contain palmitoylated cysteine residues, 73, 77 our findings provide the first evidence supporting an enzymatic cysteine residue involved in MBOAT-catalyzed protein acylation.…”

Section: Discussionmentioning

confidence: 99%

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Synthetic Triterpenoid Inhibition of Human Ghrelin O-Acyltransferase: The Involvement of a Functionally Required Cysteine Provides Mechanistic Insight into Ghrelin Acylation

et al. 2017

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The peptide hormone ghrelin plays a key role in regulating hunger and energy balance within the body. Ghrelin signaling presents a promising and unexploited target for development of small-molecule therapeutics to treat obesity, diabetes, and other health conditions. Inhibition of ghrelin O-acyltransferase (GOAT), which catalyzes an essential octanoylation step in ghrelin maturation, offers a potential avenue for controlling ghrelin signaling. Through screening a small molecule library, we have identified a class of synthetic triterpenoids that efficiently inhibit ghrelin acylation by the human isoform of GOAT (hGOAT). These compounds function as covalent reversible inhibitors of hGOAT, providing the first evidence for involvement of a nucleophilic cysteine residue in substrate acylation by a MBOAT family acyltransferase. Surprisingly, the mouse form of GOAT does not exhibit susceptibility to cysteine modifying electrophiles revealing an important distinction in the activity and behavior between these closely related GOAT isoforms. This study establishes these compounds as potent small molecule inhibitors of ghrelin acylation and provides a foundation for the development of novel hGOAT inhibitors as therapeutics targeting diabetes and obesity.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Synthetic Triterpenoid Inhibition of Human Ghrelin O-Acyltransferase: The Involvement of a Functionally Required Cysteine Provides Mechanistic Insight into Ghrelin Acylation

et al. 2017

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“…Alternatively, it is possible that MBOAT proteins themselves facilitate fatty acyl CoA transport across the ER membrane. Studies of membrane topology reveal that Hhat contains ten TMDs and two reentrant loops[141, 142], with a putative active site histidine disposed near the lumenal surface. Of note, a large fraction of the Hhat sequence is located on the cytosolic side of the ER membrane.…”

Section: Fatty Acylation Of Secreted Proteins By Mboat Enzymesmentioning

confidence: 99%

Fatty acylation of proteins: The long and the short of it

Resh

2016

Progress in Lipid Research

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Long, short and medium chain fatty acids are covalently attached to hundreds of proteins. Each fatty acid confers distinct biochemical properties, enabling fatty acylation to regulate intracellular trafficking, subcellular localization, protein-protein and protein-lipid interactions. Myristate and palmitate represent the most common fatty acid modifying groups. New insights into how fatty acylation reactions are catalyzed, and how fatty acylation regulates protein structure and function continue to emerge. Myristate is typically linked to an N-terminal glycine, but recent studies reveal that lysines can also be myristoylated. Enzymes that remove N-terminal myristoyl-glycine or myristate from lysines have now been identified. DHHC proteins catalyze S-palmitoylation, but the mechanisms that regulate substrate recognition by individual DHHC family members remain to be determined. New studies continue to reveal thioesterases that remove palmitate from S-acylated proteins. Another area of rapid expansion is fatty acylation of the secreted proteins Hedgehog, Wnt and Ghrelin, by Hhat, Porcupine and GOAT, respectively. Understanding how these membrane bound O-acyl transferases recognize their protein and fatty acyl CoA substrates is an active area of investigation, and is punctuated by the finding that these enzymes are potential drug targets in human diseases.

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“…Little is known about their enzyme mechanisms. They contain an invariant lumenal histidine and a highly conserved asparagine[8, 16–18], which are presumed to be involved in catalysis (H338 and N307 in mouse GOAT). However, in the case of HHAT, which N -palmitoylates hedgehog proteins, some activity was preserved upon mutation of the histidine to alanine[19], and for GOAT, it has been demonstrated that the conserved His and Asn are on opposite sides of the ER membrane[8].…”

Section: Introductionmentioning

confidence: 99%

Mechanistic analysis of ghrelin-O-acyltransferase using substrate analogs

Taylor

Dempsey

Hwang

et al. 2015

Bioorganic Chemistry

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Ghrelin-O-Acyltransferase (GOAT) is an 11-transmembrane integral membrane protein that octanoylates the metabolism-regulating peptide hormone ghrelin at Ser3 and may represent an attractive target for the treatment of type II diabetes and the metabolic syndrome. Protein octanoylation is unique to ghrelin in humans, and little is known about the mechanism of GOAT or of related protein-O-acyltransferases HHAT or PORC. In this study, we explored an in vitro microsomal ghrelin octanoylation assay to analyze its enzymologic features. Measurement of Km for 10-mer, 27-mer, and synthetic Tat-peptide-containing ghrelin substrates provided evidence for a role of charge interactions in substrate binding. Ghrelin substrates with amino-alanine in place of Ser3 demonstrated that GOAT can catalyze the formation of an octanoyl-amide bond at a similar rate compared with the natural reaction. A pH-rate comparison of these substrates revealed minimal differences in acyltransferase activity across pH 6.0–9.0, providing evidence that these reactions may be relatively insensitive to the basicity of the substrate nucleophile. The conserved His338 residue was required both for Ser3 and amino-Ala3 ghrelin substrates, suggesting that His338 may have a key catalytic role beyond that of a general base.

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Membrane Topology of Hedgehog Acyltransferase

Cited by 48 publications

References 31 publications

Synthetic Triterpenoid Inhibition of Human Ghrelin O-Acyltransferase: The Involvement of a Functionally Required Cysteine Provides Mechanistic Insight into Ghrelin Acylation

Synthetic Triterpenoid Inhibition of Human Ghrelin O-Acyltransferase: The Involvement of a Functionally Required Cysteine Provides Mechanistic Insight into Ghrelin Acylation

Fatty acylation of proteins: The long and the short of it

Mechanistic analysis of ghrelin-O-acyltransferase using substrate analogs

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