Yousef I. Hassan

Biotin and biotinidase deficiency

Biotin is a water-soluble vitamin that serves as an essential coenzyme for five carboxylases in mammals. Biotin-dependent carboxylases catalyze the fixation of bicarbonate in organic acids and play crucial roles in the metabolism of fatty acids, amino acids and glucose. Carboxylase activities decrease substantially in response to biotin deficiency. Biotin is also covalently attached to histones; biotinylated histones are enriched in repeat regions in the human genome and appear to play a role in transcriptional repression of genes and genome stability. Biotin deficiency may be caused by insufficient dietary uptake of biotin, drug–vitamin interactions and, perhaps, by increased biotin catabolism during pregnancy and in smokers. Biotin deficiency can also be precipitated by decreased activities of the following proteins that play critical roles in biotin homeostasis: the vitamin transporters sodium-dependent multivitamin transporter and monocarboxylate transporter 1, which mediate biotin transport in the intestine, liver and peripheral tissues, and renal reabsorption; holocarboxylase synthetase, which mediates the binding of biotin to carboxylases and histones; and biotinidase, which plays a central role in the intestinal absorption of biotin, the transport of biotin in plasma and the regulation of histone biotinylation. Symptoms of biotin deficiency include seizures, hypotonia, ataxia, dermatitis, hair loss, mental retardation, ketolactic acidosis, organic aciduria and also fetal malformations. This review focuses on the deficiencies of both biotin and biotinidase, and the medical management of such cases.

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 [(3)H]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.

A novel, enigmatic histone modification: biotinylation of histones by holocarboxylase synthetase

Holocarboxylase synthetase catalyzes the covalent binding of biotin to histones in humans and other eukaryotes. Eleven biotinylation sites have been identified in histones H2A, H3, and H4. K12-biotinylated histone H4 is enriched in heterochromatin, repeat regions, and plays a role in gene repression. About 30% of the histone H4 molecules are biotinylated at K12 in histone H4 in human fibroblast telomeres. The abundance of biotinylated histones at distinct genomic loci depends on biotin availability. Decreased histone biotinylation decreases life span and stress resistance in Drosophila. Low enrichment of biotinylated histones at transposable elements impairs repression of these elements.

The role of histone H4 biotinylation in the structure of nucleosomes.

Background Post-translational modifications of histones play important roles in regulating nucleosome structure and gene transcription. It has been shown that biotinylation of histone H4 at lysine-12 in histone H4 (K12Bio-H4) is associated with repression of a number of genes. We hypothesized that biotinylation modifies the physical structure of nucleosomes, and that biotin-induced conformational changes contribute to gene silencing associated with histone biotinylation. Methodology/Principal Findings To test this hypothesis we used atomic force microscopy to directly analyze structures of nucleosomes formed with biotin-modified and non-modified H4. The analysis of the AFM images revealed a 13% increase in the length of DNA wrapped around the histone core in nucleosomes with biotinylated H4. This statistically significant (p<0.001) difference between native and biotinylated nucleosomes corresponds to adding approximately 20 bp to the classical 147 bp length of nucleosomal DNA. Conclusions/Significance The increase in nucleosomal DNA length is predicted to stabilize the association of DNA with histones and therefore to prevent nucleosomes from unwrapping. This provides a mechanistic explanation for the gene silencing associated with K12Bio-H4. The proposed single-molecule AFM approach will be instrumental for studying the effects of various epigenetic modifications of nucleosomes, in addition to biotinylation.

N- and C-terminal domains in human holocarboxylase synthetase participate in substrate recognition

Holocarboxylase synthetase (HCS) catalyzes the binding of the vitamin biotin to carboxylases and histones. Carboxylases mediate essential steps in macronutrient metabolism. For example, propionyl-CoA carboxylase (PCC) catalyzes the carboxylation of propionyl-CoA in the metabolism of odd-chain fatty acids. HCS comprises four putative domains, i.e., the N-terminus, the biotin transfer/ATP-binding domain, a putative linker domain, and the C-terminus. Both N- and C-termini are essential for biotinylation of carboxylases by HCS, but the exact functions of these two domains in enzyme catalysis are unknown. Here we tested the hypothesis that N- and C-termini play roles in substrate recognition by HCS. Yeast-two-hybrid (Y2H) assays were used to study interactions between the four domains of human HCS with p67, a PCC-based polypeptide and HCS substrate. Both N- and C-termini interacted with p67 in Y2H assays, whereas the biotin transfer/ATP-binding and the linker domains did not interact with p67. The essentiality of N- and C-termini for interactions with carboxylases was confirmed in rescue experiments with mutant Saccharomyces cerevisiae, using constructs of truncated human HCS. Finally, a computational biology approach was used to model the 3D structure of human HCS and identify amino acid residues that interact with p67. In silico predictions were consistent with observations from Y2H assays and yeast rescue experiments, and suggested docking of p67 near Arg508 and Ser515 within the central domain of HCS.

Holocarboxylase synthetase interacts physically with euchromatic histone-lysine N-methyltransferase, linking histone biotinylation with methylation events☆

Holocarboxylase synthetase (HCS) catalyzes the binding of the vitamin biotin to histones H3 and H4, thereby creating rare histone biotinylation marks in the epigenome. These marks co-localize with K9-methylated histone H3 (H3K9me), an abundant gene repression mark. The abundance of H3K9me marks in transcriptionally competent loci decreases when HCS is knocked down and when cells are depleted of biotin. Here we tested the hypothesis that the creation of H3K9me marks is at least partially explained by physical interactions between HCS and histone-lysine N-methyltransferases. Using a novel in silico protocol, we predicted that HCS-interacting proteins contain a GGGG(K/R)G(I/M)R motif. This motif, with minor variations, is present in the histone-lysine N-methyltransferase EHMT1. Physical interactions between HCS and the N-terminal, ankyrin and SET domains in EHMT1 were confirmed using yeast-two-hybrid assays, limited proteolysis assays and co-immunoprecipitation. The interactions were stronger between HCS and the N-terminus in EHMT1 compared with the ankyrin and SET domains, consistent with the localization of the HCS-binding motif in the EHMT1 N-terminus. HCS has the catalytic activity to biotinylate K161 within the binding motif in EHMT1. Mutation of K161 weakened the physical interaction between EHMT1 and HCS, but it is unknown whether this effect was caused by loss of biotinylation or loss of the motif. Importantly, HCS knockdown decreased the abundance of H3K9me marks in repeats, suggesting that HCS plays a role in creating histone methylation marks in these loci. We conclude that physical interactions between HCS and EHMT1 mediate epigenomic synergies between biotinylation and methylation events.

Epigenetic regulation of chromatin structure and gene function by biotin: are biotin requirements being met?

Histones H2A, H3, and H4 are modified by covalent binding of the vitamin biotin to distinct lysine residues. Binding of biotin to histones is mediated by holocarboxylase synthetase (HCS) and perhaps biotinidase. Biotinylation of lysine-12 in histone H4 (K12BioH4) plays roles in gene repression, stability of repeat regions and transposable elements, and regulation of biotin transporter expression in eukaryotes. Decreased biotinylation of histones in biotin-deficient and HCS-deficient human cells and Drosophila melanogaster impairs stress resistance, life span, and biotin homeostasis. Chromatin is comprised of DNA and DNA-binding proteins, i.e., histones and non-histone proteins. Histones play a predominant role in the folding of DNA into chromatin.1 Five major classes of histones have been identified in mammals: H1, H2A, H2B, H3, and H4. Histones consist of a globular domain and a more flexible amino terminus (histone “tail”). DNA and histones form repetitive nucleoprotein units, the nucleosomes.1 Each nucleosome (“nucleosomal core particle”) consists of 146 base pairs of DNA wrapped around an octamer of core histones (one H3-H3-H4-H4 tetramer and two H2A-H2B dimers). The amino terminal tail of histones protrudes from the nucleosomal surface; covalent modifications of this tail affect the structure of chromatin and form the basis for gene regulation.1–3 Amino acid residues in histone …

Sourdough lactic acid bacteria as antifungal and mycotoxin-controlling agents

Sourdough starter cultures are rich sources of endogenous lactic acid bacteria. The extended shelf lives of sourdough breads are attributed to a large array of organic acids and low-molecular-weight metabolites produced during the fermentation process. Different species belonging to the lactic acid bacteria group of microorganisms, mainly Lactobacillus and Leuconostoc, are increasingly gaining the attention as possible means for inhibiting mold growth in animal feed and human food chains. In addition, certain lactic acid bacteria strains isolated from sourdough starters were also shown to reduce mycotoxins concentrations in contaminated products either by binding or degradation. This short review will summarize the findings in this context that pertain to lactic acid bacteria isolated specifically from sourdough starters and acquaint the reader with the most recent advancements in this bio-preservation trend.

Antifungal Activity of Lactobacillus paracasei subsp. tolerans against Fusarium proliferatum and Fusarium graminearum in a Liquid Culture Setting

Lactobacillus paracasei subsp. tolerans, isolated from a traditional sourdough bread culture and previously shown to have antifungal activity against Fusarium species, was tested for inhibition of growth of Fusarium proliferatum M 5991 and M 5689 and F. graminearum R 4053 in a liquid medium setting. This isolate completely inhibited the growth of F. proliferatum M 5689 and M 5991 and F. graminearum R 4053, whereas such growth was not inhibited in the control in a supernatant agar plate assay. When this isolate was tested using 2M medium (MRS-modified Myro media) known for supporting Fusarium growth and trichothecene production, it was found to inhibit fungal growth but promote mycotoxin production at the same time. The antifungal activity was determined to be the result of organic acids and low pH. The mechanism of the mycotoxin production promotion requires further investigation.

Sodium-Dependent Multivitamin Transporter Gene Is Regulated at the Chromatin Level by Histone Biotinylation in Human Jurkat Lymphoblastoma Cells

The sodium-dependent multivitamin transporter (SMVT) is essential for mediating and regulating biotin entry into mammalian cells. In cells, holocarboxylase synthetase (HCS) mediates covalent binding of biotin to histones; biotinylation of lysine-12 in histone H4 (K12BioH4) causes gene repression. Here we propose a novel role for HCS in sensing and regulating levels of biotin in eukaryotic cells. We hypothesize that nuclear translocation of HCS increases in response to biotin supplementation; HCS then biotinylates histone H4 at SMVT promoters, silencing biotin transporter genes. We show that nuclear translocation of HCS is a biotin-dependent process that might involve tyrosine kinases, histone deacetylases, and histone methyltransferases in human lymphoid (Jurkat) cells. The nuclear translocation of HCS correlated with biotin concentrations in cell culture media; the relative enrichment of both HCS and K12BioH4 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 K12BioH4 at the SMVT promoter was inversely linked to SMVT expression. 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 (K12BioH4 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.