Biomedical subjects

Young-Jun Park

Publications and source records attributed to Young-Jun Park.

10 recordsLinked to original sources

PubMed2006

A carboxylesterase from the thermoacidophilic archaeon Sulfolobus solfataricus P1; purification, characterization, and expression.

The carboxylesterase, a 34 kDa monomeric enzyme, was purified from the thermoacidophilic archaeon Sulfolobus solfataricus P1. The optimum temperature and pH were 85 degrees C and 8.0, respectively. The enzyme showed remarkable thermostability: 41% of its activity remained after 5 days of incubation at 80 degrees C. In addition, the purified enzyme exhibited stability against denaturing agents, including various detergents, urea, and organic solvents. The enzyme has broad substrate specificity towards various PNP esters and short acyl chain triacylglycerols such as tributyrin (C4:0). Among the PNP esters tested, the best substrate was PNP-caprylate (C8) with Km and kcat values of 71 microM and 14,700 s(-1), respectively. The carboxylesterase gene consisted of 915 bp corresponding to 305 amino acid residues. We demonstrated that active recombinant S. solfataricus carboxylesterase could be expressed in Escherichia coli. The enzyme was identified as a serine esterase belonging to mammalian hormone-sensitive lipases (HSL) family and contained a catalytic triad composed of serine, histidine, and aspartic acid in the active site.

Amino Acid Sequence↗

PubMed2006

Purifications and characterizations of a ferredoxin and its related 2-oxoacid:ferredoxin oxidoreductase from the hyperthermophilic archaeon, Sulfolobus solfataricus P1.

The coenzyme A-acylating 2-oxoacid:ferredoxin oxidoreductase and ferredoxin (an effective electron acceptor) were purified from the hyperthermophilic archaeon, Sulfolobus solfataricus P1 (DSM1616). The purified ferredoxin is a monomeric protein with an apparent molecular mass of approximately 11 kDa by SDS-PAGE and of 11,180+/-50 Da by MALDI-TOF mass spectrometry. Ferredoxin was identified to be a dicluster, [3Fe-4S][4Fe-4S], type ferredoxin by spectrophotometric and EPR studies, and appeared to be zinc-containing based on the shared homology of its N-terminal sequence with those of known zinc-containing ferredoxins. On the other hand, the purified 2-oxoacid: ferredoxin oxidoreductase was found to be a heterodimeric enzyme consisting of 69 kDa alpha and 34 kDa beta subunits by SDS-PAGE and MALDI-TOF mass spectrometry. The purified enzyme showed a specific activity of 52.6 units/mg for the reduction of cytochrome c with 2-oxoglutarate as substrate at 55 degrees C, pH 7.0. Maximum activity was observed at 70 degrees C and the optimum pH for enzymatic activity was 7.0 -8.0. The enzyme displays broad substrate specificity toward 2-oxoacids, such as pyruvate, 2-oxobutyrate, and 2-oxoglutarate. Among the 2-oxoacids tested (pyruvate, 2-oxobutyrate, and 2-oxoglutarate), 2-oxoglutarate was found to be the best substrate with Km and kcat values of 163 microM and 452 min(-1), respectively. These results provide useful information for structural studies on these two proteins and for studies on the mechanism of electron transfer between the two.

Amino Acid Sequence↗

PubMed2006

The structure of nucleosome assembly protein 1.

Nucleosome assembly protein 1 (NAP-1) is an integral component in the establishment, maintenance, and dynamics of eukaryotic chromatin. It shuttles histones into the nucleus, assembles nucleosomes, and promotes chromatin fluidity, thereby affecting the transcription of many genes. The 3.0 A crystal structure of yeast NAP-1 reveals a previously uncharacterized fold with implications for histone binding and shuttling. A long alpha-helix is responsible for homodimerization via a previously uncharacterized antiparallel non-coiled-coil, and an alpha/beta domain is implicated in protein-protein interaction. A nuclear export sequence that is embedded in the dimerization helix is almost completely masked by an accessory domain that contains several target sites for casein kinase II. The four-stranded antiparallel beta-sheet that characterizes the alpha/beta domain is found in all histone chaperones, despite the absence of homology in sequence, structural context, or quaternary structure. To our knowledge, this is the first structure of a member of the large NAP family of proteins and suggests a mechanism by which the shuttling of histones to and from the nucleus is regulated.

Amino Acid Motifs↗

PubMed2006

Structure and function of nucleosome assembly proteins.

Homologues of nucleosome assembly protein 1 (NAP1) have been identified in all eukaryotes. Although initially identified as histone chaperones and chromatin-assembly factors, additional functions include roles in tissue-specific transcription regulation, apoptosis, histone shuttling, and cell-cycle regulation, and extend beyond those of a simple chaperone and assembly factor. NAP1 family members share a structurally conserved fold, the NAP domain. Here we review current knowledge of the NAP family of proteins within the context of the recently determined crystal structure of the NAP1 family's first representative, NAP1 from yeast.

Active Transport, Cell Nucleus↗

PubMed2005

Structure and dynamic properties of nucleosome core particles.

It is now widely recognized that the packaging of genomic DNA, together with core histones, linker histones, and other functional proteins into chromatin profoundly influences nuclear processes such as transcription, replication, DNA repair, and recombination. Whereas earlier structural studies portrayed nucleosomes (the basic repeating unit of chromatin) as monolithic and static macromolecular assemblies, we now know that they are highly dynamic and capable of extensive crosstalk with the cellular machinery. Histone variants have evolved to locally alter chromatin structure, whereas histone chaperones and other cellular factors promote histone exchange and chromatin fluidity. Both of these phenomena likely facilitate interconversion between different chromatin states that show varying degrees of transcriptional activity.

Animals↗

PubMed2004

Nucleosome assembly protein 1 exchanges histone H2A-H2B dimers and assists nucleosome sliding.

Eukaryotic chromatin is highly dynamic and turns over rapidly even in the absence of DNA replication. Here we show that the acidic histone chaperone nucleosome assembly protein 1 (NAP-1) from yeast reversibly removes and replaces histone protein dimer H2A-H2B or histone variant dimers from assembled nucleosomes, resulting in active histone exchange. Transient removal of H2A-H2B dimers facilitates nucleosome sliding along the DNA to a thermodynamically favorable position. Histone exchange as well as nucleosome sliding is independent of ATP and relies on the presence of the C-terminal acidic domain of yeast NAP-1, even though this region is not required for histone binding and chromatin assembly. Our results suggest a novel role for NAP-1 (and perhaps other acidic histone chaperones) in mediating chromatin fluidity by incorporating histone variants and assisting nucleosome sliding. NAP-1 may function either untargeted (if acting alone) or may be targeted to specific regions within the genome through interactions with additional factors.

Cell Cycle Proteins↗

PubMed2004

Nucleosomes containing the histone variant H2A.Bbd organize only 118 base pairs of DNA.

H2A.Bbd is an unusual histone variant whose sequence is only 48% conserved compared to major H2A. The major sequence differences are in the docking domain that tethers the H2A-H2B dimer to the (H3-H4)(2) tetramer; in addition, the C-terminal tail is absent in H2A.Bbd. We assembled nucleosomes in which H2A is replaced by H2A.Bbd (Bbd-NCP), and found that Bbd-NCP had a more relaxed structure in which only 118+/-2 bp of DNA is protected against digestion with micrococcal nuclease. The absence of fluorescence resonance energy transfer between the ends of the DNA in Bbd-NCP indicates that the distance between the DNA ends is increased significantly. The Bbd docking domain is largely responsible for this behavior, as shown by domain-swap experiments. Bbd-containing nucleosomal arrays repress transcription from a natural promoter, and this repression can be alleviated by transcriptional activators Tax and CREB. The structural properties of Bbd-NCP described here have important implications for the in vivo function of this histone variant and are consistent with its proposed role in transcriptionally active chromatin.

Amino Acid Sequence↗

PubMed2004

A new fluorescence resonance energy transfer approach demonstrates that the histone variant H2AZ stabilizes the histone octamer within the nucleosome.

Nucleosomes are highly dynamic macromolecular complexes that are assembled and disassembled in a modular fashion. One important way in which this dynamic process can be modulated is by the replacement of major histones with their variants, thereby affecting nucleosome structure and function. Here we use fluorescence resonance energy transfer between fluorophores attached to various defined locations within the nucleosome to dissect and compare the structural transitions of a H2A.Z containing and a canonical nucleosome in response to increasing ionic strength. We show that the peripheral regions of the DNA dissociate from the surface of the histone octamer at relatively low ionic strength, under conditions where the dimer-tetramer interaction remains unaffected. At around 550 mm NaCl, the (H2A-H2B) dimer dissociates from the (H3-H4)(2) tetramer-DNA complex. Significantly, this latter transition is stabilized in nucleosomes that have been reconstituted with the essential histone variant H2A.Z. Our studies firmly establish fluorescence resonance energy transfer as a valid method to study nucleosome stability, and shed new light on the biological function of H2A.Z.

Animals↗

PubMed2003

Preferential binding of the histone (H3-H4)2 tetramer by NAP1 is mediated by the amino-terminal histone tails.

The yeast nucleosome assembly protein 1 (yNAP1) participates in many diverse activities, such as the assembly of newly synthesized DNA into chromatin and the rearrangement of nucleosomes during transcriptional activation. yNAP1 does not require ATP hydrolysis to perform these functions and is a valuable tool for in vitro chromatin assembly. Using recombinant histone complexes, we show that yNAP1 has a preference for binding the (H3-H4)2 tetramer over the (H2A-H2B) dimer. We find that the loss of the histone tails abrogates this preference for H3 and H4, and we demonstrate a direct interaction between yNAP1 and the amino-terminal tails of H3 and H4. yNAP1 binds to one histone fold domain, thus specifying the stoichiometry of the complexes formed with the histone dimer and tetramer. Finally, we provide evidence that the acidic carboxyl-terminal region of yNAP1, although dispensable for nucleosome assembly in vitro, contributes to binding via structure-independent electrostatic interactions. Our results are consistent with recent mechanistic investigations of NAP1 and expand our understanding of the histone chaperone family of assembly factors.

Animals↗

PubMed2003

Structure and dynamics of nucleosomal DNA.

The last five years have seen exciting advances in our understanding of the structure of the nucleosome core particle, the basic repeating unit in all eukaryotic chromatin. A picture emerges in which nucleosomal DNA, while distorted and compacted fivefold by tight interactions with the histone octamer core, is at the same time highly dynamic and adaptable. Here, we summarize the salient features from recent structural studies of nucleosome core particles (both published and unpublished) that concern the structure and dynamics of nucleosomal DNA, and the nature of protein-DNA interactions. Current mechanisms for chromatin remodeling and nucleosome sliding are discussed in light of new structural evidence. Finally, techniques to study nucleosome stability and ultimately dynamics are introduced.

Binding Sites↗