Ionization Patterns and Chemical Reactivity of Cytosine-Guanine Watson-Crick Pairs.

The front cover artwork is provided by Prof. Papadantonakis' group. The image shows a Watson-Crick Guanine-Cytosine pair, and the difference between vertical and adiabatic ionization potentials. Read the full text of the Research Article at 10.1002/cphc.202300946.

Ionization Patterns and Chemical Reactivity of Cytosine-Guanine Watson-Crick Pairs.

Gas-phase and aqueous vertical ionization potentials, vIPgas and vIPaq respectively and measurements of the molecular electrostatic and local ionization maps calculated at the DFT/B3LYP-D3/ 6-311+G** level of theory and the C-PCM reaction field model for single- and double-stranded CpG and 5MeCpG pairs show that the vIPaq for single- and double-stranded pairs of C-G and 5MeC-G are practically the same, in the range of 5.79 to 5.81 eV. The aqueous adiabatic ionization potentials for single-stranded CpG and 5MeCpG are 5.52 eV and 5.51 eV respectively and they reflect the nuclear reorganization that takes place after the abstraction of the electron. The aqueous adiabatic ionization energy values that correspond to the CpG+. radical cation and the hydrated electron, e-,, being at infinite distance, adIPaq+Vo, are 3.92 eV and 3.91 eV respectively with (Vo=-1.6 eV) Analysis of data suggest that the HOMO-LUMO energy gap in the hard/soft-acid/base (HSAB) concept cannot be used a priori to determine the effect of cytosine methylation on the guanine enhanced oxidative damage in DNA.

Current Challenges in Targeting Tumor Desmoplasia to Improve the Efficacy of Immunotherapy.

Desmoplasia is crucial for the development, progression and treatment of immune-resistant malignancies. Targeting desmoplasia-related metabolic pathways appears to be an interesting approach to expand our stock of disposable anti-tumor agents. CXCL12/CXCR4 axis inhibition reduces fibrosis, alleviates immunosuppression and significantly enhances the efficacy of PD-1 immunotherapy. CD40L substitute therapy may increase the activity of T-cells, downregulate CD40+, prolong patients' survival and prevent cancer progression. Although FAPα antagonists used in preclinical models did not lead to permanent cure, an alleviation of immune-resistance, modification of desmoplasia and a decrease in angiogenesis were observed. Targeting DDR2 may enhance the effect of anti-PD-1 treatment in multiple neoplasm cell lines and has the ability to overcome the adaptation to BRAF-targeted therapy in melanoma. Reprogramming desmoplasia could potentially cooperate not only with present treatment, but also other potential therapeutic targets. We present the most promising metabolic pathways related to desmoplasia and discuss the emerging strategies to improve the efficacy of immunotherapy.

CDK9: Therapeutic Perspective in HCC Therapy.

CDK9 is an important cell-cycle control enzyme essential in transcription, elongation, and mRNA maturation. Overexpression of CDK9 has been reported in several diseases, including acute lymphoblastic leukemia, chronic lymphocytic leukemia, and malignant melanoma. Recent research revealed that CDK9-inhibitors have a major impact on the induction of apoptosis in hepatocellular carcinoma (HCC) cell lines. Despite surprisingly promising results in in vitro and in vivo research, no CDK9 related therapy is currently allowed in cases of HCC. Furthermore, due to their high specificity, the inhibitors had no effects on unaltered hepatocytes and no toxic effects were shown. Considering that they were well tolerated and showed relatively few severe side-effects in mice, CDK9- inhibitors would seem to be promising targets in HCC biomarker-guided immunotherapy. Studies have verified that CDK9 has a pivotal role in c-Myc-mediated tumor growth and CDK9 inhibitors inhibit not only its progression but diametrically decrease both the mass and size of HCC nodules. CDK9-inhibitors seem to be a promising target in HCC treatment.

Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol.

Controlled distribution of lipids across various cell membranes is crucial for cell homeostasis and regulation. We developed an imaging method that allows simultaneous in situ quantification of cholesterol in two leaflets of the plasma membrane (PM) using tunable orthogonal cholesterol sensors. Our imaging revealed marked transbilayer asymmetry of PM cholesterol (TAPMC) in various mammalian cells, with the concentration in the inner leaflet (IPM) being ∼12-fold lower than that in the outer leaflet (OPM). The asymmetry was maintained by active transport of cholesterol from IPM to OPM and its chemical retention at OPM. Furthermore, the increase in the IPM cholesterol level was triggered in a stimulus-specific manner, allowing cholesterol to serve as a signaling lipid. We found excellent correlation between the IPM cholesterol level and cellular Wnt signaling activity, suggesting that TAPMC and stimulus-induced PM cholesterol redistribution are crucial for tight regulation of cellular processes under physiological conditions.

Cholesterol modulates cell signaling and protein networking by specifically interacting with PDZ domain-containing scaffold proteins.

Cholesterol is known to modulate the physical properties of cell membranes, but its direct involvement in cellular signaling has not been thoroughly investigated. Here we show that cholesterol specifically binds many PDZ domains found in scaffold proteins, including the N-terminal PDZ domain of NHERF1/EBP50. This modular domain has a cholesterol-binding site topologically distinct from its canonical protein-binding site and serves as a dual-specificity domain that bridges the membrane and juxta-membrane signaling complexes. Disruption of the cholesterol-binding activity of NHERF1 largely abrogates its dynamic co-localization with and activation of cystic fibrosis transmembrane conductance regulator, one of its binding partners in the plasma membrane of mammalian cells. At least seven more PDZ domains from other scaffold proteins also bind cholesterol and have cholesterol-binding sites, suggesting that cholesterol modulates cell signaling through direct interactions with these scaffold proteins. This mechanism may provide an alternative explanation for the formation of signaling platforms in cholesterol-rich membrane domains.

Properties of RNA polymerase bypass mutants: implications for the role of ppGpp and its co-factor DksA in controlling transcription dependent on sigma54.

The bacterial nutritional and stress alarmone ppGpp and its co-factor DksA directly bind RNA polymerase to regulate its activity at certain sigma70-dependent promoters. A number of promoters that are dependent on alternative sigma-factors function poorly in the absence of ppGpp. These include the Pseudomonas-derived sigma54-dependent Po promoter and several other sigma54-promoters, the transcription from which is essentially abolished in Escherichia coli devoid of ppGpp and DksA. However, ppGpp and DksA have no apparent effect on reconstituted in vitro sigma54-transcription, which suggests an indirect mechanism of control. Here we report analysis of five hyper-suppressor mutants within the beta- and beta'-subunits of core RNA polymerase that allow high levels of transcription from the sigma54-Po promoter in the absence of ppGpp. Using in vitro transcription and competition assays, we present evidence that these core RNA polymerase mutants are defective in one or both of two properties that could combine to explain their hyper-suppressor phenotypes: (i) modulation of competitive association with sigma-factors to favor sigma54-holoenzyme formation over that with sigma70, and (ii) reduced innate stability of RNA polymerase-promoter complexes, which mimics the essential effects of ppGpp and DksA for negative regulation of stringent sigma70-promoters. Both these properties of the mutant holoenzymes support a recently proposed mechanism for regulation of sigma54-transcription that depends on the potent negative effects of ppGpp and DksA on transcription from powerful stringent sigma70-promoters, and suggests that stringent regulation is a key mechanism by which the activity of alternative sigma-factors is controlled to meet cellular requirements.