Biomedical subjects

Shengbing Huang

Publications and source records attributed to Shengbing Huang.

3 recordsLinked to original sources

PubMed2005

Rtt106p is a histone chaperone involved in heterochromatin-mediated silencing.

Epigenetic inheritance of heterochromatin structure is an important cellular process whose mechanism remains elusive. In this article, we describe the identification of nine enhancers of the silencing defect of a Saccharomyces cerevisiae-PCNA mutant by screening a library of approximately 4,700 viable yeast deletion mutants. Of the nine mutants identified, six (hir1, hir3, sas2, sas4, sas5, and sir1) were previously known to reduce silencing synergistically with a mutation in Cac1p, the large subunit of chromatin assembly factor 1 (CAF-1). The predicted gene products that are affected in three other mutants (nam7, msh2, and rtt106) have not been implicated previously in silencing. Characterization of the rtt106Delta allele revealed that it synergistically reduced heterochromatin silencing when combined with a mutation in Cac1p but not with a mutation in Asf1p (a histone H3 and H4 chaperone). Moreover, Rtt106p interacted with histones H3 and H4 both in vitro and in vivo, and it displayed a nucleosome assembly activity in vitro. Furthermore, Rtt106p interacts with CAF-1 physically through Cac1p. These biochemical and genetic data indicate that Rtt106p is a previously uncharacterized histone chaperone connecting S phase to epigenetic inheritance.

Chromatin Assembly and Disassembly↗

PubMed2005

cDNA cloning, functional expression and cellular localization of rat liver mitochondrial electron-transfer flavoprotein-ubiquinone oxidoreductase protein.

A membrane-bound protein was purified from rat liver mitochondria. After being digested with V8 protease, two peptides containing identical 14 amino acid residue sequences were obtained. Using the 14 amino acid peptide derived DNA sequence as gene specific primer, the cDNA of correspondent gene 5'-terminal and 3'-terminal were obtained by RACE technique. The full-length cDNA that encoded a protein of 616 amino acids was thus cloned, which included the above mentioned peptide sequence. The full length cDNA was highly homologous to that of human ETF-QO, indicating that it may be the cDNA of rat ETF-QO. ETF-QO is an iron sulfur protein located in mitochondria inner membrane containing two kinds of redox center: FAD and [4Fe-4S] center. After comparing the sequence from the cDNA of the 616 amino acids protein with that of the mature protein of rat liver mitochondria, it was found that the N terminal 32 amino acid residues did not exist in the mature protein, indicating that the cDNA was that of ETF-QOp. When the cDNA was expressed in Saccharomyces cerevisiae with inducible vectors, the protein product was enriched in mitochondrial fraction and exhibited electron transfer activity (NBT reductase activity) of ETF-QO. Results demonstrated that the 32 amino acid peptide was a mitochondrial targeting peptide, and both FAD and iron-sulfur cluster were inserted properly into the expressed ETF-QO. ETF-QO had a high level expression in rat heart, liver and kidney. The fusion protein of GFP-ETF-QO co-localized with mitochondria in COS-7 cells.

Amino Acid Sequence↗

PubMed2003

Functional expression and processing of rat choline dehydrogenase precursor.

Choline dehydrogenase (CHDH, EC 1.1.99.1) was purified from rat liver mitochondria, and the amino terminal sequence was determined and used to clone a full-length cDNA encoding a protein precursor (CHDHp) of 599 amino acids (64kDa). Sequence analysis identified a possible processing site that meets the requirements of IMP in comparison to the previously determined N-terminal sequence of mature rat CHDH. This suggested that the precursor might be processed in the intermembrane space. Confocal imaging showed that expression of the CHDHp-GFP fusion gene in NIH-3T3 cells led to fusion proteins being targeted to mitochondria. In addition, expression of a recombinant version of the CHDHp gene in Saccharomyces cerevisiae led to enrichment of the target protein in the mitochondrial inner membrane. The expressed protein conferred choline dehydrogenase activity, suggesting that both functional domains (FAD and the iron sulfur cluster) were properly assembled and that the mature CHDH was appropriately located in the inner mitochondria membrane.

3T3 Cells↗