Isolation of recombinant apolipoprotein E4 N-terminal domain by foam fractionation

Lethcoe, Kyle; Fox, Colin A.; Hafiane, Anouar; Kiss, Robert S.; Ryan, Robert O.

doi:10.1016/j.pep.2023.106319

Cited by 2 publications

(1 citation statement)

References 45 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…An incubation containing AUH (0.01 µg/µL), HMG CoA (200 µM), and BSA (0.5 mg/mL) was conducted in HEPES buffer at 37 °C for 24 h. Following incubation, the sample was digested with trypsin and subjected to liquid chromatography-mass spectrometry analysis on an Orbitrap Eclipse mass spectrometer (Thermo Scientific, Waltham, MA, USA), essentially as described elsewhere [ 8 ]. Two control incubations, containing AUH and BSA only or HMG CoA and BSA only, were processed and analyzed in the same way as the test sample.…”

Section: Methodsmentioning

confidence: 99%

Characterization of trans-3-Methylglutaconyl CoA-Dependent Protein Acylation

et al. 2023

Self Cite

View full text Add to dashboard Cite

3-methylglutaconyl (3MGC) CoA hydratase (AUH) is the leucine catabolism pathway enzyme that catalyzes the hydration of trans-3MGC CoA to 3-hydroxy, 3-methylglutaryl (HMG) CoA. In several inborn errors of metabolism (IEM), however, metabolic dysfunction can drive this reaction in the opposite direction (the dehydration of HMG CoA). The recent discovery that trans-3MGC CoA is inherently unstable and prone to a series of non-enzymatic chemical reactions provides an explanation for 3MGC aciduria observed in these IEMs. Under physiological conditions, trans-3MGC CoA can isomerize to cis-3MGC CoA, which is structurally poised to undergo intramolecular cyclization with the loss of CoA, generating cis-3MGC anhydride. The anhydride is reactive and has two potential fates; (a) hydrolysis to yield cis-3MGC acid or (b) a reaction with lysine side-chain amino groups to 3MGCylate substrate proteins. An antibody elicited against a 3MGC hapten was employed to investigate protein acylation in incubations containing recombinant AUH, HMG CoA, and bovine serum albumin (BSA). The data obtained show that, as AUH dehydrates HMG CoA to trans-3MGC CoA, BSA is acylated. Moreover, α-3MGC IgG immunoblot signal intensity correlates with AUH concentration, HMG CoA substrate concentration, and incubation time. Thus, protein 3MGCylation may contribute to the phenotypic features associated with IEMs that manifest 3MGC aciduria.

show abstract

Section: Methodsmentioning

confidence: 99%