Biomedical subjects

Sheng-Chung Lee

Publications and source records attributed to Sheng-Chung Lee.

14 recordsLinked to original sources

PubMed2006

Functional cooperation between FACT and MCM helicase facilitates initiation of chromatin DNA replication.

Chromatin is suppressive in nature to cellular enzymes that metabolize DNA, mainly due to the inherent inaccessibility of the DNA template. Despite extensive understanding of the involvement of chromatin-modifying factors in transcription, roles of related activities in DNA replication remain largely elusive. Here, we show that the heterodimeric transcriptional elongation factor FACT (facilitates chromatin transcription) is functionally linked to DNA synthesis. Its involvement in DNA replication is partly mediated by the stable association with the replicative helicase complex, MCM, and further by the coexistence with MCM on replication origin. Furthermore, relying on its nucleosome-reorganizing activity, FACT can facilitate chromatin unwinding by the MCM complex, which is otherwise inert on the nucleosomal template. As a consequence, the physical and functional interaction between FACT and MCM is an important determinant in the proper initiation of DNA replication and S phase in vivo. Together, our findings identify FACT as an integral and conserved component of the endogenous replication machinery, and support a model in which the concerted action of helicase and chromatin-modifying activities promotes chromosome replication.

Chromatin↗

PubMed2006

Solid-phase extraction and elution on diamond (SPEED): a fast and general platform for proteome analysis with mass spectrometry.

This paper presents a new solid-phase extraction and elution platform based on surface-functionalized diamond nanocrystallites. Compared with conventional methods, the platform facilitates purification and concentration of intact proteins and their enzymatic digests for ensuing sodium dodecyl sulfate-polyacrylamide gel electrophoresis or matrix-assisted laser desorption/ionization mass spectrometry analysis without prior removal of the adsorbent. One-pot work flow involving reduction of disulfide bonds, protection of free cysteine residues, washing off residual chemicals, and proteolytic digestion of adsorbed proteins can be performed directly on the particles. The utility and versatility of this protein workup platform were demonstrated with liquid chromatography-electrospray ionization tandem mass spectrometry in proteome analysis of human urine. The proteome analysis of each urine sample can be completed in 8 h.

Amino Acid Sequence↗

PubMed2006

Modulation of nucleosome-binding activity of FACT by poly(ADP-ribosyl)ation.

Chromatin-modifying factors play key roles in transcription, DNA replication and DNA repair. Post-translational modification of these proteins is largely responsible for regulating their activity. The FACT (facilitates chromatin transcription) complex, a heterodimer of hSpt16 and SSRP1, is a chromatin structure modulator whose involvement in transcription and DNA replication has been reported. Here we show that nucleosome binding activity of FACT complex is regulated by poly(ADP-ribosyl)ation. hSpt16, the large subunit of FACT, is poly(ADP-ribosyl)ated by poly(ADP-ribose) polymerase-1 (PARP-1) resulting from physical interaction between these two proteins. The level of hSpt16 poly(ADP-ribosyl)ation is elevated after genotoxic treatment and coincides with the activation of PARP-1. The enhanced hSpt16 poly(ADP-ribosyl)ation level correlates with the dissociation of FACT from chromatin in response to DNA damage. Our findings suggest that poly(ADP-ribosyl)ation of hSpt16 by PARP-1 play regulatory roles for FACT-mediated chromatin remodeling.

Cell Cycle Proteins↗

PubMed2006

CDK-dependent activation of poly(ADP-ribose) polymerase member 10 (PARP10).

Proteins of the poly(ADP-ribose) polymerase (PARP) family play a wide array of functions, covering virtually every aspect of DNA metabolism and function, most notably with the response to DNA damage, transcription, and the maintenance of genomic stability. Here we report the identification and characterization of a novel PARP family member, PARP10 (FLJ14464 or hypothetical protein LOC84875). Overexpression of PARP10 results in loss of cell viability, although down-expression by short hairpin RNA leads to delayed G1 progression and concomitant cell death. PARP10 exists in both cytoplasm and nucleus, but only nucleolar PARP10 acquires CDK-dependent phosphorylation through late-G1 to S phase, and from prometaphase to cytokinesis in the nucleolar organizing regions. The PARP activity of PARP10 depends on phosphorylation by CDK2-cyclin E in vitro. CDK-phosphorylated PARP10 is absent in growth-arrested cells. These results suggest that PARP10 functions in cell proliferation and may serve as a marker for proliferating cells.

Amino Acid Sequence↗

PubMed2004

Functional interaction between nuclear matrix-associated HBXAP and NF-kappaB.

Hepatitis B virus X-associated protein (HBXAP) is a plant homeodomain (PHD) finger-containing protein implicated in transcription regulation. However, the underlying molecular mechanism remains to be defined. Here, we show that HBXAP represses NF-kappaB-mediated gene activation in a dose-dependent manner. Our results showed that HBXAP and NF-kappaB colocalize to the nuclear matrix with specific physical interaction between them. HBXAP may depend on its nuclear matrix localization for its repression of NF-kappaB-mediated gene repression. A specific nuclear matrix targeting sequence of HBXAP was identified. The sequence is included in a region encompassing amino acids 688-722 that could form a coiled-coil structure. The 18-amino acid stretch lies at the core of that structure. The present results showed that either the coiled-coil conformation or the PHD finger domain is crucial for the transcription repression activity of HBXAP on NF-kappaB-mediated gene activation. Taken together, our results suggest that HBXAP may function as a negative regulator for TNF-alpha-induced, NF-kappaB-mediated gene activation.

Amino Acid Sequence↗

PubMed2004

HCMV IE2-mediated inhibition of HAT activity downregulates p53 function.

Targeting of cellular histone acetyltransferases (HATs) by viral proteins is important in the development of virus-associated diseases. The immediate-early 2 protein (IE2) of human cytomegalovirus (HCMV) binds to the tumor suppressor, p53, and inactivates its functions by unknown mechanisms. Here, we show that IE2 binds to the HAT domain of the p53 coactivators, p300 and CREB-binding protein (CBP), and blocks their acetyltransferase activity on both histones and p53. The minimal HAT inactivation region on IE2 involves the N-terminal 98 amino acids. The in vivo DNA binding of p53 and local histone acetylation on p53-dependent promoters are all reduced by IE2, but not by mutant IE2 proteins that lack the HAT inhibition region. Furthermore, the p53 acetylation site mutant, K320/373/382R, retains both DNA binding and promoter transactivation activity in vivo and these effects are repressed by IE2 as well. Together with the finding that only wild-type IE2 exerts an antiapoptotic effect, our results suggest that HCMV IE2 downregulates p53-dependent gene activation by inhibiting p300/CBP-mediated local histone acetylation and that IE2 may have oncogenic activity.

Acetyltransferases↗

PubMed2003

Nek9, a novel FACT-associated protein, modulates interphase progression.

The heterodimeric Spt16-Pob3/DUF/FACT complex is a class of chromatin structure modulators with important roles in replication and transcription. Although regarded as a transcription elongator for chromatin template, little is known about the mode of action and involvement in other molecular processes of the mammalian FACT. Here we report the identification of a novel interacting and functional partner of FACT, Nek9. Nek9 forms a stable, approximately 600-kDa complex with FACT in the interphase nuclei. Its active form is characterized by phosphorylation-dependent electrophoretic mobility shift and phosphorylation at a conserved residue within the activation loop (Thr(210)). When complexed with FACT, Nek9 exhibits markedly elevated phosphorylation on Thr(210). Cell cycle analysis on the Nek9(dsRNAi) cells directly implicated Nek9 in maintaining proper G(1) and S progression, a role temporally correlated to the formation of a phospho-Nek9-FACT complex. Collectively, these observations provide evidence that Nek9, potentially as an active enzymatic partner of FACT, mediates certain FACT-associated cellular processes, which are ultimately essential for interphase progression.

Animals↗

PubMed2003

Differential activation of a C/EBP beta isoform by a novel redox switch may confer the lipopolysaccharide-inducible expression of interleukin-6 gene.

C/EBP beta, a member of the CCAAT/enhancer binding protein (C/EBP) family, is one of the key transcription factors responsible for the induction of a wide array of genes, some of which play important roles in innate immunity, inflammatory response, adipocyte and myeloid cell differentiation, and the acute phase response. Three C/EBP beta isoforms (i.e. LAP*, LAP, and LIP) were known to arise from differential translation initiation and display different functions in gene regulation. C/EBP beta is known to induce interleukin (IL)-6 gene when P388D1 cells are treated with lipopolysaccharide (LPS). Exactly how the transcriptional activities of C/EBP beta isoforms are involved in the regulation of the IL-6 gene remains unclear. Here we report that LPS-induced expression of IL-6 gene in P388D1 cells is mediated by a redox switch-activated LAP*. The intramolecular disulfide bonds of LAP* and LAP have been determined. Among the cysteine residues, amino acid 11 (Cys11) of LAP* plays key roles for determining the overall intramolecular disulfide bonds that form the basis for redox switch regulation. The DNA binding activity and transcriptional activity of LAP* are enhanced under reducing condition. LAP and LIP, lacking 21 and 151 amino acids, respectively, in the N-terminal region, are not regulated in a similar redox-responsive manner. Our results indicate that LAP* is the primary isoform of C/EBP beta that regulates, through a redox switch, the LPS-induced expression of the IL-6 gene.

CCAAT-Enhancer-Binding Protein-beta↗

PubMed2003

Identification and characterization of a novel p300-mediated p53 acetylation site, lysine 305.

Post-translational modifications serve as important regulatory elements in modulating the transcriptional activity of the tumor suppressor protein p53. We have previously reported a tandem mass spectrometry-based method (viz. selected ion tracing analysis) that can be applied to the identification of phosphopeptides as well as exact mapping of the phosphorylated residues within. In this study, we describe the application of the same strategy for the identification of p300 acetyltransferase-mediated acetylation sites on p53. Consistent with the previous finding, lysines 370, 372, 373, 381, and 382 were detected by this modified selected ion tracing method as the target sites of p300 in vitro. Moreover, two novel acetylation sites, Lys-292 and Lys-305, were also found. Immunoblotting using anti-acetyl-Lys-305 antibody confirmed this discovery and demonstrated that Lys-305 could be acetylated by p300 both in vitro and in vivo. We also show that an alanine or glutamine substitution at Lys-305 (K305A or K305Q) suppressed the transcriptional activity of p53, whereas an arginine mutation (K305R) increased the transcriptional activity. Thus, p300 may further regulate the transcriptional activity of p53 through a novel acetylation site, Lys-305.

Acetyltransferases↗

PubMed2003

Functional analysis of the subunits of the chromatin assembly factor RSF.

The human ISWI-containing factor RSF (for remodeling and spacing factor) is composed of two subunits: the ATPase hSNF2H and p325 (Rsf-1), a protein encoded by a novel human gene. We previously showed that RSF mediates nucleosome deposition and generates regularly spaced nucleosome arrays. Here we report the characterization of the largest subunit of RSF, Rsf-1. We found that Rsf-1 is a highly acidic protein containing a plant homology domain. The present study includes the cloning of Rsf-1, the preparation of recombinant RSF, and the dissection of the role of each subunit in the chromatin assembly reaction. The sequence of the gene for Rsf-1 includes a recently characterized cDNA, HBXAP; postulated to be involved in the transcriptional regulation of the hepatitis B virus. HBXAP actually contains a 252-amino-acid truncation of the amino terminus of Rsf-1. Finally, comparison of HBXAP and Rsf-1 properties shows that they are functionally different.

Adenosine Triphosphatases↗

PubMed2002

An RNA helicase, DDX1, interacting with poly(A) RNA and heterogeneous nuclear ribonucleoprotein K.

Heterogeneous nuclear ribonucleoprotein K (hnRNP K) is a multifunctional protein known to be involved in the regulation of transcription, translation, nuclear transport, and signal transduction. To systematically obtain insight into mechanisms of hnRNP K activities, we set out to identify protein factors that interact with hnRNP K by using glutathione S-transferase-hnRNP K affinity chromatography followed by liquid chromatography/mass spectrometry/mass spectrometry analysis. Several partner proteins in the K562 cell lysates were identified through this method. One of them is a DEAD box-containing putative RNA helicase, DDX1. In vitro binding and co-immunoprecipitation studies confirmed the protein-protein interaction between hnRNP K with DDX1, and the region spanning amino acids 1-276 of hnRNP K is apparently responsible for its physical interaction with DDX1. Interestingly, their interaction was disrupted by the addition of poly(C), poly(A), and poly(U) RNA substrates. We found that DDX1 was a homopolymeric poly(A) RNA-binding protein. On the other hand, the ATPase activity of the purified recombinant DDX1 protein was stimulated by these homopolymeric RNAs and yeast total RNA but not by DNA. Moreover, the immunoprecipitated DDX1 complex but not purified DDX1 can unwind double-stranded RNA having single-stranded poly(A) overhangs.

Adenosine Triphosphatases↗

PubMed2002

Nopp140 is a mediator of the protein kinase A signaling pathway that activates the acute phase response alpha1-acid glycoprotein gene.

The acute phase response (APR) in liver during inflammation is one of the well known examples for elucidating the signaling pathways that lead to the combinatorial regulation of gene expression. The APR is exemplified by alpha(1)-acid glycoprotein gene (agp) expression. A number of transcription factors, including CCAAT/enhancer-binding protein beta (C/EBPbeta), glucocorticoid receptor, cAMP-response element-binding protein (CREB), and Nopp140, are known to participate in its induction. The underlying mechanism of Nopp140 and other factors for regulating agp expression remains unclear. Here we demonstrate that protein kinase A (PKA)-dependent phosphorylation of Nopp140, together with C/EBPbeta, induces agp gene expression synergistically. The cooperative activation of the agp gene by Nopp140 and forskolin is sensitive to inhibition by PKI. Results from biochemical and functional characterizations of Nopp140 mutants defective in PKA phosphorylation sites suggest that PKA-dependent Nopp140 phosphorylation is important for its role in agp gene activation. Furthermore, maximal activation of the agp gene by PKA-phosphorylated Nopp140 depends on the presence of CREB and C/EBPbeta. The participation of CREB in the activation is, however, independent of its PKA-mediated phosphorylation. In summary, we demonstrate the existence of a novel Nopp140-mediated PKA signaling pathway that leads to the activation of agp, one of the major acute phase response genes.

Amino Acid Sequence↗

PubMed2002

Transcriptional activation of C/EBPbeta gene by c-Jun and ATF2.

C/EBPbeta is one of the key transcription factors responsible for the induction of a wide array of genes. Like many proto-oncogenes and transcription factors, transcription of C/EBPbeta gene can be induced by multiple extracellular signals. Using nuclear extracts from lipopolysaccharide (LPS)-stimulated mouse liver, five trans-acting factor-binding motifs, URE1 (-376 to -352), URE2 (-253 to -223), URE3 (-220 to -190), URE4 (-123 to -103), and URE5 (-72 to -45) were identified by DNAse I footprinting assays. Competition and supershift analysis of the complexes formed at the URE2 and URE4 indicated that they contain CREB/ATF and AP-1 family factors. Furthermore, recombinant ATF2 and c-Jun proteins from mammalian and bacterial cells can bind to URE2 and URE4 but not URE1. Cotransfection experiments showed that ATF2 and c-Jun activate the C/EBPbeta gene expression cooperatively through URE2 and URE4, and this activation was greatly increased under the treatment of low concentration of anisomycin. During acute phase response, the phosphorylation of c-Jun and ATF2 was found to correlate with C/EBPbeta gene expression. Taken together, our results provide the evidences that both c-Jun and ATF2 are the regulators of C/EBPbeta gene.

Activating Transcription Factor 2↗

PubMed2002

The terminal proteins of linear Streptomyces chromosomes and plasmids: a novel class of replication priming proteins.

The chromosomes of the soil bacteria Streptomyces, unlike those of most other bacteria, are linear DNA molecules. Their telomeres contain long-terminal inverted repeats and covalently bound terminal proteins (TPs). These bacteria also harbour linear plasmids that share the same structural features. In this study, we demonstrated that the TP was covalently bound to the 5' ends as proposed previously. A linear plasmid with chromosomal telomeres was constructed and used to purify the TPs of the Streptomyces coelicolor A3(2) chromosome. A 20 kDa protein and its 10 kDa degradation product were isolated and their sequences determined by mass spectrometry. The coding sequence (tpgC) was about 100 kb from the right end of the chromosome. Two tpg homologues were identified by sequencing the 50kb linear plasmid SLP2 of Streptomyces lividans: tpgSLP2 at 6 kb from the left end and a putative tpg pseudogene at 8 kb from the right. The latter was in a terminal repeat shared by the right end of SLP2 and both ends of the S. lividans chromosome. The lack of the typical Streptomyces codon preference in this open reading frame suggests that it is a pseudogene. The close physical linkage between the tpg genes and their cognate telomeres would favour their cosegregation and co-evolution. All the Tpg polypeptides are similar in length (184-185 amino acids) and sequences, which include a putative helix domain that is homologous to part of the DNA-binding 'thumb' domain of HIV reverse transcriptase, and a putative amphiphilic beta-sheet that may be involved in the observed self-aggregation of the TP and/or the proposed membrane binding.

Amino Acid Sequence↗