2-Bromopalmitoyl-CoA and 2-bromopalmitate: Promiscuous inhibitors of membrane-bound enzymes

Coleman, Rosalind A.; Rao, P. Vasanth; Fogelsong, Robert J.; Bardes, Elaine S.G.

doi:10.1016/0005-2760(92)90046-x

Cited by 90 publications

(78 citation statements)

References 26 publications

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“…Although 2-BP clearly inhibits the palmitoyltransferases, there is evidence that it inhibits the thioesterases as well (44,52,53). 2-BP also inhibits metabolic enzymes, including fatty acid CoA ligase, which reduces the cellular content of the palmitoylation substrate palmitoyl-CoA (44,52,53). We also observed a decrease in TER after treatment of mCCD cl1 cells with 2-BP, which may reflect reduced palmitoylation of claudins normally found within the tight junctions (54 -56).…”

Section: With Radiolabeling With [mentioning

confidence: 63%

“…The palmitate analog 2-BP has been regularly used in cultured cells to inhibit palmitoylation of proteins, thereby providing a complementary in vivo approach to study the function of palmitate addition to specified proteins (44,52). Although 2-BP clearly inhibits the palmitoyltransferases, there is evidence that it inhibits the thioesterases as well (44,52,53). 2-BP also inhibits metabolic enzymes, including fatty acid CoA ligase, which reduces the cellular content of the palmitoylation substrate palmitoyl-CoA (44,52,53).…”

Section: With Radiolabeling With [mentioning

confidence: 99%

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Specific Palmitoyltransferases Associate with and Activate the Epithelial Sodium Channel

Mukherjee¹,

Wang²,

Kinlough³

et al. 2017

Journal of Biological Chemistry

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Edited by Henrik G. DohlmanThe epithelial sodium channel (ENaC) has an important role in regulating extracellular fluid volume and blood pressure, as well as airway surface liquid volume and mucociliary clearance. ENaC is a trimer of three homologous subunits (␣, ␤, and ␥). We previously reported that cytoplasmic residues on the ␤ (␤Cys-43 and ␤Cys-557) and ␥ (␥Cys-33 and ␥Cys-41) subunits are palmitoylated. Mutation of Cys that blocked ENaC palmitoylation also reduced channel open probability. Furthermore, ␥ subunit palmitoylation had a dominant role over ␤ subunit palmitoylation in regulating ENaC. To determine which palmitoyltransferases (termed DHHCs) regulate the channel, mouse ENaCs were co-expressed in Xenopus oocytes with each of the 23 mouse DHHCs. ENaC activity was significantly increased by DHHCs 1, 2, 3, 7, and 14. ENaC activation by DHHCs was lost when ␥ subunit palmitoylation sites were mutated, whereas DHHCs 1, 2, and 14 still activated ENaC lacking ␤ subunit palmitoylation sites. ␤ subunit palmitoylation was increased by ENaC co-expression with DHHC 7. Both wild type ENaC and channels lacking ␤ and ␥ palmitoylation sites co-immunoprecipitated with the five activating DHHCs, suggesting that ENaC forms a complex with multiple DHHCs. RT-PCR revealed that transcripts for the five activating DHHCs were present in cultured mCCD cl1 cells, and DHHC 3 was expressed in aquaporin 2-positive principal cells of mouse aldosterone-sensitive distal nephron where ENaC is localized. Treatment of polarized mCCD cl1 cells with a general inhibitor of palmitoylation reduced ENaC-mediated Na ؉ currents within minutes. Our results indicate that specific DHHCs have a role in regulating ENaC.ENaCs 2 are amiloride-sensitive Na ϩ channels that are found in high resistance epithelia and other tissues. In the aldosterone-sensitive distal nephron (ASDN), ENaC-dependent Na ϩ transport has an important role in the maintenance of extracellular fluid volume and blood pressure, as well as extracellular K ϩ homeostasis. In the lung, ENaC has a role in regulating airway surface fluid volume, mucociliary clearance, and alveolar fluid volume (1-3). The channels are composed of three homologous subunits (termed ␣, ␤, and ␥), each with two transmembrane domains, a large extracellular loop and cytoplasmic N and C termini (3-5).ENaCs are regulated by signaling pathways that modulate its membrane trafficking, residency on the plasma membrane, and degradation (6 -8). ENaCs are also regulated by a variety of extracellular factors that affect its open probability (P o ). These include extracellular cations (H ϩ , Na ϩ , and other metals), anions (Cl Ϫ ), laminar shear stress, and proteases that cleave ENaC subunits at specific sites and release embedded inhibitory tracts (1, 2, 9 -14). Intracellular phosphorylation, inositol phospholipids, and cytoplasmic Cys palmitoylation also regulate ENaC P o (15-21).Cys palmitoylation of soluble and transmembrane proteins is a reversible post-translational modification that increases protein surface hydr...

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Section: With Radiolabeling With [mentioning

confidence: 63%

Section: With Radiolabeling With [mentioning

confidence: 99%

Specific Palmitoyltransferases Associate with and Activate the Epithelial Sodium Channel

Mukherjee¹,

Wang²,

Kinlough³

et al. 2017

Journal of Biological Chemistry

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“…2BP binds to and inhibits carnitine palmitoyl transferase-1. This reaction prevents long chain free fatty acids from entering mitochondria and thereby inhibits fatty acid β-oxidation [26,27]. 2BP also inhibits other enzymes involved in lipid metabolism, including triacylglycerol biosynthesis, Fatty Acid CoA ligase, and Glycerol-3-phosphate acyltransferase [27].…”

Section: Inhibition Of Protein Palmitoylation With 2-bromopalmitate (mentioning

confidence: 99%

Use of analogs and inhibitors to study the functional significance of protein palmitoylation

Resh

2006

Methods

146

136

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Covalent attachment of palmitate to proteins is a post-translational modification that exerts diverse effects on protein localization and function. The three key technical approaches required for an investigator to determine the role of palmitoylation of Your Favorite Palmitoylated Protein (YFPP) are methods to: 1) detect YFPP palmitoylation; 2) alter or inhibit palmitoylation of YFPP; 3) determine the functional significance of altered YFPP palmitoylation. Here, I describe experimental methods to address these three issues. Both radioactive (radiolabeling with 3 H palmitate or 125 I-IC16 palmitate) and non-radioactive (chemical labeling and mass spectrometry) methods to detect palmitoylated proteins are presented. Next, techniques to inhibit protein palmitoylation are described. These include site specific mutagenesis, and treatment of cells with inhibitors of protein palmitoylation, including 2-bromopalmitate, cerulenin, and tunicamycin. Alternative methods to replace palmitate with other fatty acids are also presented. Finally, general approaches to determining the effect of altered palmitoylation status on YFPP association with membranes and lipid rafts, as well as signal transduction, are described.

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“…Mayorek and BarTana [22] first reported the use of this compound as a specific inhibitor of DAGAT in rat hepatocytes and it has also been used to inhibit the enzyme from Brassica napus [23]. However, bromo-fatty acids have been suggested to have more general effects on membranelocated enzymes [24] so we carried out experiments at 30°C to evaluate 2-bromopalmitate as well as 2-bromooctanoate. The effect of increasing concentrations of 2-bromooctanoate on the labelling of Kennedy pathway intermediates in oil palm and olive microsomes is shown in Fig.…”

Section: Using Bromooctanoate As An Inhibitor Of Dagatmentioning

confidence: 99%

Metabolic control analysis reveals an important role for diacylglycerol acyltransferase in olive but not in oil palm lipid accumulation

et al. 2005

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We applied metabolic control analysis to the Kennedy pathway for triacylglycerol formation in tissue cultures from the important oil crops, olive (Olea europaea L.) and oil palm (Elaeis guineensis Jacq.). When microsomal fractions were incubated at 30 °C rather than 20 °C, there was an increase in triacylglycerol labelling. This increase was accompanied by a build up of diacylglycerol (DAG) radioactivity in olive but not in oil palm, suggesting that the activity of DAG acyltransferase (DAGAT) was becoming limiting in olive. We used 2‐bromooctanoate as a specific inhibitor of DAGAT and showed that the enzyme had a flux control coefficient under the experimental conditions of 0.74 in olive but only 0.12 in oil palm. These data revealed important differences in the regulation of lipid biosynthesis in cultures from different plants and suggest that changes in the endogenous activity of DAGAT is unlikely to affect oil accumulation in oil palm crops.

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2-Bromopalmitoyl-CoA and 2-bromopalmitate: Promiscuous inhibitors of membrane-bound enzymes

Cited by 90 publications

References 26 publications

Specific Palmitoyltransferases Associate with and Activate the Epithelial Sodium Channel

Specific Palmitoyltransferases Associate with and Activate the Epithelial Sodium Channel

Use of analogs and inhibitors to study the functional significance of protein palmitoylation

Metabolic control analysis reveals an important role for diacylglycerol acyltransferase in olive but not in oil palm lipid accumulation

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