Nonenzymatic biotinylation of histone H2A

Holocarboxylase synthetase (HCS, eukaryotic enzyme) and BirA (prokaryotic) are biotin protein ligases that catalyze the ATP-dependent attachment of biotin to apocarboxylases via the reactive intermediate, bio-5′-AMP. In this study, we examined the in vitro mechanism of biotin attachment to histone H2A in the presence of HCS and BirA. The experiment derives from our observations that HCS is found in the nucleus of cells in addition to the cytoplasm, and it has the ability to attach biotin to histones in vitro (Narang et al., Hum Mol Genet 2004; 13:15–23). Using recombinant HCS or BirA, the rate of biotin attachment was considerably slower with histone H2A than with the biotin binding domain of an apocarboxylase. However, on incubation of recombinant H2A with chemically synthesized bio-5′-AMP, H2A was observed to be rapidly labeled with biotin in the absence of enzyme. Nonenzymatic biotinylation of a truncated apocarboxylase (BCCP87) has been previously reported (Streaker and Beckett, Protein Sci 2006; 15:1928–1935), though at a much slower rate than we observe for H2A. The specific attachment sites of nonenzymatically biotinylated recombinant H2A at different time points were identified using mass spectrometry, and were found to consist of a similar pattern of biotin attachment as seen in the presence of HCS, with preference for lysines in the highly basic N-terminal region of the histone. None of the lysine sites within H2A resembles the biotin attachment consensus sequence seen in carboxylases, suggesting a novel mechanism for histone biotinylation.     

Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18

Holocarboxylase synthetase (HCS) mediates the binding of biotin to lysine (K) residues in histones H2A, H3 and H4; HCS knockdown disturbs gene regulation and decreases stress resistance and lifespan in eukaryotes. We tested the hypothesis that HCS interacts physically with histone H3 for subsequent biotinylation. Co-immunoprecipitation experiments were conducted and provided evidence that HCS co-localizes with histone H3 in human cells; physical interactions between HCS and H3 were confirmed using limited proteolysis assays. Yeast two-hybrid (Y2H) studies revealed that the N-terminal and C-terminal domains in HCS participate in H3 binding. Recombinant human HCS was produced and exhibited biological activity, as evidenced by biotinylation of its known substrate, recombinant p67. Recombinant histone H3.2 and synthetic H3-based peptides were also good targets for biotinylation by recombinant HCS (rHCS) in vitro, based on tracing histone-bound biotin with [³H]biotin, streptavidin and anti-biotin antibody. Biotinylation site-specific antibodies were generated and revealed that both K9 and K18 in H3 were biotinylated by HCS. Collectively, these studies provide conclusive evidence that HCS interacts directly with histone H3, causing biotinylation of K9 and K18. We speculate that the targeting of HCS to distinct regions in human chromatin is mediated by DNA sequence, biotin, RNA, epigenetic marks or chromatin proteins.     

Paradoxical regulation of biotin utilization in brain and liver and implications for inherited multiple carboxylase deficiency

Holocarboxylase synthetase (HCS) catalyzes the biotinylation of five carboxylases in human cells, and mutations of HCS cause multiple carboxylase deficiency (MCD). Although HCS also participates in the regulation of its own mRNA levels, the relevance of this mechanism to normal metabolism or to the MCD phenotype is not known. In this study, we show that mRNA levels of enzymes involved in biotin utilization, including HCS, are down-regulated during biotin deficiency in liver while remaining constitutively expressed in brain. We propose that this mechanism of regulation is aimed at sparing the essential function of biotin in the brain at the expense of organs such as liver and kidney during biotin deprivation. In MCD, it is possible that some of the manifestations of the disease may be associated with down-regulation of biotin utilization in liver because of the impaired activity of HCS and that high dose biotin therapy may in part be important to overcoming the adverse regulatory impact in such organs.     

Holocarboxylase synthetase regulates expression of biotin transporters by chromatin remodeling events at the SMVT locus

The sodium-dependent multivitamin transporter (SMVT) is essential for mediating and regulating biotin entry into mammalian cells. In cells, biotin is covalently linked to histones in a reaction catalyzed by holocarboxylase synthetase (HCS); biotinylation of lysine 12-biotinylated histone H4 (K12Bio H4) causes gene silencing. Here, we propose a novel role for HCS in sensing and regulating levels of biotin in eukaryotic cells. We hypothesized that nuclear translocation of HCS increases in response to biotin supplementation; HCS then biotinylates histone H4 at SMVT promoters, silencing biotin transporter genes. Jurkat lymphoma cells were cultured in media containing 0.025, 0.25, or 10 nmol/l biotin. The nuclear translocation of HCS correlated with biotin concentrations in media; the relative enrichment of both HCS and K12Bio H4 at SMVT promoter 1 (but not promoter 2) increased by 91% in cells cultured in medium containing 10 nmol/l biotin compared with 0.25 nmol/l biotin. This increase of K12Bio H4 at the SMVT promoter decreased SMVT expression by up to 86%. Biotin homeostasis by HCS-dependent chromatin remodeling at the SMVT promoter 1 locus was disrupted in HCS knockdown cells, as evidenced by abnormal chromatin structure (K12Bio H4 abundance) and increased SMVT expression. The findings from this study are consistent with the theory that HCS senses biotin, and that biotin regulates its own cellular uptake by participating in HCS-dependent chromatin remodeling events at the SMVT promoter 1 locus in Jurkat cells.     

Artifactual detection of biotin on histones by streptavidin

Biotinylation is a recent addition to the list of reported posttranslational modifications made to histones. Holocarboxylase synthetase (HCS) and biotinidase have been implicated as biotinylating enzymes. However, the details of the mechanism and the regulation of biotin transfer on and off histones remains unclear. Here we report that in a cell culture system low biotin availability reduces biotinylation of carboxylases, yet apparent biotinylation of histones is unaffected. This is despite biotin depletion having detrimental effects on cell viability and proliferation. Further analysis of the widely used method for detecting biotin on histones, streptavidin blotting, revealed that streptavidin interacts with histones independently of biotin binding. Preincubation of streptavidin with free biotin reduced binding to biotinylated carboxylases but did not block binding to histones. To investigate biotinylation of histones using an alternative detection method independent of streptavidin, incorporation of 14C biotin into biotinylated proteins was analyzed. Radiolabeled biotin was readily detectable on carboxylases but not on histones, implying very low levels of biotin in the nucleus attached to histone proteins (< 0.03% biotinylation). In conclusion, we would caution against the use of streptavidin for investigating histone biotinylation.     

Selectivity in post-translational biotin addition to five human carboxylases

Human holocarboxylase synthetase (HCS) catalyzes linkage of the vitamin biotin to the biotin carboxyl carrier protein (BCCP) domain of five biotin-dependent carboxylases. In the two-step reaction, the activated intermediate, bio-5'-AMP, is first synthesized from biotin and ATP, followed by covalent linkage of the biotin moiety to a specific lysine residue of each carboxylase BCCP domain. Selectivity in HCS-catalyzed biotinylation to the carboxylases was investigated in single turnover stopped flow and quench flow measurements of biotin transfer to the minimal biotin acceptor BCCP fragments of the carboxylases. The results demonstrate that biotinylation of the BCCP fragments of the mitochondrial carboxylases propionyl-CoA carboxylase, pyruvate carboxylase, and methylcrotonoyl-CoA carboxylase is fast and limited by the bimolecular association rate of the enzyme with substrate. By contrast, biotinylation of the acetyl-CoA carboxylase 1 and 2 (ACC1 and ACC2) fragments, both of which are accessible to HCS in the cytoplasm, is slow and displays a hyperbolic dependence on substrate concentration. The correlation between HCS accessibility to biotin acceptor substrates and the kinetics of biotinylation suggests that mitochondrial carboxylase sequences evolved to produce fast association rates with HCS in order to ensure biotinylation prior to mitochondrial import. In addition, the results are consistent with a role for HCS specificity in dictating biotin distribution among carboxylases.     

Microbial biotin protein ligases aid in understanding holocarboxylase synthetase deficiency

The attachment of biotin onto the biotin-dependent enzymes is catalysed by biotin protein ligase (BPL), also known as holocarboxylase synthase HCS in mammals. Mammals contain five biotin-enzymes that participate in a number of important metabolic pathways such as fatty acid biogenesis, gluconeogenesis and amino acid catabolism. All mammalian biotin-enzymes are post-translationally biotinylated, and therefore activated, through the action of a single HCS. Substrate recognition by BPLs occurs through conserved structural cues that govern the specificity of biotinylation. Defects in biotin metabolism, including HCS, give rise to multiple carboxylase deficiency (MCD). Here we review the literature on this important enzyme. In particular, we focus on the new information that has been learned about BPL's from a number of recently published protein structures. Through molecular modelling studies insights into the structural basis of HCS deficiency in MCD are discussed.     

Marginal biotin deficiency is teratogenic in mice and perhaps humans: a review of biotin deficiency during human pregnancy and effects of biotin deficiency on gene expression and enzyme activities in mouse dam and fetus

Recent studies of biotin status during pregnancy provide evidence that a marginal degree of biotin deficiency develops in a substantial proportion of women during normal pregnancy. Several lines of evidence suggest that although the degree of biotin deficiency is not severe enough to produce the classic cutaneous and behavioral manifestations of biotin deficiency, the deficiency is severe enough to produce metabolic derangements in women and may be teratogenic. In studies of mice, a similar degree of biotin deficiency induces characteristic fetal malformations at a high rate. Fetal hepatic biotin content and PCC activity decrease indicating that the fetuses also become biotin deficient. Fetal hepatic acetyl-CoA carboxylase, pyruvate carboxylase, propionyl-CoA carboxylase and beta-methylcrotonyl-CoA carboxylase abundances determined by Western blotting decreased more than the dam holocarboxylase abundances (10% of sufficient vs. 50% of sufficient); however, hepatic mRNA for the carboxylases and for HCS did not change significantly in either dams or fetuses. These observations suggest that maternal biotin deficiency results in a lack of adequate biotin to biotinylate apocarboxylases in the fetus despite the normal expression of genes coding for the apocarboxylases and holocarboxylase synthetase.     

The expression and function of β-1,4-galactosyltransferase-I in dendritic cells

β-1,4-Galactosyltransferase-I (GalTI) is unusual among the galactosyltransferase family, which has two isoforms that differ only in the length of their cytoplasmic domains [1]. In this study, we found that both the long and short isoforms of GalTI were expressed in human monocyte-derived dendritic cells (MoDCs), and localized in the cytoplasm near nucleus and cytomembrane. The expression level of GalTI and cellular adhesion ability was increased when DCs continued to mature. We also demonstrated that the cellular adhesion ability of DCs was inhibited by α-lactalbumin (α-LA) via interference with cell surface GalTI function, suggesting that the adhesion ability was positively correlated with the expression of cell surface long GalTI. α-LA also could inhibit DC-T clustering and CD4⁺ T cell proliferation. Collectively, the data suggests that GalTI might act as a key adhesion molecular participating in T cells-DCs contacts.     

Subcellular localization of adenosine kinase in mammalian cells: The long isoform of AdK is localized in the nucleus

Two isoforms of adenosine kinase (AdK) have been identified in mammalian organisms with the long isoform (AdK-long) containing extra 20-21 amino acids at the N-terminus (NTS). The subcellular localizations of these isoforms are not known and they contain no identifiable targeting sequence. Immunofluorescence labeling of mammalian cells expressing either only AdK-long or both isoforms with AdK-specific antibody showed only nuclear labeling or both nucleus and cytoplasmic labeling, respectively. The AdK-long and -short isoforms fused at the C-terminus with c-myc epitope also localized in the nucleus and cytoplasm, respectively. Fusion of the AdK-long NTS to green fluorescent protein also resulted in its nuclear localization. AdK-long NTS contains a cluster of conserved amino acids (PKPKKLKVE). Replacement of KK in this sequence with either AA or AD abolished its nuclear localization capability, indicating that this cluster likely serves as a nuclear localization signal. AdK in nucleus is likely required for sustaining methylation reactions.     

Substrate recognition characteristics of human holocarboxylase synthetase for biotin ligation

Holocarboxylase synthetase (HCS) is an essential enzyme that catalyzes the incorporation of biotin into apo carboxylase and the biotinylation of the four biotin-dependent carboxylases in the human cell. Deficiency of HCS results in decreased activity of these carboxylases and affects various metabolic processes. Despite the importance of this enzyme, the recognition mechanism of the biotinoyl domain by human HCS (hHCS) has remained unclear. We have developed a method to express hHCS in the baculovirus system and used it to purify catalytically active, full-length hHCS. NMR experiments on the biotinoyl domains from acetyl-CoA carboxylase indicate that when hHCS is added, it recognizes the MKM motif in human and in Escherichia coli with a preference to the human biotinoyl domain. In addition, hHCS can biotinylate the biotinoyl domains from human and E. coli acetyl-CoA carboxylase at similar rates compared to the E. coli biotin protein ligase, BirA, which reacts very slowly with the human biotinoyl domain. We propose that the hHCS has greater substrate acceptability, while the BirA has higher substrate specificity. These results provide insights into substrate recognition by hHCS, which can be distinguished from BirA in this respect.     

Dual targeting of Arabidopsis holocarboxylase synthetase1: a small upstream open reading frame regulates translation initiation and protein targeting

Protein biotinylation is an original and very specific posttranslational modification, compartmented in plants, between mitochondria, plastids, and the cytosol. This reaction modifies and activates few carboxylases committed in key metabolisms and is catalyzed by holocarboxylase synthetase (HCS). The molecular bases of this complex compartmentalization and the relative function of each of the HCS genes, HCS1 and HCS2, identified in Arabidopsis (Arabidopsis thaliana) are mainly unknown. Here, we showed by reverse genetics that the HCS1 gene is essential for plant viability, whereas disruption of the HCS2 gene in Arabidopsis does not lead to any obvious phenotype when plants are grown under standard conditions. These findings strongly suggest that HCS1 is the only protein responsible for HCS activity in Arabidopsis cells, including the cytosolic, mitochondrial, and plastidial compartments. A closer study of HCS1 gene expression enabled us to propose an original mechanism to account for this multiplicity of localizations. Located in the HCS1 messenger RNA 5'-untranslated region, an upstream open reading frame regulates the translation initiation of HCS1 and the subsequent targeting of HCS1 protein. Moreover, an exquisitely precise alternative splicing of HCS1 messenger RNA can regulate the presence and absence of this upstream open reading frame. The existence of these complex and interdependent mechanisms creates a rich molecular platform where different parameters and factors could control HCS targeting and hence biotin metabolism.     

Identification of holocarboxylase synthetase chromatin binding sites in human mammary cell lines using the DNA adenine methyltransferase identification technology

Holocarboxylase synthetase (HCS) is a chromatin protein that is essential for mediating the covalent binding of biotin to histones. Biotinylation of histones plays crucial roles in the repression of genes and repeats in the human genome. We tested the feasibility of DNA adenine methyltransferase identification (DamID) technology to map HCS binding sites in human mammary cell lines. Full-length HCS was fused to DNA adenine methyltransferase (Dam) for subsequent transfection into breast cancer (MCF-7) and normal breast (MCF-10A) cells. HCS docking sites in chromatin were identified by using the unique adenine methylation sites established by Dam in the fusion construct; docking sites were unambiguously identified using methylation-sensitive digestion, cloning, and sequencing. In total, 15 novel HCS binding sites were identified in the two cell lines, and the following 4 of the 15 overlapped between MCF-7 and MCF-10A cells: inositol polyphosphate-5-phosphatase A, corticotropin hormone precursor, ribosome biogenesis regulatory protein, and leptin precursor. We conclude that DamID is a useful technology to map HCS binding sites in human chromatin and propose that the entire set of HCS binding sites could be mapped by combining DamID with microarray technology.     

The polypeptide Syn67 interacts physically with human holocarboxylase synthetase, but is not a target for biotinylation

Holocarboxylase synthetase (HCS) catalyzes the binding of biotin to lysines in carboxylases and histones in two steps. First, HCS catalyzes the synthesis of biotinyl-5′-AMP; second, the biotinyl moiety is ligated to lysine residues. It has been proposed that step two is fairly promiscuous, and that protein biotinylation may occur in the absence of HCS as long as sufficient exogenous biotinyl-5′-AMP is provided. Here, we identified a novel polypeptide (Syn67) with a basic patch of lysines and arginines. Yeast-two-hybrid assays and limited proteolysis assays revealed that both N- and C-termini of HCS interact with Syn67. A potential target lysine in Syn67 was biotinylated by HCS only after arginine-to-glycine substitutions in Syn67 produced a histone-like peptide. We identified a Syn67 docking site near the active pocket of HCS by in silico modeling and site-directed mutagenesis. Biotinylation of proteins by HCS is more specific than previously assumed.