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

Abstract

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.

Keywords: 3MGC aciduria; acetyl CoA diversion; immunoblot; inborn error of metabolism; protein 3MGCylation.

Figure 1

Leucine degradation pathway in muscle tissue mitochondria. At completion, this pathway yields three acetyl CoA per leucine residue. BCAT = branched-chain aminotransferase; BCKDH = branched-chain α-keto acid dehydrogenase; IVD = isovaleryl CoA dehydrogenase; 3MCCCase = 3-methylcrotonyl CoA carboxylase; AUH = 3-methylglutaconyl CoA hydratase; HMGCL = HMG CoA lyase; SCOT = succinyl CoA:3-oxoacid CoA transferase. Pathway product acetyl CoA moieties are depicted in hatched red boxes.

Figure 2

Non-enzymatic chemical reaction scheme from trans-3MGC CoA. When hydration of trans-3MGC CoA is prevented by an inborn error of metabolism in AUH, for example, this metabolite is subject to a series of non-enzymatic chemical reactions including isomerization to cis-3MGC CoA, intramolecular cyclization to cis-3MGC anhydride plus CoA, and hydrolytic cleavage of the cyclic anhydride to yield the organic acid, cis-3MGC acid, which is excreted in urine. In addition to this outcome, cis-3MGC anhydride can react with protein lysine side-chain amino groups to covalently 3MGCylate these residues. 3MGCylated proteins may be deacylated by the NAD⁺ requiring enzyme sirtuin 4 (SIRT4), yielding cis-3MGC acid as a product. Non-enzymatic chemical reactions in this process are depicted in hatched red boxes.

Figure 3

Effect of AUH concentration on the formation of 3MGCylated BSA from HMG CoA. (A) AUH activity assays were conducted by incubating specified amounts of AUH with 250 µM HMG CoA and 50 µg BSA in 100 mM Tris HCl, pH 8.0, at 37 °C for 24 h (100 µL final volume). Following incubation, aliquots of each assay mixture were subjected to SDS-PAGE and transferred to a PVDF membrane. After blocking non-specific sites and washing, the membrane was probed with α-3MGC IgG and developed as described in Material and Methods. (B) Densitometric analysis of the immunoblot from Panel A was conducted to quantify relative band intensities. Results presented are representative of an experiment that was performed on two separate occasions.

Figure 4

Effect of HMG CoA substrate concentration on AUH-dependent 3MGCylation of BSA. (A) AUH activity assays (100 µL final volume) were conducted by incubating AUH (1 µg) and BSA (50 µg) and indicated amounts of HMG CoA in Tris buffer at 37 °C for 24 h. Following incubation, an aliquot of each assay mixture was subjected to SDS-PAGE and transferred to a PVDF membrane. After blocking non-specific sites and washing, the membrane was probed with α-3MGC IgG and developed. (B) Densitometric analysis of the immunoblot from Panel A was conducted to quantify relative band intensities. The results presented are representative of an experiment that was performed on two separate occasions.

Figure 5

Effect of buffer composition on AUH- and HMG CoA-dependent 3MGCylation of BSA. AUH activity assays were conducted in Tris buffer or HEPES buffer (100 µL final volume). Where indicated, 100 mM glycine was included in incubations. Reactions containing AUH (1 µg), HMG CoA (200 µM), and BSA (50 µg) were incubated for 24 h at 37 °C. Following incubation, an aliquot of each assay mixture was subjected to SDS-PAGE and transferred to a PVDF membrane. After blocking non-specific sites and washing, the membrane was probed with α-3MGC IgG and developed. Results presented are representative of an experiment that was performed on four separate occasions.

Figure 6

Effect of incubation time on AUH- and HMG CoA-dependent 3MGCylation of BSA. (A) AUH activity assays were conducted in HEPES buffer (100 µL final volume) containing AUH (1 µg), HMG CoA (200 µM), and BSA (50 µg) at 37 °C for specified times intervals. Following incubation, an aliquot of each assay mixture was subjected to SDS-PAGE and transferred to a PVDF membrane. After blocking non-specific sites and washing, the membrane was probed with α-3MGC IgG and developed. (B) Densitometric analysis of the immunoblot from Figure 4A was conducted to quantify relative band intensities. The results presented are representative of an experiment that was performed on four separate occasions.

Figure 7

Effect of temperature on AUH- and HMG CoA-dependent 3MGCylation of BSA. (A) AUH activity assays (100 µL final volume) were conducted in HEPES buffer containing AUH (1 µg), HMG CoA (200 µM), and BSA (50 µg) for 24 h at the indicated temperatures. Following incubation, an aliquot of each assay mixture was subjected to SDS-PAGE and transferred to a PVDF membrane. After blocking non-specific sites and washing, the membrane was probed with α-3MGC IgG and developed. Results presented are representative of an experiment that was performed on four separate occasions. (B) AUH (2.5 µg) was pre-incubated at the indicated temperatures for 15 min prior to use in assays of BSA 3MGCylation. Following pre-incubation, 1 µg aliquots of AUH were introduced into assay mixtures containing HEPES buffer, 200 µM HMG CoA and 50 µg BSA and incubated for 24 h at 37 °C. An aliquot of each assay mixture was then subjected to SDS-PAGE and transferred to a PVDF membrane. After blocking non-specific sites and washing, the membrane was probed with α-3MGC IgG and developed. Results presented are representative of an experiment that was performed on four separate occasions.