Seongman Kang

Pausing of DNA Synthesis in Vitro at Specific Loci in CTG and CGG Triplet Repeats from Human Hereditary Disease Genes

Several human hereditary neuromuscular disease genes are associated with the expansion of CTG or CGG triplet repeats. The DNA syntheses of CTG triplets ranging from 17 to 180 and CGG repeats from 9 to 160 repeats in length were studied in vitro. Primer extensions using the Klenow fragment of DNA polymerase I, the modified T7 DNA polymerase (Sequenase), or the human DNA polymerase β paused strongly at specific loci in the CTG repeats. The pausings were abolished by heating at 70°C. As the length of the triplet repeats in duplex DNA, but not in single-stranded DNA, was increased, the magnitude of pausings increased. The location of the pause sites was determined by the distance between the site of primer hybridization and the beginning of the triplet repeats. CGG triplet repeats also showed similar, but not identical, patterns of pausings. These results indicate that appropriate lengths of the triplets adopt a non-B conformation(s) that blocks DNA polymerase progression; the resultant idling polymerase may catalyze slippages to give expanded sequences and hence provide the molecular basis for this non-Mendelian genetic process. These mechanisms, if present in human cells, may be related to the etiology of certain neuromuscular diseases such as myotonic dystrophy and Fragile X syndrome.

Cloning, characterization, and properties of seven triplet repeat DNA sequences.

Several neuromuscular and neurodegenerative diseases are caused by genetically unstable triplet repeat sequences (CTG·CAG, CGG·CCG, or AAG·CTT) in or near the responsible genes. We implemented novel cloning strategies with chemically synthesized oligonucleotides to clone seven of the triplet repeat sequences (GTA·TAC, GAT·ATC, GTT·AAC, CAC·GTG, AGG·CCT, TCG·CGA, and AAG·CTT), and the adjoining paper (Ohshima, K., Kang, S., Larson, J. E., and Wells, R. D. (1996) J. Biol. Chem. 271, 16784-16791) describes studies on TTA·TAA. This approach in conjunction with in vivo expansion studies in Escherichia coli enabled the preparation of at least 81 plasmids containing the repeat sequences with lengths of ∼16 up to 158 triplets in both orientations with varying extents of polymorphisms. The inserts were characterized by DNA sequencing as well as DNA polymerase pausings, two-dimensional agarose gel electrophoresis, and chemical probe analyses to evaluate the capacity to adopt negative supercoil induced non-B DNA conformations. AAG·CTT and AGG·CCT form intramolecular triplexes, and the other five repeat sequences do not form any previously characterized non-B structures. However, long tracts of TCG·CGA showed strong inhibition of DNA synthesis at specific loci in the repeats as seen in the cases of CTG·CAG and CGG·CCG (Kang, S., Ohshima, K., Shimizu, M., Amirhaeri, S., and Wells, R. D. (1995) J. Biol. Chem. 270, 27014-27021). This work along with other studies (Wells, R. D. (1996) J. Biol. Chem. 271, 2875-2878) on CTG·CAG, CGG·CCG, and TTA·TAA makes available long inserts of all 10 triplet repeat sequences for a variety of physical, molecular biological, genetic, and medical investigations. A model to explain the reduction in mRNA abundance in Friedreichs ataxia based on intermolecular triplex formation is proposed.

FLEXIBLE DNA : GENETICALLY UNSTABLE CTG.CAG AND CGG.CCG FROM HUMAN HEREDITARY NEUROMUSCULAR DISEASE GENES

The properties of duplex CTG·CAG and CGG·CCG, which are involved in the etiology of several hereditary neurodegenerative diseases, were investigated by a variety of methods, including circularization kinetics, apparent helical repeat determination, and polyacrylamide gel electrophoresis. The bending moduli were 1.13 × 10−19 erg·cm for CTG and 1.27 × 10−19 erg·cm for CGG, ∼40% less than for random B-DNA. Also, the persistence lengths of the triplet repeat sequences were ∼60% the value for random B-DNA. However, the torsional moduli and the helical repeats were 2.3 × 10−19 erg·cm and 10.4 base pairs (bp)/turn for CTG and 2.4 × 10−19 erg·cm and 10.3 bp/turn for CGG, respectively, all within the range for random B-DNA. Determination of the apparent helical repeat by the band shift assay indicated that the writhe of the repeats was different from that of random B-DNA. In addition, molecules of 224–245 bp in length (64–71 triplet repeats) were able to form topological isomers upon cyclization. The low bending moduli are consistent with predictions from crystallographic variations in slide, roll, and tilt. No unpaired bases or non-B-DNA structures could be detected by chemical and enzymatic probe analyses, two-dimensional agarose gel electrophoresis, and immunological studies. Hence, CTG and CGG are more flexible and highly writhed than random B-DNA and thus would be expected to act as sinks for the accumulation of superhelical density.

Β-amyloid precursor protein is a direct cleavage target of HtrA2 serine protease : Implications for the physiological function of HtrA2 in the mitochondria

The processing and metabolism of amyloid precursor protein (APP) is a major interest in Alzheimer disease (AD) research, because not only amyloid β (Aβ) peptide, but also cellular or mitochondrial APP are intimately involved in cellular dysfunction and AD pathogenesis. Here we demonstrate that APP is directly and efficiently cleaved by the HtrA2 serine protease in vitro and in vivo. Using several APP mutants and N-terminal amino acid sequencing, we identified that the HtrA2-mediated APP cleavage product is the C161 fragment encompassing amino acids 535-695 of APP695. The immunofluorescence and subcellular fractionation studies indicate that APP is partly colocalized with HtrA2 in the mitochondria where HtrA2 can cleave APP under normal conditions. The HtrA2-cleaved C161 fragment was detected in the cytosolic fraction; therefore, we postulate that the C161 fragment is released into the cytosol after cleavage of APP by HtrA2. Interestingly, the level of C161 was remarkably decreased in motor neuron degeneration (mnd2) mice in which the serine protease activity of HtrA2 was greatly reduced. These results show that the protease activity of HtrA2 is essential for the production of C161 and that processing of APP into C161 is a natural event occurring under normal physiological conditions. Our study suggests that the direct cleavage of mitochondrial APP by HtrA2 may prevent mitochondrial dysfunction caused by accumulation of APP and that the regulation of HtrA2 protease activity may be a therapeutic target in AD.

Activation of Bak and Bax through c-Abl-Protein Kinase Cδ-p38 MAPK Signaling in Response to Ionizing Radiation in Human Non-small Cell Lung Cancer Cells

Intracellular signaling molecules and apoptotic factors seem to play an important role in determining the radiation response of tumor cells. However, the basis for the link between signaling pathway and apoptotic cell death machinery after ionizing irradiation remains still largely unclear. In this study, we showed that c-Abl-PKCδ-Rac1-p38 MAPK signaling is required for the conformational changes of Bak and Bax during ionizing radiation-induced apoptotic cell death in human non-small cell lung cancer cells. Ionizing radiation induced conformational changes and subsequent oligomerizations of Bak and Bax, dissipation of mitochondrial membrane potential, and cytochrome c release from mitochondria. Small interference (siRNA) targeting of Bak and Bax effectively protected cells from radiation-induced mitochondrial membrane potential loss and apoptotic cell death. p38 MAPK was found to be selectively activated in response to radiation treatment. Inhibition of p38 MAPK completely suppressed radiation-induced Bak and Bax activations, dissipation of mitochondrial membrane potential, and cell death. Moreover, expression of a dominant negative form of protein kinase Cδ (PKCδ) or siRNA targeting of PKCδ attenuated p38 MAPK activation and conformational changes of Bak and Bax. In addition, ectopic expression of RacN17, a dominant negative form of Rac1, markedly inhibited p38 MAPK activation but did not affect PKCδ activation. Upon stimulation of cells with radiation, PKCδ was phosphorylated dramatically on tyrosine. c-Abl-PKCδ complex formation was also increased in response to radiation. Moreover, siRNA targeting of c-Abl attenuated radiation-induced PKCδ and p38 MAPK activations, and Bak and Bax modulations. These data support a notion that activation of the c-Abl-PKCδ-Rac1-p38 MAPK pathway in response to ionizing radiation signals conformational changes of Bak and Bax, resulting in mitochondrial activation-mediated apoptotic cell death in human non-small cell lung cancer cells.

Spread of nontuberculous mycobacteria from 1993 to 2006 in Koreans

In Korea, the prevalence of nontuberculous mycobacterial (NTM) pulmonary disease has risen, observed primarily in immunocompetent patients with or without preexisting lung disease. The purpose of this study was to determine the frequency of various species of NTM isolates from respiratory specimens in a single institution over a 14‐year period in Korea. All samples referred to our reference laboratory over a 14‐year period in Korea were analyzed. From 1993 to 2000 our laboratory used conventional NTM identification methods, and from 2001 we adapted PCR‐restrictionfragment length polymorphism analysis(PRA). A total of 17,915 isolates were collected from 1993 to 2006. The most frequently isolated organisms were M. avium complex (n=11,705, 65%), M. abscessus (n=2,076, 11.59%), M. fortuitum complex (n=1,279, 7.14%). M. chelonae complex (n=1,134, 6.33%), M. kansasii (n=762, 4.25%), M. szulgai (n=139, 0.78%), M. celatum (n=87, 0.49%), M. scrofulaceum (n=18, 0.10%) and M. marium (n=11, 0.06%). J. Clin. Lab. Anal. 22:415–420, 2008.

USP7, a ubiquitin-specific protease, interacts with ataxin-1, the SCA1 gene product.

Spinocerebellar ataxia type 1 (SCA1) is an autosomal-dominant neurodegenerative disorder characterized by ataxia and progressive motor deterioration. SCA1 has been known to associate with elongated polyglutamine tract in ataxin-1, the SCA1 gene product. Using the yeast two-hybrid system, we have found that USP7, a ubiquitin-specific protease, binds to ataxin-1. Further experiments with deletion mutants indicated that the C-terminal region of ataxin-1 was essential for the interaction. Liquid beta-galactosidase assay and coimmunoprecipitation experiments revealed that the strength of the interaction between USP7 and ataxin-1 is influenced by the length of the polyglutamine tract in the ataxin-1; weaker interaction was observed in mutant ataxin-1 with longer polyglutamine tract and USP7 was not recruited to the mutant ataxin-1 aggregates in the Purkinje cells of SCA1 transgenic mice. Our results suggest that altered function of the ubiquitin system can be involved in the pathogenesis of spinocerebellar ataxia type 1.

Alzheimer's disease-associated amyloid beta interacts with the human serine protease HtrA2/Omi.

Amyloid beta (Abeta), a principle component of the cerebral plaques found in the brains of patients with Alzheimers disease (AD), is a pivotal factor implicated in the pathogenesis of AD. Recent reports show that not only extracellular Abeta but also intracellular Abeta induces neuronal apoptosis; however, the mechanism remains to be elucidated. Using yeast two-hybrid assays, we found that Abeta interacts with HtrA2/Omi, an essential human serine protease with proapoptotic activity. Additionally, we mapped the C-terminal region containing the PDZ domain of HtrA2/Omi as the binding determinant for Abeta? The interaction of Abeta with HtrA2/Omi was further confirmed through in vivo co-immunoprecipitation assay in HEK293 cells. This study suggests the possibility that the accumulation of intracellular Abeta and a function of proapoptotic protease, HtrA2/Omi are correlated.

Curcumin attenuates glutamate-induced HT22 cell death by suppressing MAP kinase signaling

Glutamate induces cell death by upsetting the cellular redox homeostasis, termed oxidative glutamate toxicity, in a mouse hippocampal cell line, HT22. Extracellular signal-regulated kinases (ERK) 1/2 are known key players in this process. Here we characterized the roles of both MAP kinases and cell cycle regulators in mediating oxidative glutamate toxicity and the neuroprotective mechanisms of curcumin in HT22 cells. c-Jun N-terminal kinase (JNK) and p38 kinase were activated during the glutamate-induced HT22 cell death, but at a later stage than ERK activation. Treatment with a JNK inhibitor, SP600125, or a p38 kinase inhibitor, SB203580, partly attenuated this cell death. Curcumin, a natural inhibitor of JNK signaling, protected the HT22 cells from glutamate-induced death at nanomolar concentrations more efficiently than SP600125. These doses of curcumin affected neither the level of intracellular glutathione nor the level of reactive oxygen species, but inactivated JNK and p38 significantly. Moreover, curcumin markedly upregulated a cell-cycle inhibitory protein, p21cip1, and downregulated cyclin D1 levels, which might help the cell death prevention. Our results suggest that curcumin has a neuroprotective effect against oxidative glutamate toxicity by inhibiting MAP kinase signaling and influencing cell-cycle regulation.

Report from the workshop on Pallister-Hall syndrome and related phenotypes

A one day workshop was convened on the NIH campus on March 1, 1996, in Bethesda, Maryland to discuss emerging clinical and molecular information on Pallister-Hall syndrome (PHS) and related disorders. PHS is a pleiotropic autosomal dominant disorder comprising hypothalamic hamartoma, pituitary dysfunction, central polydactyly, and visceral malformations. The goals of the meeting were to update participants in the latest clinical and research findings in the disorder, review the history and evolution of the understanding of the phenotype, determine diagnostic criteria for PHS, and make recommendations for clinical evaluation of individuals affected by PHS. These topics were addressed by several speakers and data were displayed from several of the large pedigrees of autosomal dominant PHS. 37 refs., 4 tabs.