Human holocarboxylase synthetase with a start site at methionine-58 is the predominant nuclear variant of this protein and has catalytic activity

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Abstract

Holocarboxylase synthetase (HLCS) catalyzes the covalent binding of biotin to both carboxylases in extranuclear structures and histones in cell nuclei, thereby mediating important roles in intermediary metabolism, gene regulation, and genome stability. HLCS has three putative translational start sites (methionine-1, -7, and -58), but lacks a strong nuclear localization sequence that would explain its participation in epigenetic events in the cell nucleus. Recent evidence suggests that small quantities of HLCS with a start site in methionine-58 (HLCS58) might be able to enter the nuclear compartment. We generated the following novel insights into HLCS biology. First, we generated a novel HLCS fusion protein vector to demonstrate that methionine-58 is a functional translation start site in human cells. Second, we used confocal microscopy and western blots to demonstrate that HLCS58 enters the cell nucleus in meaningful quantities, and that full-length HLCS localizes predominantly in the cytoplasm but may also enter the nucleus. Third, we produced recombinant HLCS58 to demonstrate its biological activity toward catalyzing the biotinylation of both carboxylases and histones. Collectively, these observations are consistent with roles of HLCS58 and full-length HLCS in nuclear events. We conclude this report by proposing a novel role for HLCS in epigenetic events, mediated by physical interactions between HLCS and other chromatin proteins as part of a larger multiprotein complex that mediates gene repression.

Highlights

► Unambiguous evidence is provided that methionine-58 serves as an in-frame alternative translation site for holocarboxylase synthetase (HLCS58). ► Full-length HLCS and HLCS58 enter the nucleus, but HLCS58 is the predominant variant. ► HLCS58 has biological activity as biotin protein ligase.

Introduction

Holocarboxylase synthetase (HLCS) plays a pivotal role in biotin-dependent metabolic and epigenetic phenomena in humans. In intermediary metabolism, HLCS catalyzes the covalent binding of biotin to five distinct carboxylases [1], [2], [3]. Biotinylated carboxylases are key enzymes in the metabolism of glucose, fatty acids, and leucine [4]. In the regulation of genes by epigenetic phenomena, HLCS translocates to the cell nucleus [5] where it binds to chromatin [6], [7] to catalyze the binding of biotin to histones H1, H3, H4 and, to a lesser extent, H2A [8], [9], [10], [11], [12], [13]. Moreover, evidence suggests that HLCS interacts physically with the methylated cytosine binding protein MeCP2 and the histone H3 K9-methyl transferase EHMT-1 [14] (Yong et al., unpublished).

Biotinylated histones are enriched in transcriptionally repressed loci and repeat sequences [15], [16]. Importantly, evidence suggests a role for K12-biotinylated histone H4 and HLCS in the transcriptional repression of retrotransposons, and that low abundance of K12-biotinylated histone H4 in HLCS- or biotin-deficient cells is linked with activation of retrotransposons and chromosomal abnormalities [17]. It is currently uncertain whether the effects of HLCS in epigenetic pathways are mediated by HLCS-dependent biotinylation of histones or by physical interactions of HLCS with other chromatin proteins such as MeCP2 and EHMT-1. If the latter proved correct, then biotinylation of histones would be a mark to trace loci where HLCS interacts with other chromatin proteins.

Consistent with the important roles of HLCS in intermediary metabolism and epigenetics, no living HLCS null individual has ever been reported, suggesting embryonic lethality. HLCS knockdown studies (∼30% residual activity) produced phenotypes such as decreased life span and heat resistance in Drosophila melanogaster [6] and aberrant gene regulation in human cell lines [16], [17], [18]. Mutations in the human HLCS gene cause a substantial decrease in HLCS activity and metabolic abnormalities [19], [20]. Unless diagnosed and treated early, HLCS deficiency is uniformly fatal [21].

Human HLCS is a single copy gene, which maps to chromosome 21q22.1 [1] and codes for a full-length protein of 726 amino acid with a predicted molecular weight of 81 kDa [22]. Three HLCS transcripts plus additional splicing variants originate in exons 1, 2, and 3 of the gene [22]; methionines-1, -7, and -58 in exons 6 and 7 have bene identified as possible translation start sites [23]. HLCS proteins, migrating with apparent sizes of 62, 64, 76, 82, and 86 kDa in gel electrophoresis, have been detected in human placenta and bovine liver by using anti-HLCS antibodies, but some of the bands might have been caused by protein degradation [23], [24]. Presumably, the 76-kDa, 82 kDa, and 86 kDa bands represent HLCS with translation start sites in mthionine-58, methione-7, and methionine-1, respectively.

A recent study suggests that HLCS with a translation start site in methionine-58 (HLCS58) might enter the cell nucleus [25]. In this study we sought to determine (i) whether methionine-58 in HLCS is a functional translation start site; (ii) whether HLCS58 enters the cell nucleus; and (iii) whether HLCS58 has biotin protein ligase activity in vitro. Human embryonic kidney HEK-293 cells were chosen as model in cell culture studies to facilitate comparisons with a recent report on HLCS distribution in human cells [25].

Section snippets

Plasmids

HLCS was fused to the N-terminus of enhanced green fluorescent protein (GFP) to allow for tracking the subcellular distribution of HLCS. Briefly, full-length HLCS (amino acids 1–726) was PCR amplified using primers 5′-GGGACTCGAGATGGAAGATAGACTCCACATGG-3′ (forward) and 5′-ATTTGAATTCGCCGCCGTTTGGGGAG-3′ (reverse). The PCR product was fused to the N-terminus of plasmid pEGFP-N1 (Clontech, Mountain View, CA, USA) by using XhoI and EcoRI.The plasmid was denoted HLCS-GFP and codes for a fusion protein

Translation of HLCS58 in HEK-293 cells

Studies with HLCS-GFP fusion vectors confirmed that methionine-58 can serve as a translational start site. When HEK-293 cells were transformed with HLCS-GFP (Fig. 1A), both full-length HLCS and HLCS58 were detectable by using anti-GFP as probe; methionine-1 was preferred as translational start site compared with methionine-58 (Fig. 1B, left lane). Note that these analyses were conducted in cytoplasmic extracts, and that shuttling of HLCS58 into the nucleus could also explain the relatively low

Discussion

It has long been suspected that translation of HLCS may start at methionines-1, -7, and -58 [23], [25]. This paper offers the following novel insights into HLCS biology. First, by mutating methionine-58 to leucine we provided unambiguous evidence that methionine-58 serves as an in-frame alternative translation site for HLCS transcripts. Second, we added evidence to a recent report suggesting that HLCS58 is a nuclear protein [25]. Third, full-length HLCS may also enter the nucleus, at least in

Author disclosures

No conflicts of interest.

Acknowledgments

A contribution of the University of Nebraska Agricultural Research Division, supported in part by funds provided through the Hatch Act. Additional support was provided by NIH grants DK063945, DK077816, DK082476 and ES015206, USDA CSREES grant 2006-35200-17138, and by NSF grants MCB 0615831 and EPS 0701892, and by Ministry of Education of China grants 211056 and Shanghai Municipal Education Commission grants S30701.

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