Hee-Eun Kim

A pyrene-imidazolium derivative that selectively Recognizes G-Quadruplex DNA

G-quadruplexes, formed of four stranded guanine bases stabilized by monovalent cations, serve important role in cancer cell growth and control gene expression in telomere. Since there are various types of quadruplex structures, rapid and simple screening methods with high selectivity, sensitivity and nontoxicity are required for understanding about the biological roles of quadruplex DNA as well as in designing therapeutics. Herein, we report a pyrene-imidazolium derivative, JY-1, which can with high selectivity recognize G-quadruplex using fluorescence and NMR spectroscopy. This is the first example based on the imidazolium derivative, which can detect the G-quadruplex directly to utilize the excimer/monomer emission change in pyrene fluorophore. The selectivity of strong binding to a specific sequence can allow for quadruplex sensing and the detection method presented here is very simple, using fluorescence and NMR study. Also, the groove binding characteristic of JY-1 to the G-quadruplex has a relatively low nonspecific toxicity and the structure-specific differences in fluorescent character between DNA duplex and G-quadruplex may offer more discovery and application in biological study.

Two distinct disulfide bonds formed in human heat shock transcription factor 1 act in opposition to regulate its DNA binding activity.

Under circumstances of heat stress, heat shock transcription factor 1 (HSF1) plays important roles in heat shock protein expression. In this study, an increasing concentration of dithiothreitol (DTT) was found to either enhance or inhibit the heat-induced trimerization of HSF1, suggesting the involvement of dual redox-dependent HSF1 activation mechanisms. Our in vitro experiments show that the heat-induced bonding between the cysteine C36 and C103 residues of HSF1 forms an intermolecular disulfide covalent bond (SS-I bond) and that it directly causes HSF1 to trimerize and bond to DNA. Gel filtration assays show that HSF1 can form intermolecular hydrophobic interaction-mediated (iHI-m) noncovalent oligomers. However, the lack of a trimerization domain prevents HSF1 activation, which suggests that iHI-m noncovalent trimerization is a precondition of SS-I bond formation. On the other hand, intramolecular SS-II bond (in which the C153, C373, and C378 residues of HSF1 participate) formation inhibits this iHI-m trimerization, thereby preventing SS-I bond formation and DNA binding. Thus, HSF1 activation is regulated positively by intermolecular SS-I bond formation and negatively by intramolecular SS-II bond formation. Importantly, these two SS bonds confer different DTT sensitivities (the SS-II bond is more sensitive). Therefore, a low concentration of DTT cleaves the SS-II bond but not the SS-I bond and thus improves DNA binding of HSF1, whereas a high concentration DTT cuts both SS bonds and inhibits HSF1 activation. We propose that these interesting effects further explain cellular HSF1 trimerization, DNA binding, and transcription when cells are under stress.

NMR study on the B-Z junction formation of DNA duplexes induced by Z-DNA binding domain of human ADAR1.

Z-DNA is produced in a long genomic DNA by Z-DNA binding proteins, through formation of two B-Z junctions with the extrusion of one base pair from each junction. To answer the question of how Z-DNA binding proteins induce B-Z transitions in CG-rich segments while maintaining the B-conformation of surrounding segments, we investigated the kinetics and thermodynamics of base-pair openings of a 13-bp DNA in complex with the Z-DNA binding protein, Zα(ADAR1). We also studied perturbations in the backbone of Zα(ADAR1) upon binding to DNA. Our study demonstrates the initial contact conformation as an intermediate structure during B-Z junction formation induced by Zα(ADAR1), in which the Zα(ADAR1) protein displays unique backbone conformational changes, but the 13-bp DNA duplex maintains the B-form helix. We also found the unique structural features of the 13-bp DNA duplex in the initial contact conformation: (i) instability of the AT-rich region II and (ii) longer lifetime for the opening state of the CG-rich region I. Our findings suggest a three-step mechanism of B-Z junction formation: (i) Zα(ADAR1) specifically interacts with a CG-rich DNA segment maintaining B-form helix via a unique conformation; (ii) the neighboring AT-rich region becomes very unstable, and the CG-rich DNA segment is easily converted to Z-DNA; and (iii) the AT-rich regions are base-paired again, and the B-Z junction structure is formed.

Assessment of Achilles enthesitis in the spondyloarthropathies by colour Doppler energy ultrasound in the context of the ‘enthesis organ’

Objectives: To identify the characteristic features of Achilles enthesitis of the spondyloarthropathies (SpA) detectable by colour Doppler energy ultrasound (CDEU) in the context of the ‘enthesis organ’. Methods: Seventy patients with SpA and 15 healthy subjects were clinically evaluated and underwent CDEU examination of the Achilles entheses. The CDEU images were evaluated according to five distinctive CDEU features of enthesitis in the context of the enthesis organ. Results: Fifty-six of the 70 SpA patients (80%) showed at least one abnormal finding of the enthesitis on CDEU examination, affecting 91 of 140 Achilles entheses examined (65%). Only 22 of 140 Achilles entheses (15.7%) showed abnormal vascularization in the peri-sesamoidal and periosteal areas or in the area of enthesis fibrocartilage. In addition, 67 (47.9%) and 18 (12.9%) of 140 Achilles entheses examined showed cortical bone irregularities and erosions, respectively, at areas of the periosteal and the enthesis fibrocartilage. Sixteen (88.9%) of 18 clinically detected Achilles enthesitis and six (75%) of eight Achilles entheses with swelling on clinical examination presented corresponding abnormalities on CDEU examination. The Doppler twinkling artefact (TA) was observed consistently in all normal entheses and completely disappeared upon stabilization of the probe application. The C-reactive protein (CRP) level was higher in the patients with cortical bone erosion than in those without erosion. Conclusions: This study demonstrates the characteristic features of enthesitis detectable by CDEU in the context of the enthesis organ and shows a good correlation with clinical as well as with laboratory findings.

Sequence discrimination of the Zα domain of human ADAR1 during B–Z transition of DNA duplexes

The Zα domain of human ADAR1 (ZαADAR1) preferentially binds Z‐DNA rather than B‐DNA with high binding affinity. ZαADAR1 binds to the Z‐conformation of both non‐CG‐repeat DNA duplexes and a d(CGCGCG)2 duplex similarly. We performed NMR experiments on complexes between the ZαADAR1 and non‐CG‐repeat DNA duplexes, d(CACGTG)2 or d(CGTACG)2, with a variety of protein‐DNA molar ratios. Comparison of these results with those from the analysis of d(CGCGCG)2 in the previous study suggests that ZαADAR1 exhibits the sequence preference of d(CGCGCG)2 ≫ d(CACGTG)2 > d(CGTACG)2 through multiple sequence discrimination steps during the B–Z transition.

NMR study of hydrogen exchange during the B–Z transition of a DNA duplex induced by the Zα domains of yatapoxvirus E3L

The Yaba‐like disease viruses (YLDV) are members of the Yatapoxvirus family and have double‐stranded DNA genomes. The E3L protein, which is essential for pathogenesis in the vaccinia virus, consists of two domains: an N‐terminal Z‐DNA binding domain and a C‐terminal RNA binding domain. The crystal structure of the E3L orthologue of YLDV (yabZαE3L) bound to Z‐DNA revealed that the overall structure of yabZαE3L and its interaction with Z‐DNA are very similar to those of hZαADAR1. Here we have performed NMR hydrogen exchange experiments on the complexes between yabZαE3L and d(CGCGCG)2 with a variety of protein‐to‐DNA molar ratios. This study revealed that yabZαE3L could efficiently change the B‐form helix of the d(CGCGCG)2 to left‐handed Z‐DNA via the active‐mono B–Z transition pathway like hZαADAR1.

Structural features of influenza A virus panhandle RNA enabling the activation of RIG-I independently of 5′-triphosphate

Retinoic acid-inducible gene I (RIG-I) recognizes specific molecular patterns of viral RNAs for inducing type I interferon. The C-terminal domain (CTD) of RIG-I binds to double-stranded RNA (dsRNA) with the 5′-triphosphate (5′-PPP), which induces a conformational change in RIG-I to an active form. It has been suggested that RIG-I detects infection of influenza A virus by recognizing the 5′-triphosphorylated panhandle structure of the viral RNA genome. Influenza panhandle RNA has a unique structure with a sharp helical bending. In spite of extensive studies of how viral RNAs activate RIG-I, whether the structural elements of the influenza panhandle RNA confer the ability to activate RIG-I signaling has been poorly explored. Here, we investigated the dynamics of the influenza panhandle RNA in complex with RIG-I CTD using NMR spectroscopy and showed that the bending structure of the panhandle RNA negates the requirement of a 5′-PPP moiety for RIG-I activation.

The Zβ domain of human DAI binds to Z-DNA via a novel B–Z transition pathway

The human DNA‐dependent activator of IFN‐regulatory factor (DAI) protein, which activates the innate immune response in response to DNA, contains two tandem Z‐DNA binding domains (Zα and Zβ) at the NH2 terminus. The hZβDAI structure is similar to other Z‐DNA binding proteins, although it demonstrates an unusual Z‐DNA recognition. We performed NMR experiments on complexes of hZβDAI with DNA duplex, d(CGCGCG)2, at a variety of protein‐to‐DNA molar ratios. The results suggest that hZβDAI binds to Z‐DNA via an active‐di B–Z transition mechanism, where two hZβDAI proteins bind to B‐DNA to form the hZβDAI–B‐DNA complex; the B‐DNA is subsequently converted to left‐handed Z‐DNA. This novel mechanism of DNA binding and B–Z conversion is distinct from Z‐DNA binding of the human ADAR1 protein.

Pain, xerostomia, and younger age are major determinants of fatigue in Korean patients with primary Sjögren’s syndrome: a cohort study

Objectives: Fatigue is a common clinical manifestation in patients with primary Sjögren’s syndrome (pSS). The aims of this study were to investigate the association between fatigue severity and other clinical characteristics in pSS patients and to determine the factors contributing to fatigue. Method: We analysed 257 participants from the Korean Initiative of pSS (KISS), a prospective pSS cohort. Fatigue was assessed according to the fatigue domain of the European League Against Rheumatism (EULAR) Sjögren’s Syndrome Patient-Reported Index (ESSPRI). Health-related quality of life (HRQoL) was evaluated using the EuroQol-5 dimensions (EQ-5D) questionnaire. Multiple linear regression analysis was used to estimate the effect of each variable on fatigue severity. Results: The median total ESSPRI score was 5 [interquartile range (IQR) 4–6]. Thirty-four per cent of patients reported a fatigue score > 5. Younger and premenopausal patients presented with more fatigue (p = 0.013 and p < 0.001, respectively). Higher Xerostomia Inventory (XI) scale (p < 0.001) and Ocular Surface Dryness Index (OSDI) (p < 0.001) scores were observed in patients with a fatigue score > 5. Pain, xerostomia, and age were determined to be significantly associated with fatigue severity after adjusting for depression/anxiety, OSDI score, and the presence of fibromyalgia using a multivariate general linear model. The ESSPRI fatigue score was correlated with the EQ-5D by time trade-off (TTO) values and visual analogue scale (VAS) scores. Conclusions: In Korean patients with pSS, younger age, xerostomia, and pain were correlated significantly with fatigue, and fatigue was associated with HRQoL.

Comparison of backbone dynamics of the type III antifreeze protein and antifreeze-like domain of human sialic acid synthase

Antifreeze proteins (AFPs) are found in a variety of cold-adapted (psychrophilic) organisms to promote survival at subzero temperatures by binding to ice crystals and decreasing the freezing temperature of body fluids. The type III AFPs are small globular proteins that consist of one α-helix, three 310-helices, and two β-strands. Sialic acids play important roles in a variety of biological functions, such as development, recognition, and cell adhesion and are synthesized by conserved enzymatic pathways that include sialic acid synthase (SAS). SAS consists of an N-terminal catalytic domain and a C-terminal antifreeze-like (AFL) domain, which is similar to the type III AFPs. Despite having very similar structures, AFL and the type III AFPs exhibit very different temperature-dependent stability and activity. In this study, we have performed backbone dynamics analyses of a type III AFP (HPLC12 isoform) and the AFL domain of human SAS (hAFL) at various temperatures. We also characterized the structural/dynamic properties of the ice-binding surfaces by analyzing the temperature gradient of the amide proton chemical shift and its correlation with chemical shift deviation from random coil. The dynamic properties of the two proteins were very different from each other. While HPLC12 was mostly rigid with a few residues exhibiting slow motions, hAFL showed fast internal motions at low temperature. Our results provide insight into the molecular basis of thermostability and structural flexibility in homologous psychrophilic HPLC12 and mesophilic hAFL proteins.