Cheuk Yu Lee

Molecular cloning and characterization of FHL2, a novel LIM domain protein preferentially expressed in human heart

A full-length cDNA clone encoding a novel LIM-only protein was isolated and sequenced from a human fetal heart cDNA library. This full-length clone consists of 1416 base pairs and has a predicted open reading frame (ORF) encoding 279 amino acids. The ORF of this polypeptide codes for the human heart-specific four and a half LIM-only protein 2 (FHL2). It possesses an extra zinc finger that is a half LIM domain and four repeats of LIM domain. When the human FHL2 cDNA probe was used to hybridize with poly-A RNA of various human tissues, a very strong signal could be seen in heart tissues, and only moderately low signals could be detected in placenta, skeletal muscle and ovary. Virtually no signal could be detected in brain, lung, liver, kidney, pancreas, spleen, thymus, prostate, testis, small intestine, colon or peripheral blood leukocyte. FHL2 was mapped to chromosome 2q12-q13 by fluorescent in-situ hybridization (FISH).

Paeoniae Radix, a Chinese herbal extract, inhibit hepatoma cells growth by inducing apoptosis in a p53 independent pathway.

Paeoniae Radix (PR) is the root of traditional Chinese Herb named Paeonia lactiflora Pallas, which is commonly used to treat liver diseases in China for centuries. Several earlier studies have indicated that PR has anticancer growth activities, however the mechanism underlying these activities was unclear and remained to be elucidated. In this study, we evaluated the molecular mechanism of the effect of PR on human hepatoma cell lines, HepG2 and Hep3B. Our results showed that the water-extract of Paeoniae Radix (PRE) had inhibitory effect on the growth of both HepG2 and Hep3B cell lines. The induction of internucleosomal DNA fragmentation and chromatin condensation appearance, and accumulation of sub-G1 phase of cell cycle profile in PRE treated hepatoma cells evidenced that the cytotoxicity of PRE to the hepatoma cells is through activation of the cell death program, apoptosis. The activation of apoptosis by PRE is independent of the p53 pathway as Hep3B cell is p53-deficient. In addition, the differential gene expression of PRE treated HepG2 was examined by cDNA microarray technology and RT-PCR analysis. We found that the gene expression of BNIP3 was up-regulated while ZK1, RAD23B, and HSPD1 were down-regulated during early apoptosis of the hepatoma cell mediated by PRE. The elucidation of the drug targets of PR on inhibition of tumor cells growth should enable further development of PR for liver cancer therapy.

Chromosomal mapping, tissue distribution and cDNA sequence of four-and-a-half LIM domain protein 1 (FHL1).

We have isolated and sequenced a human heart cDNA clone encoding a novel LIM-only protein. This full-length cDNA clone has a predicted open reading frame (ORF) encoding 280 amino acids. The ORF of this cDNA codes for a LIM-only protein that possesses four repeats of LIM domain and an extra zinc finger and this putative protein is named four-and-a-half LIM domain protein 1 (FHL1). FHL1 is unique when compared with other LIM-only proteins because it possesses an odd number of zinc fingers. When the FHL1 cDNA probe was used to hybridize with poly-(A) RNA of various human tissues, a very strong signal was detected in skeletal muscle, a moderate one in the heart; only weak signals were associated with the placenta, ovary, prostate, testis, small intestine, colon and spleen, and virtually no signal could be detected in brain, lung, liver, kidney, pancreas, thymus and peripheral blood leukocytes. The FHL1 gene was located to human chromosome at Xq27.2 by somatic cell hybrid mapping, fluorescent in situ hybridization (FISH) and radiation hybrid mapping.

Protein–protein interaction of FHL3 with FHL2 and visualization of their interaction by green fluorescent proteins (GFP) two-fusion fluorescence resonance energy transfer (FRET)

LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation and remodeling of the cell cytoskeleton. Human Four‐and‐a‐half LIM‐only protein 3 (FHL3) is a type of LIM‐only protein that contains four tandemly repeated LIM motifs with an N‐terminal single zinc finger (half LIM motif). FHL3 expresses predominantly in human skeletal muscle. In this report, FHL3 was shown to be a novel interacting partner of FHL2 using the yeast two‐hybrid assay. Furthermore, site‐directed mutagenesis of FHL3 indicated that the LIM2 of FHL3 is the essential LIM domain for interaction with FHL2. Green fluorescent protein (GFP) was used to tag FHL3 in order to study its distribution during myogenesis. Our result shows that FHL3 was localized in the focal adhesions and nucleus of the cells. FHL3 mainly stayed in the focal adhesion during myogenesis. Moreover, using site‐directed mutagenesis, the LIM1 of FHL3 was identified as an essential LIM domain for its subcellular localization. Mutants of GFP have given rise to a novel technique, two‐fusion fluorescence resonance energy transfer (FRET), in the determination of protein–protein interaction at particular subcellular locations of eukaryotic cells. To determine whether FHL2 and FHL3 can interact with one another and to locate the site of this interaction in a single intact mammalian cell, we fused FHL2 and FHL3 to different mutants of GFP and studied their interactions using FRET. BFP/GFP fusion constructs were cotransfected into muscle myoblast C2C12 to verify the colocalization and subcellular localization of FRET. We found that FHL2 and FHL3 were colocalized in the mitochondria of the C2C12 cells and FRET was observed by using an epi‐fluorescent microscope equipped with an FRET specific filter set. J. Cell. Biochem. 80:293–303, 2001.

High-Dose Oral Erythromycin Decreased the Incidence of Parenteral Nutrition-Associated Cholestasis in Preterm Infants

Background & Aims: Feeding intolerance because of functional gastrointestinal dysmotility and parenteral nutrition-associated cholestasis (PNAC) are common problems in preterm, very-low-birth-weight (VLBW) infants. This double-blind, randomized, placebo-controlled study aimed to assess the effectiveness of “high-dose” oral erythromycin as a prokinetic agent in decreasing the incidence of PNAC. Two secondary end points, including the time to achieve full enteral feeding and the duration of parenteral nutrition, were also evaluated. Methods: Infants consecutively admitted to the neonatal unit were randomized to receive erythromycin (12.5 mg/kg/dose every 6 hours for 14 days) or an equivalent volume of normal saline (placebo) if they attained less than half the total daily fluid intake (<75 mL/kg/day) as milk feeds on day 14 of life. Results: Of 182 VLBW infants enrolled, 91 received erythromycin. The incidence of PNAC was significantly lower in erythromycin-treated infants (18/91) compared with placebo infants (37/91; P = .003). Treated infants achieved full enteral nutrition significantly earlier (mean, 10.1; SE, 1.7 days; P < .001), and the duration of parenteral nutrition was also significantly decreased by 10 days (P < .001). Importantly, fewer infants receiving erythromycin had 2 or more episodes of septicemia (n = 4) compared with placebo patients (n = 13, P = .03). No serious adverse effect was associated with erythromycin treatment. Conclusions: High-dose oral erythromycin can be considered as a rescue measure for VLBW infants who fail to establish adequate enteral nutrition and in whom anatomically obstructive pathologies of the gastrointestinal tract have been excluded.

Translocation of a human focal adhesion LIM‐only protein, FHL2, during myofibrillogenesis and identification of LIM2 as the principal determinants of FHL2 focal adhesion localization

LIM domain proteins are found to be important regulators in cell growth, cell fate determination, cell differentiation, and remodeling of the cell cytoskeleton. Human Four-and-a-half LIM-only protein 2 (FHL2) is expressed predominantly in human heart and is only slightly expressed in skeletal muscle. Since FHL2 is an abundant protein in human heart, it may play an important role in the regulation of cell differentiation and myofibrillogenesis of heart at defined subcellular compartment. Therefore, we hypothesized that FHL2 act as a multi-functional protein by the specific arrangement of the LIM domains of FHL2 and that one of the LIM domains of FHL2 can function as an anchor and localizes it into a specific subcellular compartment in a cell type specific manner to regulate myofibrillogenesis. From our results, we observed that FHL2 is localized at the focal adhesions of the C2C12, H9C2 myoblast as well as a nonmyogenic cell line, HepG2 cells. Colocalization of vinculin-CFP and FHL2-GFP at focal adhesions was also observed in cell lines. Site-directed mutagenesis, in turn, suggested that the second LIM domain-LIM2 is essential for its specific localization to focal adhesions. Moreover, FHL2 was observed along with F-actin and focal adhesion of C2C12 and H9C2 myotubes. Finally, we believe that FHL2 moves from focal adhesions and then stays at the Z-discs of terminally differentiated heart muscle.

Interaction of the heart‐specific LIM domain protein, FHL2, with DNA‐binding nuclear protein, hNP220

Using a yeast two‐hybrid library screen, we have identified that the heart specific FHL2 protein, four‐and‐a‐half LIM protein 2, interacted with human DNA‐binding nuclear protein, hNP220. Domain studies by the yeast two‐hybrid interaction assay revealed that the second LIM domain together with the third and the fourth LIM domains of FHL2 were responsible to the binding with hNP220. Using green fluorescent protein (GFP)‐FHL2 and blue fluorescent protein (BFP)‐hNP220 fusion proteins co‐expressed in the same cell, we demonstrated a direct interaction between FHL2 and hNP220 in individual nucleus by two‐fusion Fluorescence Resonance Energy Transfer (FRET) assay. Besides, Western blot analysis using affinity‐purified anti‐FHL2 antipeptide antibodies confirmed a 32‐kDa protein of FHL2 in heart only. Virtually no expression of FHL2 protein was detected in brain, liver, lung, kidney, testis, skeletal muscle, and spleen. Moreover, the expression of FHL2 protein was also detectable in the human diseased heart tissues. Our results imply that FHL2 protein can shuttle between cytoplasm and nucleus and may act as a molecular adapter to form a multicomplex with hNP220 in the nucleus, thus we speculate that FHL2 may be particularly important for heart muscle differentiation and the maintenance of the heart phenotype. J. Cell. Biochem. 84: 556–566, 2002.

Isolation and characterization of a human heart cDNA encoding a new member of the small heat shock protein family — HSPL27☆

A novel cDNA clone was isolated from a human adult heart cDNA library. This cDNA clone is similar to the small heat shock protein (smhsp) in both DNA and amino acid sequences, especially in the conserved region. Sequence analysis has shown that the putative novel smhsp, named 27 kDa heat-shock-protein-like protein (HSPL27) is a protein of 241 amino acids with a deduced molecular mass of 26.7 kDa and a deduced pI of 8.0. We have expressed the HSPL27 in E. coli and the expressed protein was found to be present in the soluble fraction of the bacterial cell lysate. Chromosomal mapping data shows that the HSPL27 gene is located at human chromosome 5q11.2.

A novel cDNA encoding for a lim domain protein located at human chromosome 14q32 as a candidate for leukemic translocation

A full‐length cDNA clone encoding a zinc finger protein was isolated and sequenced. This full‐length clone consists of 728 bp and has a predicted open reading frame (ORF) encoding 208 amino acids. The ORF of this polypeptide codes for the human cysteine‐rich protein 2 (HCRP2) and has an amino acid sequence that is 92.8% identical to its rat homolog (RCRP2). HCRP2 was mapped to chromosome 14q32, which is a hot spot of translocation in tumor development, by fluorescent in situ hybridization (FISH).

Identification of a cDNA encoding 6-phosphogluconate dehydrogenase from a human heart cDNA library

A full-length cDNA clone for human 6-phosphogluconate dehydrogenase (PGD) was isolated from a human adult heart cDNA library. The clone encoded an open reading frame of 483 amino acids. When the amino acid sequences of human PGD and sheep PGD were aligned, 94.2% identity between these two proteins was found. Its calculated molecular weight is 53,149 daltons. The predicted isoelectric point is 6.85. When the secondary structure of human PGD was examined by the PROSIS software, 36% α-helix and 9% β-sheet were found.