2-bromohexadecanoic acid, or 2-bromopalmitate, was introduced nearly 50 years ago as a non-selective inhibitor of lipid metabolism. More recently, 2-bromopalmitate re-emerged as a general inhibitor of protein S-palmitoylation. Here, we investigate the cellular targets of 2-bromopalmitate through the synthesis and application of click-enabled analogues. In cells, 2-bromopalmitate is converted to 2-bromopalmitoyl-CoA, although less efficiently than free palmitate. Once conjugated to CoA, probe reactivity is dramatically enhanced. Importantly, both 2-bromopalmitate and 2-bromopalmitoyl-CoA label DHHC palmitoyl acyl transferases (PATs), the enzymes that catalyze protein S-palmitoylation. Mass spectrometry analysis of enriched 2-bromopalmitate targets identified PAT enzymes, transporters, and many palmitoylated proteins, with no observed preference for CoA-dependent enzymes. These data question whether 2-bromopalmitate (or 2-bromopalmitoyl-CoA) blocks S-palmitoylation by inhibiting protein acyl transferases, or by blocking palmitate incorporation by direct covalent competition. Overall, these findings highlight the promiscuous reactivity of 2BP, and validate clickable 2BP analogues as activity-based probes of diverse membrane associated enzymes.

Edited by Luke O'NeillItaconic acid is an important metabolite produced by macrophages after stimulation with LPS. the cell culture medium leads to elevated intracellular levels of unlabeled succinate, with no evidence of intracellular uptake. The goal of this study is to encourage the development of effective pro-drug strategies to increase the intracellular levels of itaconate, which will enable more conclusive analysis of its action on macrophages and other cell and tissue types.Itaconic acid is a dicarboxylic acid polar metabolite originally characterized in Aspergillus terreus, but is also produced in mammalian cells (1). After LPS stimulation, itaconic acid is secreted by macrophages, where it can inhibit bacterial cell growth (1). In macrophages, itaconate synthesis is catalyzed by the immune-responsive gene 1 (IRG1) protein, which mediates the decarboxylation of cis-aconitate to itaconate (2). Metabolomic and fluxomic analysis of LPS-stimulated macrophages demonstrated reduced Isocitrate dehydrogenase-1 (IDH1) expression and increased IRG1 expression, resulting in a diversion of citrate from the TCA cycle 2 toward itaconate production (3). This metabolic remodeling results in glutamate serving as the anaplerotic substrate to maintain or elevate succinate levels (3). Moreover, elevated succinate acts as an inflammatory signal that induces secretion of IL-1␤ and stabilization of HIF-1␣ (4). Based on the similarity between itaconate (methylene succinic acid) and succinate, recent investigations have focused on the link between itaconate synthesis and succinate accumulation. Two complementary studies reported that itaconate inhibits succinate dehydrogenase and drives succinate accumulation (5, 6). Studying the role of itaconate requires either lowering its intracellular concentration by using IRG1 knock-out mouse models (5, 6) or increasing its intracellular concentration by using either itaconate (5) or a "cell-permeable" analog, dimethyl itaconate (DMI) (6). However, there is no direct evidence that itaconate or DMI can cross cell membranes and increase intracellular itaconate. Without direct evidence of intracellular delivery, it remains unclear whether itaconate-mediated metabolic and inflammatory effects are induced by increasing intracellular itaconate or by an extracellular mechanism.Here we synthesized isotopically labeled [ 13 C]itaconate and dimethyl [13 C]itaconate ([ 13 C]DMI) to directly profile itaconate metabolism and uptake (Fig. 1). This analysis suggests that exogenous itaconate is not taken up into cells and [13 C]DMI is not metabolized into [13 C]itaconate in bone marrow-derived macrophages. We also report that [13 C]itaconate in the cell culture medium leads to elevated intracellular levels of unlabeled succinate, yet there is no evidence of intracellular uptake. Overall, this study highlights current limitations in intracellular itaconate delivery, and emphasizes the development of effective pro-drug strategies to conclusively define its action on macrophages and other cell and tissue t...

The scanning electron microscopic study of day 9 embryos reported here documents differences among normal mouse strains in morphology of cranial neural tube closure. The site of initiation of contact and fusion of the cranial neural folds, previously defined as Closure 2 (Macdonald et al., '89), is located in the region of the junction between the forebrain (prosencephalon) and midbrain (mesencephalon) in three normal strains: LM/Bc, AEJ/RkBc, and ICR/Bc. However in a fourth normal strain, SWV/Bc, Closure 2 is initiated much further rostral, in the prosencephalic region. In addition, the anterior neuropore, rostral to Closure 2, closes late in ICR/Bc embryos, relative to the posterior progress of development of the Closure 2 seam. Initiation of closure from the most rostral end of the neural tube (Closure 3) appears to be relatively delayed in ICR/Bc embryos. We hypothesize that the observed genetic polymorphism in location of the first site of fusion between the cranial neural folds in normal mouse embryos may be one basis for differences among normal strains in liability to exencephaly induced by teratogens.

Cysteine S-nitrosation and S-sulfination are naturally occurring post-translational modifications (PTMs) on proteins induced by physiological signals and redox stress. Here we demonstrate that sulfinic acids and nitrosothiols react to form a stable thiosulfonate bond, and leverage this reactivity using sulfinate-linked probes to enrich and annotate hundreds of endogenous S-nitrosated proteins. In physiological buffers, sulfinic acids do not react with iodoacetamide or disulfides, enabling selective alkylation of free thiols and site-specific analysis of S-nitrosation. In parallel, S-nitrosothiol-linked probes enable enrichment and detection of endogenous S-sulfinated proteins, confirming that a single sulfinic acid can react with a nitrosothiol to form a thiosulfonate linkage. Using this approach, we find that hydrogen peroxide addition increases S-sulfination of human DJ-1 (PARK7) at Cys106, whereas Cys46 and Cys53 are fully oxidized to sulfonic acids. Comparative gel-based analysis of different mouse tissues reveals distinct profiles for both S-nitrosation and S-sulfination. Quantitative proteomic analysis demonstrates that both S-nitrosation and S-sulfination are widespread, yet exhibit enhanced occupancy on select proteins, including thioredoxin, peroxiredoxins, and other validated redox active proteins. Overall, we present a direct, bidirectional method to profile select redox cysteine modifications based on the unique nucleophilicity of sulfinic acids.

Protein palmitoylation describes the post-translational fatty acyl thioesterification of cellular cysteine residues, and is critical for the localization, trafficking, and compartmentalization of a large number of membrane proteins. This labile thioester modification facilitates a dynamic acylation cycle that directionally traffics key signaling complexes, receptors, and channels to select membrane compartments. Chemical enrichment and advanced mass spectrometry-based proteomics methods have highlighted a pervasive role for palmitoylation across all eukaryotes, including animals, plants, and parasites. Emerging chemical tools promise to open new avenues to study the enzymes, substrates, and dynamics of this distinct post-translational modification.