The mitochondrial proteome comprises 1000 to 1500 proteins, in addition to proteins for which the mitochondrial localization is uncertain. About 800 diseases have been linked with mutations in mitochondrial proteins. We devised a cell survival assay for assessing the mitochondrial localization in a high-throughput format. This protocol allows us to assess the mitochondrial localization of proteins and their mutants, and to identify drugs and nutrients that modulate the mitochondrial targeting of proteins. The assay works equally well for proteins directed to the outer mitochondrial membrane, inner mitochondrial membrane mitochondrial and mitochondrial matrix, as demonstrated by assessing the mitochondrial targeting of the following proteins: carnitine palmitoyl transferase 1 (consensus sequence and R123C mutant), acetyl-CoA carboxylase 2, uncoupling protein 1 and holocarboxylase synthetase. Our screen may be useful for linking the mitochondrial proteome with rare diseases and for devising drug- and nutrition-based strategies for altering the mitochondrial targeting of proteins.

Holocarboxylase synthetase (HLCS) is the sole protein-biotin ligase in the human proteome. HLCS has key regulatory functions in intermediary metabolism, including fatty acid metabolism, and in gene repression through epigenetic mechanisms. The objective of this study was to identify food-borne inhibitors of HLCS that alter HLCS-dependent pathways in metabolism and gene regulation. When libraries of extracts from natural products and chemically pure compounds were screened for HLCS inhibitor activity, resveratrol compounds in grape materials caused an HLCS inhibition of >98% in vitro. The potency of these compounds was piceatannol>resveratrol>piceid. Grape-borne compounds other than resveratrol metabolites also contributed toward HLCS inhibition, e.g., p-coumaric acid and cyanidin chloride. HLCS inhibitors had meaningful effects on body fat mass. When Drosophila melanogaster brummer mutants, which are genetically predisposed to storing excess amounts of lipids, were fed diets enriched with grape leaf extracts and piceid, body fat mass decreased by more than 30% in males and females. However, Drosophila responded to inhibitor treatment with an increase in the expression of HLCS, which elicited an increase in the abundance of biotinylated carboxylases in vivo. We conclude that mechanisms other than inhibition of HLCS cause body fat loss in flies. We propose that the primary candidate is the inhibition of the insulin receptor/Akt signaling pathway.

Acetyl‐CoA carboxylases (ACC) 1 and 2 are key enzymes in fatty acid (FA) synthesis and repression of FA degradation, respectively. Holocarboxylase synthetase (HLCS) catalyzes the covalent binding of the essential coenzyme biotin to ACCs. We hypothesized that dietary inhibitors of HLCS cause a loss of biotinylated ACCs, leading to a lean phenotype. The PEKISH library of natural compounds was screened for HLCS inhibitor activity in vitro; the screen suggested that resveratrol and its metabolites piceatannol and piceid inhibited HLCS by >90% at nutritionally relevant low micromolar levels (P<0.05 vs. vehicle, by ANOVA & posthoc, n=3). When NIH3T3 fibroblasts were treated with 25 µM piceatannol the levels of biotinylated ACC 1 and 2 decreased by 22% compared to controls. When Drosophila melanogaster brummer mutants, which are genetically predisposed to accumulate body fat, were fed diets supplemented with resveratrol compounds, body fat mass decreased by 50% (P<0.05, n=4) in male and female flies. We conclude that resveratrol compounds are potent inhibitors of HLCS‐dependent biotinylation of ACC 1 and 2, thereby contributing toward body fat loss in Drosophila melanogaster brummer mutants. Future studies will include mammalian models.Grant Funding Source: Supported by ARD Hatch, NIH, and BMBF.

The role of holocarboxylase synthetase (HLCS) in catalyzing the covalent binding of biotin to the five biotin-dependent carboxylases in humans is well established, as are the essential roles of these carboxylases in the metabolism of fatty acids, the catabolism of leucine, and gluconeogenesis. This review examines recent discoveries regarding the roles of HLCS in assembling a multiprotein gene repression complex in chromatin. In addition, emerging evidence suggests that the number of biotinylated proteins is far larger than previously assumed and includes members of the heat-shock superfamily of proteins and proteins coded by the ENO1 gene. Evidence is presented linking biotinylation of heat-shock proteins HSP60 and HSP72 with redox biology and immune function, respectively, and biotinylation of the two ENO1 gene products MBP-1 and ENO1 with tumor suppression and glycolysis, respectively.

About 40% of the hotspots for meiotic recombination contain the degenerate consensus sequence 5'-CCNCCNTNNCCNC-3'. Here we present a novel protocol for enriching hotspot sequences from digested genomic DNA by using biotinylated oligonucleotides and streptavidin-coated magnetic beads. The captured hotspots can be released by simple digestion with restriction enzymes for subsequent characterization by second generation sequencing or PCR. The capture protocol specifically enriches hotspot sequences, judged by using fluorophore-conjugated synthetic oligonucleotides and synthetic double-stranded oligonucleotides in combination with PCR. The capture protocol enriches single-stranded DNA, denatured double-stranded DNA, and large fragments (>3000 bp) of digested plasmid DNA with good efficacy. No false positive and false negatives were detected when enriching digested DNA from human cell cultures and primary human cells. The protocol can probably be adapted to enriching sequences other than the hotspot sequence by altering the sequence in the capture oligonucleotide. We intend to apply this protocol in studies assessing effects of micronutrient status on meiotic recombination events in human sperm.

Acetyl‐CoA carboxylases (ACC) 1 and 2 depend on biotin as a coenzyme and catalyze the carboxylation of acetyl‐CoA to malonyl‐CoA. Malonyl‐CoA produced by cytoplasmic ACC1 and mitochondrial ACC2 is a precursor in fatty acid (FA) synthesis and an inhibitor of mitochondrial FA oxidation, respectively. We hypothesized that ACCs are checkpoints in adipocyte differentiation, and used human mesenchymal stem cells (hMSC) and murine 3T3‐L1 preadipocytes to test our hypothesis. The expression of ACC2 increased by 1500% in differentiating hMSC compared with non‐differentiating hMSC, judged by qRT‐PCR and western blot. This increase preceded the increase of adipocyte marker genes FABP4 and PPARy. Treatment of hMSCs with grape leaf extract (GLE) inhibited the differentiation into adipocytes judged by the abundance of PPARy/FABP4 mRNA and staining of lipid droplets with Oil Red‐O. Likewise, treatment of 3T3‐L1 cells with the microbial ACC inhibitor soraphen A inhibited differentiation, judged by decreased lipid accumulation. Treatment of transgenic fruit flies, predisposed to storing excess body lipids, with GLE decreased body lipids by ~50%. In future studies, we will use mutagenesis to determine which of the two ACCs is the critical checkpoint in differentiation. We conclude that ACCs are checkpoints in adipocyte differentiation and that manipulation of ACC activity decreases body fat.Grant Funding Source: ARD Hatch, NIFA, and NIH

Biotin serves as a covalently bound coenzyme in five human carboxylases; biotin is also attached to histones H2A, H3, and H4, although the abundance of biotinylated histones is low. Biotinylation of both carboxylases and histones is catalyzed by holocarboxylase synthetase. Human biotin requirements are unknown. Recommendations for adequate intake of biotin are based on the typical intake of biotin in an apparently healthy population, which is only a crude estimate of the true intake due to analytical problems. Importantly, intake recommendations do not take into account possible effects of biotin deficiency on impairing genome stability. Recent studies suggest that biotin deficiency causes de-repression of long terminal repeats, thereby causing genome instability. While it was originally proposed that these effects are caused by loss of biotinylated histones, more recent evidence suggests a more immediate role of holocarboxylase synthetase in forming multiprotein complexes in chromatin that are important for gene repression. Holocarboxylase synthetase appears to interact physically with the methyl-CpG-binding domain protein 2 and, perhaps, histone methyl transferases, thereby creating epigenetic synergies between biotinylation and methylation events. These observations might offer a mechanistic explanation for some of the birth defects seen in biotin-deficient animal models.