The ribosomal protein S26 regulates p53 activity in response to DNA damage.

Ribosomal proteins have emerged as novel regulators of the Mdm2-p53 feedback loop, especially in the context of ribosomal stress. RPS26 is a recently identified Diamond-Blackfan Anemia-related ribosomal protein and its role in p53 activation has not been previously explored. In this study we found knockdown of RPS26 induced p53 stabilization and activation via a RPL11-dependent mechanism, resulting in p53-dependent cell growth inhibition. Moreover, RPS26 has the ability to interact with Mdm2 and inhibits Mdm2-mediated p53 ubiquitination that leads to p53 stabilization upon overexpression. Importantly, we discovered that RPS26 knockdown impaired p53's ability to transcriptionally activate its target genes in response to DNA damage, without affecting its stability. Accordingly, the cells lost the ability to induce G2/M cell cycle arrest. We further found that upon RPS26 knockdown, the DNA damage induced recruitment of p53 to the promoters of its target genes and p53 acetylation were both greatly reduced. In addition, RPS26 can interact with p53 independent of Mdm2 and coexist in a complex with p53 and p300. These data establish a role of RPS26 in DNA damage response by directly influencing p53 transcriptional activity, and suggest that RPS26 acts distinctively in different scenarios of p53 activation. Our finding also implicates p53 transcriptional activity control as an important mechanism of p53 regulation by ribosomal proteins.

T-cell response to human papillomavirus type 58 L1, E6, And E7 peptides in women with cleared infection, cervical intraepithelial neoplasia, or invasive cancer.

Human papillomavirus type 58 (HPV-58) exists in a relatively high prevalence in certain parts of the world, including East Asia. This study examined the T-cell response to HPV-58 L1, E6, and E7 peptides among women with cleared infection, cervical intraepithelial neoplasia grade 2 (CIN2) or CIN3, or invasive cervical cancer (ICC). Peptides found to be reactive in the in vitro peptide binding assay or mouse-stimulating study were tested with a gamma interferon (IFN-gamma) enzyme-linked immunospot (ELISPOT) assay to detect peptide-specific responses from the peripheral blood mononuclear cells (PBMC) collected from 91 HPV-58-infected women (32 with cleared infection, 16 CIN2, 15 CIN3, and 28 ICC). Four HLA-A11-restricted HPV-58 L1 peptides, located at amino acid positions 296 to 304, 327 to 335, 101 to 109, and 469 to 477, showed positive IFN-gamma ELISPOT results and were mainly from women with cleared infection. Two HLA-A11-restricted E6 peptides (amino acid positions 64 to 72 and 94 to 102) and three HLA-A11-restricted E7 peptides (amino acid positions 78 to 86, 74 to 82, and 88 to 96) showed a positive response. A response to E6 and E7 peptides was mainly observed from subjects with CIN2 or above. One HLA-A2-restricted E6 peptide, located at amino acid position 99 to 107, elicited a positive response in two CIN2 subjects. One HLA-A24-restricted L1 peptide, located at amino acid position 468 to 476, also elicited a positive response in two CIN2 subjects. In summary, this study has identified a few immunogenic epitopes for HPV-58 E6 and E7 proteins. It is worthwhile to further investigate whether responses to these epitopes have a role in clearing an established cervical lesion.

Identification of hemopexin in tear film.

Human tear fluid is a complex mixture of aqueous lipids, proteins, enzymes, and other biochemical and cellular elements. By conventional comparative proteomic approaches, we investigated the proteome in human tear fluid and compared the tear protein profile of normal control subjects with that of patients suffering from the ocular inflammatory disease vernal keratoconjunctivitis (VKC). Collected tear samples were directed to two-dimensional polyacrylamide gel electrophoresis protein separation and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry peptide identification. Six differentially expressed proteins-interleukin 4, phospholipase A2, albumin, lactoferrin, hemopexin, and lipocalin-were displayed. Hemopexin had not been reported previously in tear film. Enzyme-linked immunosorbent assay confirmed that hemopexin concentrations were significantly higher in VKC tear samples and increased with disease stages. The results implied clinical interest of hemopexin in the tear proteome and eye diseases.

Novel hypoglycemic effects of Ganoderma lucidum water-extract in obese/diabetic (+db/+db) mice.

In this study, we evaluated the pharmacological effects of Ganoderma lucidum (G. lucidum) (water-extract) (0.003, 0.03 and 0.3g/kg, 4-week oral gavage) consumption using the lean (+db/+m) and the obese/diabetic (+db/+db) mice. Different physiological parameters (plasma glucose and insulin levels, lipoproteins-cholesterol levels, phosphoenolpyruvate carboxykinase (PEPCK), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA reductase) and isolated aorta relaxation of both species were measured and compared. G. lucidum (0.03 and 0.3g/kg) lowered the serum glucose level in +db/+db mice after the first week of treatment whereas a reduction was observed in +db/+m mice only fed with 0.3g/kg of G. lucidum at the fourth week. A higher hepatic PEPCK gene expression was found in +db/+db mice. G. lucidum (0.03 and 0.3g/kg) markedly reduced the PEPCK expression in +db/+db mice whereas the expression of PEPCK was attenuated in +db/+m mice (0.3g/kg G. lucidum). HMG CoA reductase protein expression (in both hepatic and extra-hepatic organs) and the serum insulin level were not altered by G. lucidum. These data demonstrate that G. lucidum consumption can provide beneficial effects in treating type 2 diabetes mellitus (T2DM) by lowering the serum glucose levels through the suppression of the hepatic PEPCK gene expression.

Characterization of the biologically important interaction between troponin C and the N-terminal region of troponin I.

The N-terminal regulatory region of Troponin I, residues 1-40 (TnI 1-40, regulatory peptide) has been shown to have a biologically important function in the interactions of troponin I and troponin C. Truncated analogs corresponding to shorter versions of the N-terminal region (1-30, 1-28, 1-26) were synthesized by solid-phase methodology. Our results indicate that residues 1-30 of TnI comprises the minimum sequence to retain full biological activity as measured in the acto-S1-TM ATPase assay. Binding of the TnI N-terminal regulatory peptides (TnI 1-30 and the N-terminal regulatory peptide (residues 1-40) labeled with the photoprobe benzoylbenzoyl group, BBRp) were studied by gel electrophoresis and photochemical cross-linking experiments under various conditions. Fluorescence titrations of TnI 1-30 were carried out with TnC mutants that carry a single tryptophan fluorescence probe in either the N- or C-domain (F105W, F105W/C domain (88-162), F29W and F29W/N domain (1-90)) (Fig. 1). Low Kd values (Kd < 10(-7) M) were obtained for the interaction of F105W and F105W/C domain (88-162) with TnI 1-30. However, there was no observable change in fluorescence when the fluorescence probe was located at the N-domain of the TnC mutant (F29W and F29W/N domain (1-90)). These results show that the regulatory peptide binds strongly to the C-terminal domain of TnC.

Structural and functional studies on Troponin I and Troponin C interactions.

Troponin I (TnI) peptides (TnI inhibitory peptide residues 104-115, Ip; TnI regulatory peptide resides 1-30, TnI1-30), recombinant Troponin C (TnC) and Troponin I mutants were used to study the structural and functional relationship between TnI and TnC. Our results reveal that an intact central D/E helix in TnC is required to maintain the ability of TnC to release the TnI inhibition of the acto-S1-TM ATPase activity. Ca(2+)-titration of the TnC-TnI1-30 complex was monitored by circular dichroism. The results show that binding of TnI1-30 to TnC caused a three-folded increase in Ca(2+) affinity in the high affinity sites (III and IV) of TnC. Gel electrophoresis and high performance liquid chromatography (HPLC) studies demonstrate that the sequences of the N- and C-terminal regions of TnI interact in an anti-parallel fashion with the corresponding N- and C-domain of TnC. Our results also indicate that the N- and C-terminal domains of TnI which flank the TnI inhibitory region (residues 104 to 115) play a vital role in modulating the Ca(2+)- sensitive release of the TnI inhibitory region by TnC within the muscle filament. A modified schematic diagram of the TnC/TnI interaction is proposed.

Characterization of tissue-specific LIM domain protein (FHL1C) which is an alternatively spliced isoform of a human LIM-only protein (FHL1).

We have cloned and characterized another alternatively spliced isoform of the human four-and-a-half LIM domain protein 1 (FHL1), designated FHL1C. FHL1C contains a single zinc finger and two tandem repeats of LIM domains at the N-terminus followed by a putative RBP-J binding region at the C-terminus. FHL1C shares the same N-terminal two-and-a-half LIM domains with FHL1 but different C-terminal protein sequences. Due to the absence of the exon 4 in FHL1C, there is a frame-shift in the 3' coding region. Sequence analysis indicated that FHL1C is the human homolog of murine KyoT2. The Northern blot and RT-PCR results revealed that FHL1 is widely expressed in human tissues, including skeletal muscle and heart at a high level, albeit as a relatively low abundance transcript in brain, placenta, lung, liver, kidney, pancreas, and testis. In contrast, FHL1C is specifically expressed in testis, skeletal muscle, and heart at a relatively low level compared with FHL1. The expression of FHL1C transcripts was also seen in aorta, left atrium, left, and right ventricles of human heart at low level. Immunoblot analysis using affinity-purified anti-FHL1C antipeptide antibodies confirmed a 20 kDa protein of FHL1C in human skeletal muscle and heart. Unlike FHL1B, which is another FHL1 isoform recently reported by our group and localized predominantly in the nucleus [Lee et al., 1999], FHL1C is localized both in the nucleus and cytoplasm of mammalian cell.

In silico studies of energy metabolism of normal and diseased heart.

Biotechnology research is developing into genomic analyses that involve the simultaneous monitoring of thousands of genes. The development of various bioinformatics resources that provide efficient access to information is necessary. We have used single-pass sequencing of randomly selected cDNA clones to generate expressed sequence tags (ESTs). These ESTs data has been widely used to study gene expression in a variety of heart libraries [1, 21]. Data annotation on our recent finding allows us to construct the profiles of genes in the energy metabolizing pathways (glycolysis and glycogen metabolism) that are expressed in heart cDNA libraries. In silico studies of genes of energy metabolism yields data that are consistent with results derived from conventional metabolic experiments. The change in gene profiles describing the metabolism of diseased hearts is also presented here.

Protein-protein interaction of FHL2, a LIM domain protein preferentially expressed in human heart, with hCDC47.

In the yeast two-hybrid library screening, the heart-specific FHL2 protein was found to interact with hCDC47. In vitro interaction study between FHL2 protein and hCDC47 was demonstrated. From the results of domain studies by the yeast two-hybrid assay, the second and third LIM domains in conjunction with the first half LIM domain of FHL2 were identified to be important in binding with hCDC47. Besides, in Northern blot hybridization of human cancer cell lines, the highest FHL2 mRNA expression was detected in colorectal adenocarcinoma SW480 and HeLa cell S3. Our results imply that FHL2 protein may associate with cancer development and may act as a molecular adapter to form a multicomplex with hCDC47 in the nucleus, thus it plays an important role in the specification or maintenance of the terminal differentiated phenotype of heart muscle cells.

In vivo and in vitro association of 14-3-3 epsilon isoform with calmodulin: implication for signal transduction and cell proliferation.

Using a yeast two-hybrid screen, human 14-3-3 epsilon protein was found to interact with human calmodulin. In vitro binding assay between human 14-3-3 epsilon protein/peptide and calmodulin was demonstrated by native gel electrophoresis, and the interaction was shown to be calcium dependent. Our results, along with the association of the 14-3-3 epsilon protein with other signaling proteins, suggest that the 14-3-3 protein could provide a link between signal transduction and cell proliferation.

Characterization of the human 36-kDa carboxyl terminal LIM domain protein (hCLIM1).

We characterized a human cDNA clone encoding a 36-kDa carboxyl terminal LIM domain protein with a PDZ domain at the amino terminal. This full-length cDNA clone has a predicted open reading frame (ORF) of 329 amino-acid residues. The ORF of this cDNA encodes the human homolog of rat CLP36, and the putative protein is named human 36-kDa carboxyl terminal LIM domain protein (hCLIM1, nomenclature approved by the HUGO/GDB Nomenclature Committee). The hCLIM1 probe was used to hybridize with poly(A)+ RNA of various human tissues. Strong signals were detected in heart and skeletal muscle; moderate signals were detected in spleen, small intestine, colon, placenta, and lung; weaker levels were detected in liver, thymus, kidney, prostate, and pancreas; and no observable signals were detected in brain, testis, ovary, and peripheral blood leukocytes. The hCLIM1 gene was studied by fluorescence in situ hybridization (FISH), somatic cell hybrid analysis, and radiation hybrid mapping, and it is located at the human chromosome 10q26.

Developmental regulation of 14-3-3 epsilon isoform in rat heart.

Human heart cDNA sequencing yielded a cDNA clone that is similar in DNA and amino acid sequences to that of mouse 14-3-3 epsilon isoform. The 6xHis-tagged H1433 epsilon recombinant protein was expressed in Escherichia coli and its size was approximately 30 kDa. From Northern blot results with human multiple tissues, human skeletal muscle was found to have the highest level of h1433 epsilon mRNA expression, whereas Northern blots of human cancer cell lines detected the highest mRNA level of h1433 epsilon in colorectal adenocarcinoma SW480. The protein expression level of h1433 epsilon and Raf-1 is found to be regulated coordinately during rat heart development, and their protein expression was highest from 14.5 to 16.5 days postcoitum.

Photochemical cross-linking between native rabbit skeletal troponin C and benzoylbenzoyl-troponin I inhibitory peptide, residues 104-115.

The troponin I (TnI) inhibitory region (residues 104-115) was synthesized with alpha-14C-labeled Gly-104 and a covalently linked benzoylbenzoyl (BB) moiety at the N terminus to yield a photoactivatable radioactive peptide (BBIp). BBIp was cross-linked to rabbit skeletal muscle troponin C (TnC) to locate the binding site on TnC. The TnC/BBIp mixture was subjected to photolysis in aqueous buffer at pH 7.5 in the presence or absence of Ca2+. A covalent (1:1) cross-linked protein-peptide complex (TnC.BBIp) was isolated in both cases. The cross-linked complex was digested with trypsin, and the peptide fragments were separated by reversed-phase high performance liquid chromatography. The radioactive cross-linked peptide was isolated and further characterized by peptide sequencing and mass spectrometry before and after cyanogen bromide cleavage. The results indicated that Met-155 of TnC was cross-linked to the BB moiety of BBIp in either the presence or absence of Ca2+. The biological activity of both the BBIp peptide and the cross-linked TnC.BBIp complex was studied and a model of the TnC.inhibitory peptide complex was derived using molecular dynamic and energy minimization calculations.

Biologically important interactions between synthetic peptides of the N-terminal region of troponin I and troponin C.

The interaction between troponin I and troponin C plays a critical role in the regulation of muscle contraction. In this study the interaction between troponin C (TnC) and the N-terminal region of TnI was investigated by the synthesis of three TnI peptides (residues 1-40/Rp, 10-40, and 20-40). The regulatory peptide (Rp) on binding to TnC prevents the ability of TnC to release the inhibition of the acto-S1-tropomyosin ATPase activity caused by TnI or the TnI inhibitory peptide (Ip), residues 104-115. A stable complex between TnC and Rp in the presence of Ca2+ was demonstrated by polyacrylamide gel electrophoresis in the presence of 6 M urea. Rp was able to displace TnI from a preformed TnI.TnC complex. In the absence of Ca2+, Rp was unable to maintain a complex with TnC in benign conditions of polyacrylamide gel electrophoresis which demonstrates the Ca(2+)-dependent nature of this interaction. Size-exclusion chromatography demonstrated that the TnC.Rp complex consisted of a 1:1 complex. The results of these studies have shown that the N-terminal region of TnI (1-40) plays a critical role in modulating the Ca(2+)-sensitive release of TnI inhibition by TnC.