Attila Ambrus

Human telomeric sequence forms a hybrid-type intramolecular G-quadruplex structure with mixed parallel/antiparallel strands in potassium solution

Human telomeric DNA consists of tandem repeats of the sequence d(TTAGGG). The formation and stabilization of DNA G-quadruplexes in the human telomeric sequence have been shown to inhibit the activity of telomerase, thus the telomeric DNA G-quadruplex has been considered as an attractive target for cancer therapeutic intervention. However, knowledge of the intact human telomeric G-quadruplex structure(s) formed under physiological conditions is a prerequisite for structure-based rational drug design. Here we report the folding structure of the human telomeric sequence in K+ solution determined by NMR. Our results demonstrate a novel, unprecedented intramolecular G-quadruplex folding topology with hybrid-type mixed parallel/antiparallel G-strands. This telomeric G-quadruplex structure contains three G-tetrads with mixed G-arrangements, which are connected consecutively with a double-chain-reversal side loop and two lateral loops, each consisting of three nucleotides TTA. This intramolecular hybrid-type telomeric G-quadruplex structure formed in K+ solution is distinct from those reported on the 22 nt Tel22 in Na+ solution and in crystalline state in the presence of K+, and appears to be the predominant conformation for the extended 26 nt telomeric sequence Tel26 in the presence of K+, regardless of the presence or absence of Na+. Furthermore, the addition of K+ readily converts the Na+-form conformation to the K+-form hybrid-type G-quadruplex. Our results explain all the reported experimental data on the human telomeric G-quadruplexes formed in the presence of K+, and provide important insights for understanding the polymorphism and interconversion of various G-quadruplex structures formed within the human telomeric sequence, as well as the effects of sequence and cations. This hybrid-type G-quadruplex topology suggests a straightforward pathway for the secondary structure formation with effective packing within the extended human telomeric DNA. The hybrid-type telomeric G-quadruplex is most likely to be of pharmacological relevance, and the distinct folding topology of this G-quadruplex suggests that it can be specifically targeted by G-quadruplex interactive small molecule drugs.

Structure of the intramolecular human telomeric G-quadruplex in potassium solution: a novel adenine triple formation

We report the NMR solution structure of the intramolecular G-quadruplex formed in human telomeric DNA in K+. The hybrid-type telomeric G-quadruplex consists of three G-tetrads linked with mixed parallel–antiparallel G-strands, with the bottom two G-tetrads having the same G-arrangement (anti:anti:syn:anti) and the top G-tetrad having the reversed G-arrangement (syn:syn:anti:syn). The three TTA loop segments adopt different conformations, with the first TTA assuming a double-chain-reversal loop conformation, and the second and third TTA assuming lateral loop conformations. The NMR structure is very well defined, including the three TTA loops and the two flanking sequences at 5′- and 3′-ends. Our study indicates that the three loop regions interact with the core G-tetrads in a specific way that defines and stabilizes the unique human telomeric G-quadruplex structure in K+. Significantly, a novel adenine triple platform is formed with three naturally occurring adenine residues, A21, A3 and A9, capping the top tetrad of the hybrid-type telomeric G-quadruplex. This adenine triple is likely to play an important role in the formation of a stable human telomeric G-quadruplex structure in K+. The unique human telomeric G-quadruplex structure formed in K+ suggests that it can be specifically targeted for anticancer drug design.

Inhibition of the alpha-ketoglutarate dehydrogenase-mediated reactive oxygen species generation by lipoic acid.

Dihydrolipoamide dehydrogenase (LADH) is a flavo‐enzyme that serves as a subunit of α‐ketoglutarate dehydrogenase complex (α‐KGDHC). Reactive oxygen species (ROS) generation by α‐KGDHC has been assigned to LADH (E3 subunit) and explained by the diaphorase activity of E3. Dysfunctions of α‐KGDHC and concurrent ROS production have been implicated in neurodegeneration, ischemia‐reperfusion, and other pathological conditions. In this work we investigated the in‐depth details of ROS generation by isolated LADH and α‐KGDHC. We found a parallel generation of superoxide and hydrogen peroxide by the E3 subunit of α‐KGDHC which could be blocked by lipoic acid (LA) acting on a site upstream of the E3 subunit. The pathologically relevant ROS generation (at high NADH/NAD+ ratio and low pH) in the reverse mode of α‐KGDHC could also be inhibited by LA. Our results contradict the previously proposed mechanism for pH‐dependent ROS generation by LADH, showing no disassembling of the E3 functional homodimer at acidic pH using a physiologically relevant method for the examination. It is also suggested that LA could be beneficial in reducing the cell damage related to excessive ROS generation under pathological conditions.

A direct and nondestructive approach to determine the folding structure of the I-motif DNA secondary structure by NMR

I-motifs are four-stranded DNA secondary structures formed in C-rich DNA sequences and consist of parallel-stranded DNA duplexes zipped together in an antiparallel orientation by intercalated, hemiprotonated cytosine(+)-cytosine base pairs. I-motif structures have been indicated to form in various regions of the human genome as well as in nanotechnological applications. While NMR is a major tool for structural studies of I-motifs, the determination of the folding topologies of unimolecular I-motifs has been a challenging and arduous task using conventional NMR spectral assignment strategies, due to the inherent sequence redundancy of the C-rich strands in the formation of unimolecular I-motif structures. We report here a direct and nondestructive method that can be utilized to unambiguously determine the hemiprotonated C(+)-C base pairs and thus the folding topology of unimolecular I-motif structures formed from native C-rich DNA sequences. The reported approach uses affordable low-enrichment site-specific labeling. More significantly, the reported method can directly and unambiguously determine the equilibrating multiple conformations coexisting in a single DNA sequence, which would be a very difficult task using conventional assignment strategies. Additionally, this method can be applied to the direct detection of the base-paired thymines that are involved in the capping structures.

Stimulation of reactive oxygen species generation by disease-causing mutations of lipoamide dehydrogenase

We investigated pathogenic mutations relevant in dihydrolipoamide dehydrogenase (LADH; gene: Dld) deficiency, a severe human disease, to elucidate how they alter reactive oxygen species (ROS) generation and associated biophysical characteristics of LADH. Twelve known disease-causing mutants of human LADH have been expressed and purified to homogeneity from E. coli. Detailed biophysical and biochemical characterization of the mutants has been performed applying circular dichroism (CD) spectroscopy, nano-spray mass spectrometry (MS), calibrated gel filtration and flavin adenine dinucleotide-content analysis. Functional analyses revealed that four of the pathogenic mutations significantly stimulated the ROS-generating activity of LADH and also increased its sensitivity to an acidic shift in pH. LADH activity was reduced by variable extents in the mutants exhibiting excessive ROS generation. It is remarkable that in the P453L mutant, enzyme activity was nearly completely lost with a ROS-forming activity becoming dominant, whereas the G194C mutation, common among Ashkenazi Jews, resulted in no alteration in LADH activity but a gain in the ROS-generating activity. There have been neither major conformational alterations nor monomerization of the functional homodimer of LADH associated with the higher ROS-generating capacity as measured by CD spectroscopy and size-exclusion chromatography combined with nano-spray MS, respectively. The excessive ROS generation of selected LADH mutants could be an important factor in the pathology and clinical presentation of human LADH deficiency and raises the possibility of an antioxidant therapy in the treatment of this condition.

Calcium binding of transglutaminases: A 43Ca NMR study combined with surface polarity analysis

Abstract Transglutaminases (TGases) form cross-links between glutamine and lysine side-chains of polypeptides in a Ca2+-dependent reaction. The structural basis of the Ca2+-effect is poorly defined. 43Ca NMR, surface polarity analysis combined with multiple sequence alignment and the construction of a new homology model of human tissue transglutaminase (tTGase) were used to obtain structural information about Ca2+ binding properties of factor XIII-A2, tTGase and TGase 3 (each of human origin). 43Ca NMR provided higher average dissociation constants titrating on a wide Ca2+-concentration scale than previous studies with equilibrium dialysis performed in shorter ranges. These results suggest the existence of low affinity Ca2+ binding sites on both FXIII-A and tTGase in addition to high affinity ones in accordance with our surface polarity analysis identifying high numbers of negatively charged clusters. Upon increasing the salt concentration or activating with thrombin, FXIII-A2 partially lost its original Ca2+ affinity; the NMR data suggested different mechanisms for the two activation processes. The NMR provided structural evidence of GTP-induced conformational changes on the tTGase molecule diminishing all of its Ca2+ binding sites. NMR data on the Ca2+ binding properties of the TGase 3 are presented here; it binds Ca2+ the most tightly, which is weakened after its proteolytic activation. The investigated TGases seem to have very symmetric Ca2+ binding sites and no EF-hand motifs.

Human 2-Oxoglutarate Dehydrogenase Complex E1 Component Forms a Thiamin-derived Radical by Aerobic Oxidation of the Enamine Intermediate

Background: The human 2-oxoglutarate dehydrogenase complex, comprising E1o, E2o, and E3 components, catalyzes conversion of 2-oxoglutarate to succinyl-CoA. Results: Human E1o generates both a thiamin-enamine-derived radical and the reactive oxygen species, superoxide, and hydrogen peroxide. Conclusion: Human E1o produces reactive oxygen species at a rate of <1% of succinyl-CoA under physiological conditions. Significance: This work presents the novel discovery that the 5-carboxyl group affects enzymatic reactivity of 2-oxoglutarate. Herein are reported unique properties of the human 2-oxoglutarate dehydrogenase multienzyme complex (OGDHc), a rate-limiting enzyme in the Krebs (citric acid) cycle. (a) Functionally competent 2-oxoglutarate dehydrogenase (E1o-h) and dihydrolipoyl succinyltransferase components have been expressed according to kinetic and spectroscopic evidence. (b) A stable free radical, consistent with the C2-(C2α-hydroxy)-γ-carboxypropylidene thiamin diphosphate (ThDP) cation radical was detected by electron spin resonance upon reaction of the E1o-h with 2-oxoglutarate (OG) by itself or when assembled from individual components into OGDHc. (c) An unusual stability of the E1o-h-bound C2-(2α-hydroxy)-γ-carboxypropylidene thiamin diphosphate (the “ThDP-enamine”/C2α-carbanion, the first postdecarboxylation intermediate) was observed, probably stabilized by the 5-carboxyl group of OG, not reported before. (d) The reaction of OG with the E1o-h gave rise to superoxide anion and hydrogen peroxide (reactive oxygen species (ROS)). (e) The relatively stable enzyme-bound enamine is the likely substrate for oxidation by O2, leading to the superoxide anion radical (in d) and the radical (in b). (f) The specific activity assessed for ROS formation compared with the NADH (overall complex) activity, as well as the fraction of radical intermediate occupying active centers of E1o-h are consistent with each other and indicate that radical/ROS formation is an “off-pathway” side reaction comprising less than 1% of the “on-pathway” reactivity. However, the nearly ubiquitous presence of OGDHc in human tissues, including the brain, makes these findings of considerable importance in human metabolism and perhaps disease.

Molecular dynamics study of the structural basis of dysfunction and the modulation of reactive oxygen species generation by pathogenic mutants of human dihydrolipoamide dehydrogenase.

Human dihydrolipoamide dehydrogenase (LADH, E3) is a component in the pyruvate-, alpha-ketoglutarate- and branched-chain ketoacid dehydrogenase complexes and in the glycine cleavage system. The pathogenic mutations of LADH cause severe metabolic disturbances, called E3 deficiency that often involve cardiological and neurological symptoms and premature death. Our laboratory has recently shown that some of the known pathogenic mutations augment the reactive oxygen species (ROS) generation capacity of LADH, which may contribute to the clinical presentations. A recent report concluded that elevated oxidative stress generated by the above mutants turns the lipoic acid cofactor on the E2 subunits dysfunctional. In the present contribution we generated by molecular dynamics (MD) simulation the conformation of LADH that is proposed to be compatible with ROS generation. We propose here for the first time the structural changes, which are likely to turn the physiological LADH conformation to its ROS-generating conformation. We also created nine of the pathogenic mutants of the ROS-generating conformation and again used MD simulation to detect structural changes that the mutations induced in this LADH conformation. We propose the structural changes that may lead to the modulation in ROS generation of LADH by the pathogenic mutations.

Periplasmic cold expression and one-step purification of human dihydrolipoamide dehydrogenase

Dihydrolipoamide dehydrogenase (LADH) is a FAD-linked subunit of alpha-ketoglutarate, pyruvate and branched-chain amino acid dehydrogenases and the glycine cleavage system. As an oxidoreductase it transfers electrons from the dihydrolipoic acid prosthetic group to the NAD(+) cofactor via its FAD center. Besides its physiological function it is capable of generating harmful reactive oxygen species (ROS) in pathological settings therefore it is implicated in neurodegeneration, ischemia-reperfusion, cancer and several other disorders. Pathological mutants of the enzyme cause severe, sometimes lethal syndromes like hypotonia, metabolic acidosis or inefficiency in development. Recently it has been revealed that LADH is a moonlighting protease when specific mutations in the dimerization surface destabilize the functional homodimer and expose a serine-protease-like catalytic dyad. As the basis of versatile functions of LADH is far from elucidation, there is a constant need for a pure and functional enzyme product for investigations. Several studies used recombinant human LADH before, however, it was generated by more complicated and/or physiologically less compatible protocols than reported here; most papers on functional and structural studies do not even report detailed protocols and characteristics (most importantly the purity) of their protein products. Here we describe the details of an optimized, easy-to-use periplasmic expression and one-step purification protocol for obtaining a highly pure, active and authentic (tag-cleaved) enzyme with the characterization of the protein product. The purified LADH can be used in biophysical and structural studies while the published protocol is easily convertible to a protein labeling procedure.

A re-evaluation of the role of matrix acidification in uncoupler-induced Ca2+ release from mitochondria.

Massive amounts of Ca2+ can accumulate in mitochondria, owing to its complexation with matrix phosphate. Under conditions in which the mitochondrial uniporter is the foremost pathway for Ca2+ efflux, the release of sequestered Ca2+ by protonophoric uncouplers is invariably demonstrated. This has been recently ascribed to matrix acidification, which results in the dissociation of the Ca2+–phosphate complex. In the present study, we compared the effect of stepwise depolarization on Ca2+ release induced by either the complex III inhibitor stigmatellin or an uncoupler in energized Ca2+‐loaded rat liver mitochondria in the presence of phosphate, at extramitochondrial pH (pHo) 6.8 and pHo 7.8. Both poisons were examined in the presence and absence of oligomycin. Prior to Ca2+ loading, mitochondria were allowed to phosphorylate 0.5 mm ADP. Opening of the permeability transition pore was additionally hampered by cyclosporin A, and was monitored by changes in light scattering. Na+ was excluded from the medium, preventing Na+/Ca2+ exchange. At both pHo values, ΔpH was in the range 0.11–0.15. Complete depolarization by uncoupling with or without oligomycin resulted in an approximately pH 0.05 acidic shift, but there was none in the case of stigmatellin plus oligomycin. At pHo 6.8 and in the presence of oligomycin, the uncoupler‐induced Ca2+ release started in the −80 to −50 mV range, whereas in the absence of oligomycin, the release occurred at approximately −15 mV. Stigmatellin induced minor Ca2+ release only in the presence of oligomycin, starting at approximately −4 mV. At pHo 7.8, the uncoupler‐induced Ca2+ release started at approximately −11 mV, irrespective of the presence or absence of oligomycin. Unexpectedly, at this alkaline pH and in the presence of oligomycin, stigmatellin induced substantial Ca2+ release, starting at approximately −10 mV. From the above findings, we conclude that matrix acidification cannot be the sole explanation for uncoupler‐induced Ca2+ release from mitochondria.