Heon Man Lim

A “master” in base unpairing during isomerization of a promoter upon RNA polymerase binding

Isomerization of a closed to open complex of a promoter upon RNA polymerase binding involves base unpairing at the −10 region. After potassium permanganate sensitivity of unpaired thymine residues, we studied base unpairing at the −10 region during isomerization upon RNA polymerase binding at the P1 and P3 promoters of the gal operon. Substitution of adenine by 2-amino purine (2-AP) at the invariable A⋅T base pair at the −11 position of P1 and P3 prevented unpairing not only at that position but also at the other downstream positions, suggesting a “master” role of the adenine base at −11 of the template strand in overall base unpairing. 2-AP at −11 did not inhibit the formation of RNA polymerase⋅promoter complex and subsequent isomerization of the polymerase. Substitution of adenine by 2-AP at several other positions did not affect thymine unpairing. Changing the position of the amino group from C6 in adenine to C2 in 2-AP is mutational only at the master switch position, −11.

Extracellular hydrogen peroxide contributes to oxidative glutamate toxicity

Oxidative glutamate toxicity is characterized by the inhibition of cystine uptake, the depletion of intracellular glutathione, and increased levels of intracellular reactive oxygen species, factors that lead to neuronal injury. We found that the presence of extracellular catalase protected cultured neuronal cells, such as HT22, SH-SY5Y and PC12 cells, from glutamate-induced cytotoxicity. Extracellular hydrogen peroxide (H₂O₂) accumulated in a time- and concentration-dependent manner in HT22 cells during prolonged exposure to glutamate. To investigate the involvement of NADPH oxidase in glutamate-induced H₂O₂ generation, we used small interference RNA (siRNA). Knockdown of Nox2 and Nox4 expression reduced H₂O₂ accumulation and increased cell survival. siRNA specific for Nox4 reduced the production of H₂O₂ by ~74% compared with control siRNA. Furthermore, H₂O₂ accumulation was also suppressed by U0126, a MEK/ERK inhibitor, in a concentration-dependent manner. These results suggest that glutamate triggers the Nox-dependent generation of extracellular H₂O₂ via ERK1/2 activation, which contributes to oxidative glutamate toxicity.

Cnu, a Novel oriC-Binding Protein of Escherichia coli

We have found, using a newly developed genetic method, a protein (named Cnu, for oriC-binding nucleoid-associated) that binds to a specific 26-base-pair sequence (named cnb) in the origin of replication of Escherichia coli, oriC. Cnu is composed of 71 amino acids (8.4 kDa) and shows extensive amino acid identity to a group of proteins belonging to the Hha/YmoA family. Cnu was previously discovered as a protein that, like Hha, complexes with H-NS in vitro. Our in vivo and in vitro assays confirm the results and further suggest that the complex formation with H-NS is involved in Cnu/Hha binding to cnb. Unlike the hns mutants, elimination of either the cnu or hha gene did not disturb the growth rate, origin content, and synchrony of DNA replication initiation of the mutants compared to the wild-type cells. However, the cnu hha double mutant was moderately reduced in origin content. The Cnu/Hha complex with H-NS thus could play a role in optimal activity of oriC.

Asynchronous basepair openings in transcription initiation: CRP enhances the rate-limiting step

The mechanism of isomerization (basepair openings) during transcription initiation by RNA polymerase at the galP1 promoter of Escherichia coli was investigated by 2‐aminopurine (2,AP) fluorescence. The fluorescence of 2,AP is quenched in DNA duplex and enhanced when the basepair is distorted or deformed. The increase of 2,AP fluorescence was used to monitor basepair distortion at several individual positions in the promoter. We observed that basepair distortions during isomerization are a multi‐step process. Three distinct hitherto unresolved steps in kinetic terms were observed, where significant fluorescence change occurs: a fast step with a half‐life of around 1 s, which is followed by two slower steps occurring with a half‐life in the range of minutes at 25°C. Contrary to commonly held expectations, basepairs at different positions opened by 2,AP assays without any obvious pattern, suggesting that basepair opening is an asynchronous multi‐step process. cAMP·CRP, which activates transcription at galP1, enhanced the rate‐limiting step.

Involvement of PTP-RQ in differentiation during adipogenesis of human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are self-renewable multipotent progenitor cells with the capacity to differentiate into several distinct mesenchymal lineages. While MSCs display significant potential in tissue engineering and therapeutic applications, the regulatory mechanisms underlying the differentiation of these cells are yet to be established. Phosphorylation is a post-translational modification that plays a significant role in diverse biological phenomena. In this study, to mine the protein tyrosine phosphatases (PTPs) involved in adipogenesis of human MSCs, differential expression of human PTPs was examined using RT-PCR analysis. Among the 107 human PTPs, PTP-RQ was dramatically downregulated during the early phase of adipogenesis. PTP-RQ is classified as a receptor-type III PTP with phosphatidylinositol phosphatase (PIPase) activity. Overexpression of PTP-RQ consistently led to reduced differentiation of MSCs into adipocytes via decreasing the phosphatidyl inositol phosphate level in cells, and consequently downregulating Akt/PKB phosphorylation. Our results collectively suggest that PTP-RQ is a useful target protein for regulating the differentiation of MSCs into adipocytes, and may be used to develop novel drugs for the treatment of obesity.

Differential in vitro and cellular effects of iron chelators for hypoxia inducible factor hydroxylases

Hypoxia inducible factor 1α (HIF‐1α), an essential transcriptional factor, is negatively regulated by two different types of oxygen and Fe2+‐dependent HIF hydroxylases, proline hydroxylase (PHD) and factor inhibiting HIF (FIH), under normoxia. Iron chelators have therefore been used for inducing HIF‐1α expression by inhibiting the hydroxylases. In this study, the iron chelators displayed differential effects for PHD and FIH in cells depending on their iron specificity and membrane permeability rather than their in vitro potencies. The membrane permeability of the strict Fe2+‐chelator potentially inhibited both hydroxylases, whereas the membrane impermeable one showed no inhibitory effect in cells. In contrast, the depletion of the extracellular Fe3+ ion was mainly correlated to PHD inhibition, and the membrane permeable one elicited low efficacy for both enzymes in cells. The 3′‐hydroxyl group of quercetin, a natural flavonoid, was critical for inhibition of intracellular hydroxylases. Since the 3′‐methylation of quercetin is induced by catechol‐O‐methyl transferase, the enzyme may regulate the intracellular activity of quercetin. These data suggest that the multiple factors of iron‐chelators may be responsible for regulating the intracellular activity HIF hydroxylases. J. Cell. Biochem. 114: 864–873, 2013.

Structure of the nucleoid-associated protein Cnu reveals common binding sites for H-NS in Cnu and Hha.

Cnu is a nucleoid protein that has a high degree of sequence homology with Hha/YmoA family proteins, which bind to chromatin and regulate the expression of Escherichia coli virulence genes in response to changes in temperature or ionic strength. Here, we determined its solution structure and dynamic properties and mapped H-NS binding sites. Cnu consists of three alpha helices that are comparable with those of Hha, but it has significant flexibility in the C-terminal region and lacks a short alpha helix present in Hha. Upon increasing ionic strength, the helical structure of Cnu is destabilized, especially at the ends of the helices. The dominant H-NS binding sites, located at helix 3 as in Hha, reveal a common structural platform for H-NS binding. Our results may provide structural and dynamic bases for the similarity and dissimilarity between Cnu and Hha functions.

PI3K-ERK1/2 activation contributes to extracellular H2O2 generation in amyloid β toxicity.

Amyloid β peptide (Aβ) induces hydrogen peroxide (H2O2) and superoxide generation, leading to neuronal death. Many studies have shown the involvement of NADPH oxidase, but the isotype-specific role was not assessed. Moreover, the activation status of phosphoinositide 3-kinase (PI3K) and extracellular signal-regulated kinase (ERK) 1/2 is unclear in extracellular H2O2 generation. In this paper, we showed that Aβ1-42 induced extracellular H2O2 generation and the resulting cytotoxicity in a concentration-dependent manner. Nox2- and Nox4-specific siRNAs suppressed H2O2 and superoxide generation. LY294002 and U0126, inhibitors of PI3K and ERK1/2, respectively, reduced H2O2 generation in concentration-dependent manners. Furthermore, PI3K activation is responsible for ERK1/2 phosphorylation. An additional increase in H2O2 generation and corresponding cytotoxicity was observed after treatment with Aβ1-42 and glutamate. These results suggest that Aβ1-42 enhances the neuronal vulnerability to oxidative injury in Alzheimers disease (AD) by increasing H2O2 generation.

Structural and dynamic basis of a supercoiling-responsive DNA element

In both eukaryotes and prokaryotes, negative supercoiling of chromosomal DNA acts locally to regulate a variety of cellular processes, such as transcription, replication, recombination and response to environmental stresses. While studying the interaction between the Hin recombinase and mutated versions of its cognate DNA-binding site, we identified a mutated DNA site that binds Hin only when the DNA is supercoiled. To understand the mechanism of this supercoiling-responsive DNA site, we used NMR spectroscopy and fluorescence resonance energy transfer to determine the solution structures and dynamics of three related DNA oligonucleotides. The supercoiling-responsive DNA site formed a partially unwound and stretched helix and showed significant flexibility and base pair opening kinetics. The single CAG/CTG triplet contained in this DNA sequence displayed the same characteristics as do multiple CAG/CTG repeats, which are associated with several hereditary neuromuscular diseases. It is known that short DNA sequence motifs that have either very high or low bending flexibility occur preferentially at supercoiling-sensitive bacterial and eukaryotic promoters. From our results and these previous data, we propose a model in which supercoiling utilizes the intrinsic flexibility of a short DNA site to switch the local DNA structure from an inefficient conformation for protein binding to an efficient one, or vice versa.

Purification, characterization, and cloning of trimethylamine dehydrogenase fromMethylophaga sp. strain SK1

Trimethylamine dehydrogenase (TMADH, EC 1.5.99.7), an iron-sulfur flavoprotein that catalyzes the oxidative demethylation of trimethylamine to form dimethylamine and formaldehyde, was purified fromMethylophaga sp. strain SK1. The active TMADH was purified 12.3-fold through three purification steps. The optimal pH and temperature for enzyme activity was determined to be 8.5 and 55°C, respectively. TheVmax andKm values were 7.9 nmol/min/mg protein and 1.5 mM. A genomic DNA of 2,983 bp fromMethylophaga sp. strain SK1 was cloned, and DNA sequencing revealed the open reading frame (ORF) of the gene coding for TMADH. The ORF contained 728 amino acids with extensive identity (82%) to that ofMethylophilus methylotrophus W3A1.