Structural characterization of the heme‐based oxygen sensor,AfGcHK, its interactions with the cognate response regulator,and their combined mechanism of action in a bacterial two‐component signaling system

The oxygen sensor histidine kinase AfGcHK from the bacterium Anaeromyxobacter sp. Fw 109‐5 forms a two‐component signal transduction system together with its cognate response regulator (RR). The binding of oxygen to the heme iron of its N‐terminal sensor domain causes the C‐terminal kinase domain of AfGcHK to autophosphorylate at His183 and then transfer this phosphate to Asp52 or Asp169 of the RR protein. Analytical ultracentrifugation revealed that AfGcHK and the RR protein form a complex with 2:1 stoichiometry. Hydrogen‐deuterium exchange coupled to mass spectrometry (HDX‐MS) suggested that the most flexible part of the whole AfGcHK protein is a loop that connects the two domains and that the heme distal side of AfGcHK, which is responsible for oxygen binding, is the only flexible part of the sensor domain. HDX‐MS studies on the AfGcHK:RR complex also showed that the N‐side of the H9 helix in the dimerization domain of the AfGcHK kinase domain interacts with the helix H1 and the β‐strand B2 area of the RR protein's Rec1 domain, and that the C‐side of the H8 helix region in the dimerization domain of the AfGcHK protein interacts mostly with the helix H5 and β‐strand B6 area of the Rec1 domain. The Rec1 domain containing the phosphorylable Asp52 of the RR protein probably has a significantly higher affinity for AfGcHK than the Rec2 domain. We speculate that phosphorylation at Asp52 changes the overall structure of RR such that the Rec2 area containing the second phosphorylation site (Asp169) can also interact with AfGcHK. Proteins 2016; 84:1375–1389. © 2016 Wiley Periodicals, Inc.     

Effect of cadmium on the roots of beetroot (Beta vulgaris L.)

Abstract

The article dwells upon identifying the effect of cadmium on the roots of beetroot. The exposure effects of various concentrations of cadmium were studied at different levels of the plant organization (tissue pieces, organelles, membrane vesicles). The effect was noted only at a concentration of 100?μm. The negative effect of cadmium on the roots tissues of beetroot appeared with an increase in permeability and a decrease in the stability of cell membranes due to a change in the composition of fatty acids of membrane lipids and an increase in oxidation processes. The effect of cadmium in model experiments on the activity of the proton pumps of the vacuolar membrane has been evaluated. The pumps provide for the transport of heavy metals into the vacuole, which is one of the effective mechanisms for phytoremediation. The influence of cadmium in model experiments on the activity of the proton pump of a vacuolar membrane was evaluated. Under the influence of cadmium, a decrease in the activity of V-ATPase was observed, while the activity of V-PPase did not change. The results obtained complement our understanding of the damaging effects that occur in plant cells under cadmium stress.     

Surface wax esters contribute to drought tolerance in Arabidopsis

Waxes are components of the cuticle covering the aerial organs of plants. Accumulation of waxes has previously been associated with protection against water loss, therefore contributing to drought tolerance. However, not much information is known about the function of individual wax components during water deficit. We studied the role of wax ester synthesis during drought. The wax ester load on Arabidopsis leaves and stems was increased during water deficiency. Expression of three genes, WSD1, WSD6 and WSD7 of the wax ester synthase/diacylglycerol acyltransferase (WS/DGAT or WSD) family was induced during drought, salt stress and abscisic acid treatment. WSD1 has previously been identified as the major wax ester synthase of stems. wsd1 mutants have shown reduced wax ester coverage on leaves and stems during normal or drought condition, while wax ester loads of wsd6, wsd7 and of the wsd6wsd7 double mutant were unchanged. The growth and relative water content of wsd1 plants were compromised during drought, while leaf water loss of wsd1 was increased. Enzyme assays with recombinant proteins expressed in insect cells revealed that WSD6 and WSD7 contain wax ester synthase activity, albeit with different substrate specificity compared with WSD1. WSD6 and WSD7 localize to the endoplasmic reticulum (ER)/Golgi. These results demonstrated that WSD1 is involved in the accumulation of wax esters during drought, while WSD6 and WSD7 might play other specific roles in wax ester metabolism during stress.     

Sequential development of several RT-qPCR tests using LNA nucleotides and dual probe technology to differentiate SARS-CoV-2 from influenza A and B

Sensitive and accurate RT-qPCR tests are the primary diagnostic tools to identify SARS-CoV-2-infected patients. While many SARS-CoV-2 RT-qPCR tests are available, there are significant differences in test sensitivity, workflow (e.g. hands-on-time), gene targets and other functionalities that users must consider. Several publicly available protocols shared by reference labs and public health authorities provide useful tools for SARS-CoV-2 diagnosis, but many have shortcomings related to sensitivity and laborious workflows. Here, we describe a series of SARS-CoV-2 RT-qPCR tests that are originally based on the protocol targeting regions of the RNA-dependent RNA polymerase (RdRp) and envelope (E) coding genes developed by the Charité Berlin. We redesigned the primers/probes, utilized locked nucleic acid nucleotides, incorporated dual probe technology and conducted extensive optimizations of reaction conditions to enhance the sensitivity and specificity of these tests. By incorporating an RNase P internal control and developing multiplexed assays for distinguishing SARS-CoV-2 and influenza A and B, we streamlined the workflow to provide quicker results and reduced consumable costs. Some of these tests use modified enzymes enabling the formulation of a room temperature-stable master mix and lyophilized positive control, thus increasing the functionality of the test and eliminating cold chain shipping and storage. Moreover, a rapid, RNA extraction-free version enables high sensitivity detection of SARS-CoV-2 in about an hour using minimally invasive, self-collected gargle samples. These RT-qPCR assays can easily be implemented in any diagnostic laboratory and can provide a powerful tool to detect SARS-CoV-2 and the most common seasonal influenzas during the vaccination phase of the pandemic.     

Protease signalling in cell death: caspases versus cysteine cathepsins

Proteases were, for a long time, mainly considered as protein degrading enzymes. However, in the last decade this view has changed dramatically, and the focus is now on proteases as signalling molecules. One of the best examples is apoptosis, the major mechanism used by eukaryotes to remove superfluous, damaged and potentially dangerous cells, in which a number of proteases have been found to play a central role. Of these the caspases have been considered to be the major players. However, more recently, other proteases have been increasingly suggested as being important in apoptosis, in particular the cysteine cathepsins. In this review the roles of caspases and cysteine cathepsins in apoptosis signalling are compared and discussed.     

Third activity of Bordetella adenylate cyclase (AC) toxin-hemolysin. Membrane translocation of AC domain polypeptide promotes calcium influx into CD11b+ monocytes independently of the catalytic and hemolytic activities

The Bordetella adenylate cyclase toxin-hemolysin (CyaA) targets phagocytes expressing the alpha(M)beta2 integrin (CD11b/CD18), permeabilizes their membranes by forming small cation-selective pores, and delivers into cells a calmodulin-activated adenylate cyclase (AC) enzyme that dissipates cytosolic ATP into cAMP. We describe here a third activity of CyaA that yields elevation of cytosolic calcium concentration ([Ca2+]i) in target cells. The CyaA-mediated [Ca2+]i increase in CD11b+ J774A.1 monocytes was inhibited by extracellular La3+ ions but not by nifedipine, SK&F 96365, flunarizine, 2-aminoethyl diphenylborinate, or thapsigargin, suggesting that influx of Ca2+ into cells was not because of receptor signaling or opening of conventional calcium channels by cAMP. Compared with intact CyaA, a CyaA-AC- toxoid unable to generate cAMP promoted a faster, albeit transient, elevation of [Ca2+]i. This was not because of cell permeabilization by the CyaA hemolysin pores, because a mutant exhibiting a strongly enhanced pore-forming activity (CyaA-E509K/E516K), but unable to deliver the AC domain into cells, was also unable to elicit a [Ca2+]i increase. Further mutations interfering with AC translocation into cells, such as proline substitutions of glutamate residues 509 or 570 or deletion of the AC domain as such, reduced or ablated the [Ca2+]i-elevating capacity of CyaA. Moreover, structural alterations within the AC domain, because of insertion of various oligopeptides, differently modulated the kinetics and extent of Ca2+ influx elicited by the respective AC- toxoids. Hence, the translocating AC polypeptide itself appears to participate in formation of a novel type of membrane path for calcium ions, contributing to action of CyaA in an unexpected manner.     

C1 inhibitor serpin domain structure reveals the likely mechanism of heparin potentiation and conformational disease

C1 inhibitor, a member of the serpin family, is a major down-regulator of inflammatory processes in blood. Genetic deficiency of C1 inhibitor results in hereditary angioedema, a dominantly inheritable, potentially lethal disease. Here we report the first crystal structure of the serpin domain of human C1 inhibitor, representing a previously unreported latent form, which explains functional consequences of several naturally occurring mutations, two of which are discussed in detail. The presented structure displays a novel conformation with a seven-stranded beta-sheet A. The unique conformation of the C-terminal six residues suggests its potential role as a barrier in the active-latent transition. On the basis of surface charge pattern, heparin affinity measurements, and docking of a heparin disaccharide, a heparin binding site is proposed in the contact area of the serpin-proteinase encounter complex. We show how polyanions change the activity of the C1 inhibitor by a novel "sandwich" mechanism, explaining earlier reaction kinetic and mutagenesis studies. These results may help to improve therapeutic C1 inhibitor preparations used in the treatment of hereditary angioedema, organ transplant rejection, and heart attack.     

O6-methylguanine-DNA-methyltransferase (MGMT) gene therapy targeting haematopoietic stem cells: studies addressing safety issues

As haematopoietic stem cell gene therapy utilizing O(6)-methylguanine-DNA-methyltransferase has reached the clinical stage, safety-related questions become increasingly important. These issues concern insertional mutagenesis of viral vectors, the acute toxicity of pre-transplant conditioning protocols and in vivo selection regimens as well as potential genotoxic side effects of the alkylating drugs administered in this context. To address these questions, we have investigated toxicity-reduced conditioning regimens combining low-dose alkylator application with sublethal irradiation and have analysed their influence on engraftment and subsequent selectability of transduced haematopoietic stem cells. In addition, a strategy to monitor the acute and long-term genotoxic effects of drugs with high guanine-O(6) alkylating potential, such as chloroethylnitrosoureas or temozolomide is introduced. For this purpose, assays were implemented which allow an assessment of the generation and fate of primary drug-induced adducts as well as their long-term effect on chromosomal integrity at the single cell level.     

Von Vogelkot zu grünen Teppichen

This study tested whether potash tailing piles can be restored with biological soil crusts using an additive of the company upi. A biocrust community consists of different organisms, such as microalgae, lichens, and mosses. An established biocrust stabilizes the ground and traps rain water, which could in return reduce salt leachate into the environment. This pioneer community promotes formation of a new habitat that can be recolonized by higher plants. For this study microalgae were isolated from biological soil crusts collected at potash tailing pile sites. We characterized their salt tolerance and established first artificial biocrusts on the heap material. Upcoming experiments will focus on the establishment of artificial biocrusts in selected heap areas.     

Membrane skeleton detachment in spherical and cylindrical microexovesicles

We observed that amphiphile-induced microexovesicles may be spherical or cylindrical, depending on the species of the added amphiphile. The spherical microexovesicle corresponds to an extreme local difference between the two monolayer areas of the membrane segment with a fixed area, while the cylindrical microexovesicle corresponds to an extreme local area difference if the area of the budding segment is increased due to lateral influx of anisotropic membrane constituents. Protein analysis showed that both types of vesicles are highly depleted in the membrane skeleton. It is suggested that a partial detachment of the skeleton in the budding region is favoured due to accumulated skeleton shear deformations in this region.