The role of the ubiquitin ligase E6-AP in human papillomavirus E6-mediated degradation of PDZ domain-containing proteins

The E6 oncoprotein of human papillomaviruses associated with cervical cancer targets the tumor suppressor p53 and several other cellular proteins including the human homologs of Dlg and Scribble for degradation via the ubiquitin-proteasome system. Similar to p53 degradation, E6-induced degradation of Scribble is mediated by the ubiquitin ligase E6-AP. In contrast, degradation of Dlg in vitro and within cells has been reported to be independent of E6-AP, suggesting that the E6 oncoprotein has the ability to interact with ubiquitin ligases other than E6-AP. Furthermore, the ability of the E6 oncoprotein to interact with these yet unidentified ubiquitin ligases may be shared by the E6 protein of so-called low risk human papillomaviruses that are not associated with cervical cancer. In this study, we used the RNA interference technology and mouse embryo fibroblasts derived from E6-AP-deficient mice to obtain information about the identity of the ubiquitin ligase(s) involved in E6-mediated degradation of Dlg. We report that, within cells, E6-mediated degradation of Dlg depends on the presence of functional E6-AP and provide evidence that the E6 protein of low risk human papillomaviruses functionally interacts with E6-AP. Based on these data, we propose that, in general, the proteolytic properties of human papillomavirus E6 proteins are mediated by interaction with E6-AP.     

UBE1L2, a novel E1 enzyme specific for ubiquitin

UBE1 is known as the human ubiquitin-activating enzyme (E1), which activates ubiquitin in an ATP-dependent manner. Here, we identified a novel human ubiquitin-activating enzyme referred to as UBE1L2, which also shows specificity for ubiquitin. The UBE1L2 sequence displays a 40% identity to UBE1 and also contains an ATP-binding domain and an active site cysteine conserved among E1 family proteins. UBE1L2 forms a covalent link with ubiquitin in vitro and in vivo, which is sensitive to reducing conditions. In an in vitro polyubiquitylation assay, recombinant UBE1L2 could activate ubiquitin and transfer it onto the ubiquitin-conjugating enzyme UbcH5b. Ubiquitin activated by UBE1L2 could be used for ubiquitylation of p53 by MDM2 and supported the autoubiquitylation of the E3 ubiquitin ligases HectH9 and E6-AP. The UBE1L2 mRNA is most abundantly expressed in the testis, suggesting an organ-specific regulation of ubiquitin activation.     

Subunit-dependent cadmium and nickel inhibition of acid-sensing ion channels

Acid-sensing ion channels (ASIC) are ligand-gated cation channels that are highly expressed in peripheral sensory and central neurons. ASIC are transiently activated by decreases in extracellular pH and are thought to play important roles in sensory perception, neuronal transmission, and excitability, and in the pathology of neurological conditions, such as brain ischemia. We demonstrate here that the heavy metals Ni(2+) and Cd(2+) dose-dependently inhibit ASIC currents in hippocampus CA1 neurons and in Chinese hamster ovary (CHO) cells heterologously expressing these channels. The effects of both Ni(2+) and Cd(2+) were voltage-independent, fast, and reversible. Neither metal affected activation and desensitization kinetics but rather decreased pH-sensitivity. Moreover, distinct ASIC isoforms were differentially inhibited by Ni(2+) and Cd(2+). External application of 1 mM Ni(2+) rapidly inhibited homomeric ASIC1a and heteromeric ASIC1a/2a channels without affecting ASIC1b, 2a, and ASIC3 homomeric channels and ASIC1a/3 and 2a/3 heteromeric channels. In contrast, external Cd(+) (1 mM) inhibited ASIC2a and ASIC3 homomeric channels and ASIC1a/2a, 1a/3, and 2a/3 heteromeric channels but not ASIC1a homomeric channels. The acid-sensing current in isolated rat hippocampus CA1 neurons, thought to be carried primarily by ASIC1a and 1a/2a, was inhibited by 1 mM Ni(2+). The current study identifies ASIC as a novel target for the neurotoxic heavy metals Cd(2+) and Ni(2+).     

Effect of bedaquiline on the functions of rat liver mitochondria

The paper considers the effects of bedaquiline (BDQ), an antituberculous preparation of the new generation, on rat liver mitochondria. It was shown that 50?μM BDQ inhibited mitochondrial respiration measured with substrates of complexes I and II (glutamate/malate and succinate/rotenone systems respectively) in the states V₃ and V_DNP. At the same time, at concentrations below 50?μM, BDQ slightly stimulated respiration with substrates of complex I in the state V₂. BDQ was also found to suppress, in a dose-dependent manner, the activity of complex II and the total activity of complexes II?+?III of the mitochondrial transport chain. It was discovered that at concentrations up to 10?μM, BDQ inhibited H₂O₂ production in mitochondria. BDQ (10–50?μM) suppressed the opening of Ca²⁺-dependent CsA-sensitive mitochondrial permeability transition pore. The latter was revealed experimentally as the inhibition of Ca²⁺/P_i-dependent swelling of mitochondria, suppression of cytochrome c release, and an increase in the Ca²⁺ capacity of the organelles. BDQ also decreased the rate of mitochondrial energy-dependent K⁺ transport, which was evaluated by the energy-dependent swelling of mitochondria in a K⁺ buffer and DNP-induced K⁺ efflux from the organelles. The possible mechanisms of BDQ effect of rat liver mitochondria are discussed.     

Desiccation‐induced alterations in surface topography of thylakoids from resurrection plant Haberlea rhodopensis studied by atomic force microscopy,electrokinetic and optical measurements

With their ability to survive complete desiccation, resurrection plants are a suitable model system for studying the mechanisms of drought tolerance. In the present study, we investigated desiccation‐induced alterations in surface topography of thylakoids isolated from well‐hydrated, moderately dehydrated, severely desiccated and rehydrated Haberlea rhodopensis plants by means of atomic force microscopy (AFM), electrokinetic and optical measurements. According to our knowledge, so far, there were no reports on the characterization of surface topography and polydispersity of thylakoid membranes from resurrection plants using AFM and dynamic light scattering. To study the physicochemical properties of thylakoids from well‐hydrated H. rhodopensis plants, we used spinach thylakoids for comparison as a classical model from higher plants. The thylakoids from well‐hydrated H. rhodopensis had a grainy surface, significantly different from the well‐structured spinach thylakoids with distinct grana and lamella, they had twice smaller cross‐sectional area and were 1.5 times less voluminous than that of spinach. Significant differences in their physicochemical properties were observed. The dehydration and subsequent rehydration of plants affected the size, shape, morphology, roughness and therefore the structure of the studied thylakoids. Drought resulted in significant enhancement of negative charges on the outer surface of thylakoid membranes which correlated with the increased roughness of thylakoid surface. This enhancement in surface charge density could be due to the partial unstacking of thylakoids exposing more negatively charged groups from protein complexes on the membrane surface that prevent from possible aggregation upon drought stress.     

Ameliorating effect of lipo-ATRA treatment on the expression of TIG3 and its suppressing effect on PPARγ gene expression in lung cancer animal model

Vitamin D₃ deficiency was found to be tightly linked to many health problems including metabolic syndrome, cancer, cardiovascular diseases, and type 2 diabetes mellitus. In our study, we tested the possible antidiabetic effects of one of vitamin D₃ analogs, alfacalcidol, solely or in a combination with metformin on type 2 diabetic rats. Type 2 diabetic model rats were induced by feeding high-fat diet for 4 weeks followed by intraperitoneal injection of streptozotocin. In addition to the control group, the diabetic rats were divided into four groups: untreated, metformin-treated, alfacalcidol-treated, and combination-treated group (metformin?+?alfacalcidol) for 4 weeks. The level of fasting blood glucose, fasting serum insulin, homeostatic model of insulin resistance, serum lipid profile, liver enzymes, calcium, phosphorus, and 25-hydroxyvitamin D₃ were also determined. Besides, sterol regulatory element binding protein-1c (SREBP-1c) and vitamin D receptors (VDR) gene expression at mRNA and protein levels were evaluated. The level of significance was fixed at P?≤?0.05 for all statistical tests. Alfacalcidol, solely or combined with metformin, significantly ameliorated glucose homeostasis and lipid profile parameters (P?<?0.001) with a neutral effect on calcium and phosphorus levels. Significant downregulation of mRNA expression of SREBP-1c in the liver, white as well as brown adipose tissues (P?<?0.001) and different patterns of mRNA expression of VDR gene in pancreas and white adipose tissue were observed in rats treated with alfacalcidol solely or in combination with metformin. Vitamin D₃ analogs can modulate glucose parameters and lipid metabolism in a diabetic rat model and it provides additional protective effects when combined with metformin.

     

Targeted genomic integration of EGFP under tubulin beta 3 class III promoter and mEos2 under tryptophan hydroxylase 2 promoter does not produce sufficient levels of reporter gene expression

Neuronal tracing is a modern technology that is based on the expression of fluorescent proteins under the control of cell type–specific promoters. However, random genomic integration of the reporter construct often leads to incorrect spatial and temporal expression of the marker protein. Targeted integration (or knock-in) of the reporter coding sequence is supposed to provide better expression control by exploiting endogenous regulatory elements. Here we describe the generation of two fluorescent reporter systems: enhanced green fluorescent protein (EGFP) under pan-neural marker class III β-tubulin (Tubb3) promoter and mEos2 under serotonergic neuron-specific tryptophan hydroxylase 2 (Tph2) promoter. Differentiation of Tubb3-EGFP embryonic stem (ES) cells into neurons revealed that though Tubb3-positive cells express EGFP, its expression level is not sufficient for the neuronal tracing by routine fluorescent microscopy. Similarly, the expression levels of mEos2-TPH2 in differentiated ES cells was very low and could be detected only on messenger RNA level using polymerase chain reaction-based methods. Our data shows that the use of endogenous regulatory elements to control transgene expression is not always beneficial compared with the random genomic integration.     

Efficient target cleavage by Type V Cas12a effectors programmed with split CRISPR RNA

CRISPR RNAs (crRNAs) that direct target DNA cleavage by Type V Cas12a nucleases consist of constant repeat-derived 5′-scaffold moiety and variable 3′-spacer moieties. Here, we demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by a Cas12a ortholog from Acidaminococcus sp. (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer moiety only. crRNAs split into separate scaffold and spacer RNAs catalyzed highly specific and efficient cleavage of target DNA by AsCas12a in vitro and in lysates of human cells. In addition to dsDNA target cleavage, AsCas12a programmed with split crRNAs also catalyzed specific ssDNA target cleavage and non-specific ssDNA degradation (collateral activity). V-A effector nucleases from Francisella novicida (FnCas12a) and Lachnospiraceae bacterium (LbCas12a) were also functional with split crRNAs. Thus, the ability of V-A effectors to use split crRNAs appears to be a general property. Though higher concentrations of split crRNA components are needed to achieve efficient target cleavage, split crRNAs open new lines of inquiry into the mechanisms of target recognition and cleavage and may stimulate further development of single-tube multiplex and/or parallel diagnostic tests based on Cas12a nucleases.