Metallo-β-lactamase domain-containing protein 2 is S-palmitoylated and exhibits acyl-CoA hydrolase activity

Abstract

Members of the metallo-β-lactamase (MBL) superfamily of enzymes harbor a highly conserved αββα MBL-fold domain and were first described as inactivators of common β-lactam antibiotics. In humans, these enzymes have been shown to exhibit diverse functions, including hydrolase activity towards amides, esters, and thioesters. An uncharacterized member of the human MBL family, MBLAC2, was detected in multiple palmitoylproteomes, identified as a zDHHC20 S-acyltransferase interactor, and annotated as a potential thioesterase. In this study, we confirmed that MBLAC2 is palmitoylated and identified the likely S-palmitoylation site as Cys254. S-palmitoylation of MBLAC2 is increased in cells when expressed with zDHHC20 and MBLAC2 is a substrate for purified zDHHC20 in vitro. To determine its biochemical function, we tested the ability of MBLAC2 to hydrolyze a variety of small molecules and acylprotein substrates. MBLAC2 has acyl-CoA thioesterase activity with kinetic parameters and acyl-CoA selectivity comparable to acyl-CoA thioesterase 1 (ACOT1). Two predicted zinc-binding residues, Asp87 and His88 are required for MBLAC2 hydrolase activity. Consistent with a role in fatty acid metabolism in cells, MBLAC2 was cross-linked to a photoactivatable fatty acid in a manner that was independent of its S-fatty acylation at Cys254. Our study adds to previous investigations demonstrating the versatility of the MBL-fold domain in supporting a variety of enzymatic reactions.

Target deconvolution of HDAC pharmacopoeia reveals MBLAC2 as common off-target

“…Moreover, the quantitative data revealed that drugs interacting with HDAC1 as part of gene regulatory CoREST complexes 13 containing either RCOR1 or RCOR3 show a larger than ten-fold difference in HDAC1 binding affinity. Surprisingly, about half of the HDACis, including clinically advanced molecules, inhibit the acyl-CoA hydrolase metallo-beta-lactamase domain-containing protein 2 (MBLAC2) 14 at nano-molar concentrations. We further demonstrate that pharmacological inhibition or knockdown of MBLAC2 triggers accumulation of extracellular vesicles in cell culture, placing this poorly characterized protein into the context of extracellular vesicle biology.…”

Target deconvolution of HDAC pharmacopoeia highlights MBLAC2 as common off-target

“…To ascertain that inhibition of binding equals inhibition of enzymatic activity, we deployed a recombinant enzyme activity assay based on the MBLAC2-catalyzed hydrolysis of 3 H-labeled palmitoyl-CoA (Fig. 4b) 23 . MBLAC2 activity was affected by nearly all binders at 300 nM drug concentration and reduced to background for 18 HDACis (Fig.…”

“…Fifth, among the non-HDAC off-targets, MBLAC2 stands out for its potent and frequent binding to hydroxamic acid HDAC inhibitors. This protein has recently been shown to hydrolyse acyl-CoA and just like HDACs utilizes Zn 2+ for catalysis 23 . Our data shows for the rst time that HDACis can inhibit the palmitoyl-CoA hydrolase function of the enzyme in vitro and several do so with single digit nano-molar EC 50 s (pEC 50 > 8.0).…”

“…Moreover, the quantitative data revealed that drugs interacting with the CoREST complex variants containing either RCOR1 or RCOR3 show larger than ten-fold difference in HDAC1 binding a nity. Surprisingly, about half of the HDACis, including clinically advanced molecules, inhibit metallo-betalactamase domain-containing protein 2 (MBLAC2) 23 at nano-molar concentrations. We further demonstrate that pharmacological inhibition or knockdown of MBLAC2 triggers accumulation of extracellular vesicles in cell culture, placing this poorly characterized protein into the context of extracellular vesicle biology.…”

“…protein. Yet, it has been shown to interact with the palmitoyltransferase ZDHHC20 23 , a regulator of endocytosis-mediated EGFR internalization 40 , as well as other proteins (BioPlex) [41][42][43] whose GO term enrichment hints at roles in endo-or exocytosis (Table S3). Concurring GO annotations were also found when analysing the localisation and roles of MBLAC2-coregulated proteins (proteomeHD) 44 (Table S3).…”