Structural and biochemical impact of C8-aryl-guanine adducts within the NarI recognition DNA sequence: influence of aryl ring size on targeted and semi-targeted mutagenicity

Chemical mutagens with an aromatic ring system may be enzymatically transformed to afford aryl radical species that preferentially react at the C8-site of 2′-deoxyguanosine (dG). The resulting carbon-linked C8-aryl-dG adduct possesses altered biophysical and genetic coding properties compared to the precursor nucleoside. Described herein are structural and in vitro mutagenicity studies of a series of fluorescent C8-aryl-dG analogues that differ in aryl ring size and are representative of authentic DNA adducts. These structural mimics have been inserted into a hotspot sequence for frameshift mutations, namely, the reiterated G₃-position of the NarI sequence within 12mer (NarI(12)) and 22mer (NarI(22)) oligonucleotides. In the NarI(12) duplexes, the C8-aryl-dG adducts display a preference for adopting an anti-conformation opposite C, despite the strong syn preference of the free nucleoside. Using the NarI(22) sequence as a template for DNA synthesis in vitro, mutagenicity of the C8-aryl-dG adducts was assayed with representative high-fidelity replicative versus lesion bypass Y-family DNA polymerases, namely, Escherichia coli pol I Klenow fragment exo⁻ (Kf⁻) and Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4). Our experiments provide a basis for a model involving a two-base slippage and subsequent realignment process to relate the miscoding properties of C-linked C8-aryl-dG adducts with their chemical structures.     

Photosystem II and the unique role of bicarbonate: A historical perspective

In photosynthesis, cyanobacteria, algae and plants fix carbon dioxide (CO(2)) into carbohydrates; this is necessary to support life on Earth. Over 50years ago, Otto Heinrich Warburg discovered a unique stimulatory role of CO(2) in the Hill reaction (i.e., O(2) evolution accompanied by reduction of an artificial electron acceptor), which, obviously, does not include any carbon fixation pathway; Warburg used this discovery to support his idea that O(2) in photosynthesis originates in CO(2). During the 1960s, a large number of researchers attempted to decipher this unique phenomenon, with limited success. In the 1970s, Alan Stemler, in Govindjee's lab, perfected methods to get highly reproducible results, and observed, among other things, that the turnover of Photosystem II (PSII) was stimulated by bicarbonate ions (hydrogen carbonate): the effect would be on the donor or the acceptor, or both sides of PSII. In 1975, Thomas Wydrzynski, also in Govindjee's lab, discovered that there was a definite bicarbonate effect on the electron acceptor (the plastoquinone) side of PSII. The most recent 1.9? crystal structure of PSII, unequivocally shows HCO(3)(-) bound to the non-heme iron that sits in-between the bound primary quinone electron acceptor, Q(A), and the secondary quinone electron acceptor Q(B). In this review, we focus on the historical development of our understanding of this unique bicarbonate effect on the electron acceptor side of PSII, and its mechanism as obtained by biochemical, biophysical and molecular biological approaches in many laboratories around the World. We suggest an atomic level model in which HCO(3)(-)/CO(3)(2-) plays a key role in the protonation of the reduced Q(B). In addition, we make comments on the role of bicarbonate on the donor side of PSII, as has been extensively studied in the labs of Alan Stemler (USA) and Vyacheslav Klimov (Russia). We end this review by discussing the uniqueness of bicarbonate's role in oxygenic photosynthesis and its role in the evolutionary development of O(2)-evolving PSII. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Identification of Specific Hemopexin-like Domain Residues That Facilitate Matrix Metalloproteinase Collagenolytic Activity

Collagen serves as a structural scaffold and a barrier between tissues, and thus collagen catabolism (collagenolysis) is required to be a tightly regulated process in normal physiology. In turn, the destruction or damage of collagen during pathological states plays a role in tumor growth and invasion, cartilage degradation, or atherosclerotic plaque formation and rupture. Several members of the matrix metalloproteinase (MMP) family catalyze the hydrolysis of collagen triple helical structure. This study has utilized triple helical peptide (THP) substrates and inhibitors to dissect MMP-1 collagenolytic behavior. Analysis of MMP-1/THP interactions by hydrogen/deuterium exchange mass spectrometry followed by evaluation of wild type and mutant MMP-1 kinetics led to the identification of three noncatalytic regions in MMP-1 (residues 285–295, 302–316, and 437–457) and two specific residues (Ile-290 and Arg-291) that participate in collagenolysis. Ile-290 and Arg-291 contribute to recognition of triple helical structure and facilitate both the binding and catalysis of the triple helix. Evidence from this study and prior studies indicates that the MMP-1 catalytic and hemopexin-like domains collaborate in collagen catabolism by properly aligning the triple helix and coupling conformational states to facilitate hydrolysis. This study is the first to document the roles of specific residues within the MMP-1 hemopexin-like domain in substrate binding and turnover. Noncatalytic sites, such as those identified here, can ultimately be utilized to create THP inhibitors that target MMPs implicated in disease progression while sparing proteases with host-beneficial functions.The mechanism of collagenolysis, by which proteases catalyze the hydrolysis of amide bonds within triple helical structures, has been investigated for over 30 years. Despite this lengthy period, few inroads have been made in the identification of specific enzyme residues that facilitate collagenolysis. The primary mammalian collagenases have been identified as cathepsin K and several members of the matrix metalloproteinase (MMP)³ family. Most of the early work on MMP collagenolysis focused on analysis of the sites of hydrolysis, and how unique features within these sites may direct collagen catabolism (1). More recent work has evaluated the active sites and domains of MMPs to better understand the dynamic role that the enzyme plays in collagen hydrolysis (2–4).Collagenolytic members of the MMP family possess similar domain organizations, including propeptide, catalytic (CAT), linker, and hemopexin-like (HPX) domains (5). Several of these domains and/or regions within them have been implicated in collagenolysis. For example, MMP-1 residues 183–191, which are on the V-B loop between the fifth β-strand and the second α-helix in the CAT domain, as well as the active site cleft itself, have substantial roles in collagenolysis (6, 7). MMP-1 residue Gly-233 has been implicated as necessary for conformational flexibility of the active site (8). Within the MMP-1 linker domain, residues 262–276 were proposed to form a polyproline type II helix and interact with and destabilize the MMP cleavage site in collagen (9), whereas Gly-272 may allow bending of the linker domain to aid in interaction between the CAT and HPX domains (10).The HPX domain has a critical role in collagenolysis, as removal of the MMP-1, MMP-8, MMP-13, or MMP-14 (MT1-MMP) HPX domain results in a loss of collagenolytic activity (11–16). However, no information has been obtained as to the identity of specific residues within the HPX domain that participate in collagenolysis. Secondary binding sites (exosites) may promote interaction of proteases with large, macromolecular substrates, such as collagen. The identification of exosites involved in collagenolysis may aid in the design of selective MMP inhibitors (17–20). Ultimately, as exosites are identified, the manner in which the CAT, linker, and HPX domains work together to facilitate collagenolysis can be revealed.One approach for the rapid analysis of protein structure and identification of binding sites within proteins involves hydrogen/deuterium exchange (HDX) of protein backbone amide hydrogens with detection by mass spectrometry (MS) (21–23). A protein or protein/ligand pair is incubated for defined intervals in a deuterated environment. After rapid quenching of the HDX reaction, the partially deuterated protein is digested, and the resulting peptide fragments are analyzed by LC-MS. The deuterium buildup curve measured for each fragment yields an average amide exchange rate that reflects the environment of the peptide in the intact protein. HDX MS has been used previously to monitor the interaction between doxycycline and MMP-7 (24). The interaction sites identified were consistent with other biophysical studies mapping doxycycline binding outside of the catalytic Zn²⁺ (24). This present study has utilized HDX MS with a triple helical peptide (THP) substrate to identify nonactive site MMP-1 regions involved in collagenolysis. Subsequently, site-specific mutagenesis of MMP-1 in combination with THP inhibitors and substrates was utilized to identify, for the first time, specific HPX domain residues that participate in collagenolysis and to provide insight as to how these residues function mechanistically.     

An improved fluorescent substrate for assaying soluble and membrane-associated ADAM family member activities

A fluorescent resonance energy transfer substrate with improved sensitivity for ADAM17, −10, and −9 (where ADAM represents a disintegrin and metalloproteinase) has been designed. The new substrate, Dabcyl-Pro-Arg-Ala-Ala-Ala-Homophe-Thr-Ser-Pro-Lys(FAM)-NH₂, has specificity constants of 6.3 (±0.3) × 10⁴ M⁻¹ s⁻¹ and 2.4 (±0.3) × 10³ M⁻¹ s⁻¹ for ADAM17 and ADAM10, respectively. The substrate is more sensitive than widely used peptides based on the precursor tumor necrosis factor-alpha (TNF-alpha) cleavage site, PEPDAB010 or Dabcyl-Ser-Pro-Leu-Ala-Gln-Ala-Val-Arg-Ser-Ser-Lys(FAM)-NH₂ and Mca-Pro-Leu-Ala-Gln-Ala-Val-Dpa-Arg-Ser-Ser-Arg-NH₂. ADAM9 also processes the new peptide more than 18-fold better than the TNF-alpha-based substrates. The new substrate has a unique selectivity profile because it is processed less efficiently by ADAM8 and MMP1, −2, −3, −8, −9, −12, and −14. This substrate provides a unique tool in which to assess ADAM17, −10, and −9 activities.     

Oncolytic specificity of Newcastle disease virus is mediated by selectivity for apoptosis-resistant cells

Newcastle disease virus (NDV) is a negative-sense RNA virus that has been shown to possess oncolytic activity. NDV's selective replication in tumor cells has been previously suggested to be due to the lack of a proper antiviral response in these cells. Here we demonstrate that NDV possesses oncolytic activity in tumor cells capable of a robust type I interferon (IFN) response, suggesting that another mechanism underlies NDV's tumor specificity. We show that the oncolytic selectivity of NDV for tumor cells is dependent upon tumor cell resistance to apoptosis. Utilizing the human non-small-cell lung cancer cell line A549 overexpressing the antiapoptotic protein Bcl-xL, we show significant enhancement of oncolytic activity and NDV replication. Interestingly, while the Bcl-xL-overexpressing cells were resistant to apoptotic stimuli induced by chemotherapeutic agents and early viral replication, during the subsequent viral cycles, we observed a paradoxical increase in apoptosis in response to NDV. The increased oncolytic activity seen was secondary to enhanced viral replication and syncytium formation. The induction of a type I IFN response was enhanced in Bcl-xL cells. Overall, these findings propose a new mechanism for cancer cell specificity for NDV, making it an attractive anticancer agent for chemoresistant tumors with enhanced antiapoptotic activity.     

Tissue-Specific Functional Networks for Prioritizing Phenotype and Disease Genes

Integrated analyses of functional genomics data have enormous potential for identifying phenotype-associated genes. Tissue-specificity is an important aspect of many genetic diseases, reflecting the potentially different roles of proteins and pathways in diverse cell lineages. Accounting for tissue specificity in global integration of functional genomics data is challenging, as “functionality” and “functional relationships” are often not resolved for specific tissue types. We address this challenge by generating tissue-specific functional networks, which can effectively represent the diversity of protein function for more accurate identification of phenotype-associated genes in the laboratory mouse. Specifically, we created 107 tissue-specific functional relationship networks through integration of genomic data utilizing knowledge of tissue-specific gene expression patterns. Cross-network comparison revealed significantly changed genes enriched for functions related to specific tissue development. We then utilized these tissue-specific networks to predict genes associated with different phenotypes. Our results demonstrate that prediction performance is significantly improved through using the tissue-specific networks as compared to the global functional network. We used a testis-specific functional relationship network to predict genes associated with male fertility and spermatogenesis phenotypes, and experimentally confirmed one top prediction, Mbyl1. We then focused on a less-common genetic disease, ataxia, and identified candidates uniquely predicted by the cerebellum network, which are supported by both literature and experimental evidence. Our systems-level, tissue-specific scheme advances over traditional global integration and analyses and establishes a prototype to address the tissue-specific effects of genetic perturbations, diseases and drugs.     

A parturition-associated nonsynaptic coherent activity pattern in the developing hippocampus

Correlated neuronal activity is instrumental in the formation of networks, but its emergence during maturation is poorly understood. We have used multibeam two-photon calcium microscopy combined with targeted electrophysiological recordings in order to determine the development of population coherence from embryonic to postnatal stages in the hippocampus. At embryonic stages (E16-E19), synchronized activity is absent, and neurons are intrinsically active and generate L-type channel-mediated calcium spikes. At birth, small cell assemblies coupled by gap junctions spontaneously generate synchronous nonsynaptic calcium plateaus associated to recurrent burst discharges. The emergence of coherent calcium plateaus at birth is controlled by oxytocin, a maternal hormone initiating labour, and progressively shut down a few days later by the synapse-driven giant depolarizing potentials (GDPs) that synchronize the entire network. Therefore, in the developing hippocampus, delivery is an important signal that triggers the first coherent activity pattern, which is silenced by the emergence of synaptic transmission.     

1,3,7-trimethylxanthine (caffeine) may exacerbate acute inflammatory liver injury by weakening the physiological immunosuppressive mechanism

The genetic elimination of A2A adenosine receptors (A2AR) was shown to disengage the critical immunosuppressive mechanism and cause the dramatic exacerbation of acute inflammatory tissue damage by T cells and myeloid cells. This prompted the evaluation of the proinflammatory vs the anti-inflammatory effects of the widely consumed behavioral drug caffeine, as the psychoactive effects of caffeine are mediated largely by its antagonistic action on A2AR in the brain. Because caffeine has other biochemical targets besides A2AR, it was important to test whether the consumption of caffeine during an acute inflammation episode would lead to the exacerbation of immune-mediated tissue damage. We examined acute and chronic treatment with caffeine for its effects on acute liver inflammation. It is shown that caffeine at lower doses (10 and 20 mg/kg) strongly exacerbated acute liver damage and increased levels of proinflammatory cytokines. Because caffeine did not enhance liver damage in A2AR-deficient mice, we suggest that the potentiation of liver inflammation was mediated by interference with the A2AR-mediated tissue-protecting mechanism. In contrast, a high dose of caffeine (100 mg/kg) completely blocked both liver damage and proinflammatory cytokine responses through an A2AR-independent mechanism. Furthermore, caffeine administration exacerbated liver damage even when mice consumed caffeine chronically, although the extent of exacerbation was less than in "naive" mice that did not consume caffeine before. This study suggests an unappreciated "man-made" immunological pathogenesis whereby consumption of the food-, beverage-, and medication-derived adenosine receptor antagonists may modify an individual's inflammatory status and lead to excessive organ damage during acute inflammation.