A toolbox for class I HDACs reveals isoform specific roles in gene regulation and protein acetylation

The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes. Using this genetic toolbox we compare the effect of individual HDAC inhibition with the effects of class I specific inhibitors on cell viability, protein acetylation and gene expression. Individual inactivation of HDAC1 or HDAC2 has only mild effects on cell viability, while HDAC3 inactivation or loss results in DNA damage and apoptosis. Inactivation of HDAC1/HDAC2 led to increased acetylation of components of the COREST co-repressor complex, reduced deacetylase activity associated with this complex and derepression of neuronal genes. HDAC3 controls the acetylation of nuclear hormone receptor associated proteins and the expression of nuclear hormone receptor regulated genes. Acetylation of specific histone acetyltransferases and HDACs is sensitive to inactivation of HDAC1/HDAC2. Over a wide range of assays, we determined that in particular HDAC1 or HDAC2 catalytic inactivation mimics class I specific HDAC inhibitors. Importantly, we further demonstrate that catalytic inactivation of HDAC1 or HDAC2 sensitizes cells to specific cancer drugs. In summary, our systematic study revealed isoform-specific roles of HDAC1/2/3 catalytic functions. We suggest that targeted genetic inactivation of particular isoforms effectively mimics pharmacological HDAC inhibition allowing the identification of relevant HDACs as targets for therapeutic intervention.     

The overlooked role of a biotin precursor for marine bacteria - desthiobiotin as an escape route for biotin auxotrophy

Biotin (vitamin B₇) is involved in a wide range of essential biochemical reactions and a crucial micronutrient that is vital for many pro- and eukaryotic organisms. The few biotin measurements in the world’s oceans show that availability is subject to strong fluctuations. Numerous marine microorganisms exhibit biotin auxotrophy and therefore rely on supply by other organisms. Desthiobiotin is the primary precursor of biotin and has recently been detected at concentrations similar to biotin in seawater. The last enzymatic reaction in the biotin biosynthetic pathway converts desthiobiotin to biotin via the biotin synthase (BioB). The role of desthiobiotin as a precursor of biotin synthesis in microbial systems, however, is largely unknown. Here we demonstrate experimentally that bacteria can overcome biotin auxotrophy if they retain the bioB gene and desthiobiotin is available. A genomic search of 1068 bacteria predicts that the biotin biosynthetic potential varies greatly among different phylogenetic groups and that 20% encode solely bioB and thus can potentially overcome biotin auxotrophy. Many Actino- and Alphaproteobacteria cannot synthesize biotin de novo, but some possess solely bioB, whereas the vast majority of Gammaproteobacteria and Flavobacteriia exhibit the last four crucial biotin synthesis genes. We detected high intra- and extracellular concentrations of the precursor relative to biotin in the prototrophic bacterium, Vibrio campbellii, with extracellular desthiobiotin reaching up to 1.09 ± 0.15*10⁶ molecules per cell during exponential growth. Our results provide evidence for the ecological role of desthiobiotin as an escape route to overcome biotin auxotrophy for bacteria in the ocean and presumably in other ecosystems.Subject terms: Water microbiology, Ecosystem ecology, Marine microbiology     

N6-substituted 9-methyladenines: a new class of adenosine receptor antagonists

A series of 15 N6-substituted 9-methyladenines have been assessed as antagonists of A2-adenosine receptor-mediated stimulation of adenylate cyclase in membranes of human platelets and rat PC12 cells and of A1-adenosine receptor-mediated inhibition of adenylate cyclases in membranes of rat fat cells and as inhibitors of binding of N6-R-[3H]phenylisopropyladenosine to A1-adenosine receptors in rat brain membranes. N6 substitution can markedly increase the potency of 9-methyladenine at A1 receptors, while having lesser effects or even decreasing potency at A2 receptors. Effects of N6 substituents on adenosine receptor activity of the 9-methyladenines are reminiscent of effects of N6 substituents on activity of adenosine, suggesting that N6 substituted 9-methyladenines bind to adenosine receptors in the same orientation as do N6-substituted adenosines. N6-Cyclopentyl-9-methyladenine with Ki values at the A1 receptors of 1.3 microM (fat cells) and 0.5 microM (brain) is at least 100-fold more potent than 9-methyladenine (Ki 100 microM, both receptors), while at the A2 receptors KB values of 5 microM (platelets) and 25 microM (PC12 cells) make it 5-fold more potent and equipotent, respectively, compared to 9-methyladenine (KB 24 microM, both receptors). N6-Cyclopentyl and several other N6-alkyl and N6-cycloalkyl analogs are selective for A1 receptors while 9-methyladenine is the most A2 receptor selective antagonist. The N6-R- and N6-S-(1-phenyl-2-propyl)-9-methyladenines, analogous to N6-R- and N6-S-phenylisopropyladenosines, exhibit stereoselectivity at both A1 and A2 receptors. Marked differences in potency of certain N6-substituted 9-methyladenines at the A2 receptors of human platelets and rat PC12 cells provide evidence that these are not identical receptors.     

Uncertainty in predicting range dynamics of endemic alpine plants under climate warming

Correlative species distribution models have long been the predominant approach to predict species’ range responses to climate change. Recently, the use of dynamic models is increasingly advocated for because these models better represent the main processes involved in range shifts and also simulate transient dynamics. A well‐known problem with the application of these models is the lack of data for estimating necessary parameters of demographic and dispersal processes. However, what has been hardly considered so far is the fact that simulating transient dynamics potentially implies additional uncertainty arising from our ignorance of short‐term climate variability in future climatic trends. Here, we use endemic mountain plants of Austria as a case study to assess how the integration of decadal variability in future climate affects outcomes of dynamic range models as compared to projected long‐term trends and uncertainty in demographic and dispersal parameters. We do so by contrasting simulations of a so‐called hybrid model run under fluctuating climatic conditions with those based on a linear interpolation of climatic conditions between current values and those predicted for the end of the 21st century. We find that accounting for short‐term climate variability modifies model results nearly as differences in projected long‐term trends and much more than uncertainty in demographic/dispersal parameters. In particular, range loss and extinction rates are much higher when simulations are run under fluctuating conditions. These results highlight the importance of considering the appropriate temporal resolution when parameterizing and applying range‐dynamic models, and hybrid models in particular. In case of our endemic mountain plants, we hypothesize that smoothed linear time series deliver more reliable results because these long‐lived species are primarily responsive to long‐term climate averages.     

Real‐time Raman and SRS imaging of living human macrophages reveals cell‐to‐cell heterogeneity and dynamics of lipid uptake

Monitoring living cells in real‐time is important in order to unravel complex dynamic processes in life sciences. In particular the dynamics of initiation and progression of degenerative diseases is intensely studied. In atherosclerosis the thickening of arterial walls is related to high lipid levels in the blood stream, which trigger the lipid uptake and formation of droplets as neutral lipid reservoirs in macrophages in the arterial wall. Unregulated lipid uptake finally results in foam cell formation, which is a hallmark of atherosclerosis. In previous studies, the uptake and storage of different fatty acids was monitored by measuring fixed cells. Commonly employed fluorescence staining protocols are often error prone because of cytotoxicity and unspecific fluorescence backgrounds. By following living cells with Raman spectroscopic imaging, lipid uptake of macrophages was studied with real‐time data acquisition. Isotopic labeling using deuterated palmitic acid has been combined with spontaneous and stimulated Raman imaging to investigate the dynamic process of fatty acid storage in human macrophages for incubation times from 45 min to 37 h. Striking heterogeneity in the uptake rate and the total concentration of deuterated palmitic acid covering two orders of magnitude is detected in single as well as ensembles of cultured human macrophages.

SRS signal of deuterated palmitic acid measured at the CD vibration band after incorporation into living macrophages.     

DARPins recognizing the tumor-associated antigen EpCAM selected by phage and ribosome display and engineered for multivalency

Designed Ankyrin Repeat Proteins (DARPins) represent a novel class of binding molecules. Their favorable biophysical properties such as high affinity, stability and expression yields make them ideal candidates for tumor targeting. Here, we describe the selection of DARPins specific for the tumor-associated antigen epithelial cell adhesion molecule (EpCAM), an approved therapeutic target on solid tumors. We selected DARPins from combinatorial libraries by both phage display and ribosome display and compared their binding on tumor cells. By further rounds of random mutagenesis and ribosome display selection, binders with picomolar affinity were obtained that were entirely monomeric and could be expressed at high yields in the cytoplasm of Escherichia coli. One of the binders, denoted Ec1, bound to EpCAM with picomolar affinity (K_d = 68 pM), and another selected DARPin (Ac2) recognized a different epitope on EpCAM. Through the use of a variety of bivalent and tetravalent arrangements with these DARPins, the off-rate on cells was further improved by up to 47-fold. All EpCAM-specific DARPins were efficiently internalized by receptor-mediated endocytosis, which is essential for intracellular delivery of anticancer agents to tumor cells. Thus, using EpCAM as a target, we provide evidence that DARPins can be conveniently selected and rationally engineered to high-affinity binders of various formats for tumor targeting.     

Transport of nucleosides across the Plasmodium falciparum parasite plasma membrane has characteristics of PfENT1

Like all parasitic protozoa, the human malaria parasite Plasmodium falciparum lacks the enzymes required for de novo synthesis of purines and it is therefore reliant upon the salvage of these compounds from the external environment. P. falciparum equilibrative nucleoside transporter 1 (PfENT1) is a nucleoside transporter that has been localized to the plasma membrane of the intraerythrocytic form of the parasite. In this study we have characterized the transport of purine and pyrimidine nucleosides across the plasma membrane of 'isolated' trophozoite-stage P. falciparum parasites and compared the transport characteristics of the parasite with those of PfENT1 expressed in Xenopus oocytes. The transport of nucleosides into the parasite: (i) was, in the case of adenosine, inosine and thymidine, very fast, equilibrating within a few seconds; (ii) was of low affinity [K(m) (adenosine) = 1.45 +/- 0.25 mM; K(m) (thymidine) = 1.11 +/- 0.09 mM]; and (iii) showed 'cross-competition' for adenosine, inosine and thymidine, but not cytidine. The kinetic characteristics of nucleoside transport in intact parasites matched very closely those of PfENT1 expressed in Xenopus oocytes [K(m) (adenosine) = 1.86 +/- 0.28 mM; K(m) (thymidine) = 1.33 +/- 0.17 mM]. Furthermore, PfENT1 transported adenosine, inosine and thymidine, with a cross-competition profile the same as that seen for isolated parasites. The data are consistent with PfENT1 serving as a major route for the uptake of nucleosides across the parasite plasma membrane.     

Chimeric IgG-binding receptors engineered from staphylococcal protein A and streptococcal protein G

Chimeric Fc receptors, consisting of the IgG-binding domains of both staphylococcal protein A and streptococcal protein G, were constructed. An efficient bacterial expression system was used to produce the recombinant proteins, which vary in size and number of IgG-binding domains. The purified receptors were analyzed by immunodiffusion and a competitive enzyme-linked immunosorbent assay to establish the relative binding strength to various polyclonal and monoclonal immunoglobulins from different species. The results demonstrate that protein A and protein G have complementary binding patterns and that the chimeric receptors retain the binding capacities of both the parental constituents. This suggests that these novel chimeric receptors might be versatile reagents for immunochemical assays.