Biochemie, Chemie und Pharmazie
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Acinetobacter baumannii is an important nosocomial pathogen that requires thoughtful consideration in the antibiotic prescription strategy due to its multidrug resistant phenotype. Tetracycline antibiotics have recently been re-administered as part of the combination antimicrobial regimens to treat infections caused by A. baumannii. We show that the TetA(G) efflux pump of A. baumannii AYE confers resistance to a variety of tetracyclines including the clinically important antibiotics doxycycline and minocycline, but not to tigecycline. Expression of tetA(G) gene is regulated by the TetR repressor of A. baumannii AYE (AbTetR). Thermal shift binding experiments revealed that AbTetR preferentially binds tetracyclines which carry a O-5H moiety in ring B, whereas tetracyclines with a 7-dimethylamino moiety in ring D are less well-recognized by AbTetR. Confoundingly, tigecycline binds to AbTetR even though it is not transported by TetA(G) efflux pump. Structural analysis of the minocycline-bound AbTetR-Gln116Ala variant suggested that the non-conserved Arg135 interacts with the ring D of minocycline by cation-π interaction, while the invariant Arg104 engages in H-bonding with the O-11H of minocycline. Interestingly, the Arg135Ala variant exhibited a binding preference for tetracyclines with an unmodified ring D. In contrast, the Arg104Ala variant preferred to bind tetracyclines which carry a O-6H moiety in ring C except for tigecycline. We propose that Arg104 and Arg135, which are embedded at the entrance of the AbTetR binding pocket, play important roles in the recognition of tetracyclines, and act as a barrier to prevent the release of tetracycline from its binding pocket upon AbTetR activation. The binding data and crystal structures obtained in this study might provide further insight for the development of new tetracycline antibiotics to evade the specific efflux resistance mechanism deployed by A. baumannii.
The formation of oligomers of the amyloid-β peptide plays a key role in the onset of Alzheimer's disease. We describe herein the investigation of disease-relevant small amyloid-β oligomers by mass spectrometry and ion mobility spectrometry, revealing functionally relevant structural attributes. In particular, we can show that amyloid-β oligomers develop in two distinct arrangements leading to either neurotoxic oligomers and fibrils or non-toxic amorphous aggregates. Comprehending the key-attributes responsible for those pathways on a molecular level is a pre-requisite to specifically target the peptide's tertiary structure with the aim to promote the emergence of non-toxic aggregates. Here, we show for two fibril inhibiting ligands, an ionic molecular tweezer and a hydrophobic peptide that despite their different interaction mechanisms, the suppression of the fibril pathway can be deduced from the disappearance of the corresponding structure of the first amyloid-β oligomers.
The development of super-resolution microscopy (SRM) has widened our understanding of biomolecular structure and function in biological materials. Imaging multiple targets within a single area would elucidate their spatial localization relative to the cell matrix and neighboring biomolecules, revealing multi-protein macromolecular structures and their functional co-dependencies. SRM methods are, however, limited to the number of suitable fluorophores that can be imaged during a single acquisition as well as the loss of antigens during antibody washing and restaining for organic dye multiplexing. We report the visualization of multiple protein targets within the pre- and postsynapse in 350–400 nm thick neuronal tissue sections using DNA-assisted single-molecule localization microscopy (SMLM). In a single labeling step, antibodies conjugated with short DNA oligonucleotides visualized multiple targets by sequential exchange of fluorophore-labeled complementary oligonucleotides present in the imaging buffer. This approach avoids potential effects on structural integrity when using multiple rounds of immunolabeling and eliminates chromatic aberration, because all targets are imaged using a single excitation laser wavelength. This method proved robust for multi-target imaging in semi-thin tissue sections with a lateral resolution better than 25 nm, paving the way toward structural cell biology with single-molecule SRM.
Bioactive lipid mediators play a major role in regulating inflammatory processes. Herein, early pro-inflammatory phases are characterized and regulated by prostanoids and leukotrienes, whereas specialized pro-resolving mediators (SPM), including lipoxins, resolvins, protectins, and maresins, dominate during the resolution phase. While pro-inflammatory properties of prostanoids have been studied extensively, their impact on later phases of the inflammatory process has been attributed mainly to their ability to initiate the lipid-mediator class switch towards SPM. Yet, there is accumulating evidence that prostanoids directly contribute to the resolution of inflammation and return to homeostasis. In this mini review, we summarize the current knowledge of the resolution-regulatory properties of prostanoids and discuss potential implications for anti-inflammatory, prostanoid-targeted therapeutic interventions.
Ubiquitin fold modifier 1 (UFM1) is a member of the ubiquitin-like protein family. UFM1 undergoes a cascade of enzymatic reactions including activation by UBA5 (E1), transfer to UFC1 (E2) and selective conjugation to a number of target proteins via UFL1 (E3) enzymes. Despite the importance of ufmylation in a variety of cellular processes and its role in the pathogenicity of many human diseases, the molecular mechanisms of the ufmylation cascade remains unclear. In this study we focused on the biophysical and biochemical characterization of the interaction between UBA5 and UFC1. We explored the hypothesis that the unstructured C-terminal region of UBA5 serves as a regulatory region, controlling cellular localization of the elements of the ufmylation cascade and effective interaction between them. We found that the last 20 residues in UBA5 are pivotal for binding to UFC1 and can accelerate the transfer of UFM1 to UFC1. We solved the structure of a complex of UFC1 and a peptide spanning the last 20 residues of UBA5 by NMR spectroscopy. This structure in combination with additional NMR titration and isothermal titration calorimetry experiments revealed the mechanism of interaction and confirmed the importance of the C-terminal unstructured region in UBA5 for the ufmylation cascade.
11,12-Dihydrodibenzo[c,g]-1,2-diazocines have been established as a viable alternative to azobenzene for photoswitching, in particular, as they show an inverted switching behavior: the ground state is the Z isomer. In this paper, we present an improved method to obtain dibenzodiazocine and its derivatives from the respective 2-nitrotoluenes in two reaction steps, each proceeding in minutes. This fast access to a variety of derivatives permitted the study of substitution effects on the synthesis and on the photochemical properties. With biochemical applications in mind, methanol was chosen as a protic solvent system for the photochemical investigations. In contrast to the azobenzene system, none of the tested substitution patterns resulted in more efficient switching or in significantly prolonged half-lives, showing that the system is dominated by the ring strain.
Membrane-suspended nanopores in microchip arrays for stochastic transport recording and sensing
(2021)
The transport of nutrients, xenobiotics, and signaling molecules across biological membranes is essential for life. As gatekeepers of cells, membrane proteins and nanopores are key targets in pharmaceutical research and industry. Multiple techniques help in elucidating, utilizing, or mimicking the function of biological membrane-embedded nanodevices. In particular, the use of DNA origami to construct simple nanopores based on the predictable folding of nucleotides provides a promising direction for innovative sensing and sequencing approaches. Knowledge of translocation characteristics is crucial to link structural design with function. Here, we summarize recent developments and compare features of membrane-embedded nanopores with solid-state analogues. We also describe how their translocation properties are characterized by microchip systems. The recently developed silicon chips, comprising solid-state nanopores of 80 nm connecting femtoliter cavities in combination with vesicle spreading and formation of nanopore-suspended membranes, will pave the way to characterize translocation properties of nanopores and membrane proteins in high-throughput and at single-transporter resolution.
The p63 gene encodes a master regulator of epidermal commitment, development, and differentiation. Heterozygous mutations in the DNA binding domain cause Ectrodactyly, Ectodermal Dysplasia, characterized by limb deformation, cleft lip/palate, and ectodermal dysplasia while mutations in in the C-terminal domain of the α-isoform cause Ankyloblepharon-Ectodermal defects-Cleft lip/palate (AEC) syndrome, a life-threatening disorder characterized by skin fragility, severe, long-lasting skin erosions, and cleft lip/palate. The molecular disease mechanisms of these syndromes have recently become elucidated and have enhanced our understanding of the role of p63 in epidermal development. Here we review the molecular cause and functional consequences of these p63-mutations for skin development and discuss the consequences of p63 mutations for female fertility.
In this review, we focus on the ubiquitination process within the endoplasmic reticulum associated protein degradation (ERAD) pathway. Approximately one third of all synthesized proteins in a cell are channeled into the endoplasmic reticulum (ER) lumen or are incorporated into the ER membrane. Since all newly synthesized proteins enter the ER in an unfolded manner, folding must occur within the ER lumen or co-translationally, rendering misfolding events a serious threat. To prevent the accumulation of misfolded protein in the ER, proteins that fail the quality control undergo retrotranslocation into the cytosol where they proceed with ubiquitination and degradation. The wide variety of misfolded targets requires on the one hand a promiscuity of the ubiquitination process and on the other hand a fast and highly processive mechanism. We present the various ERAD components involved in the ubiquitination process including the different E2 conjugating enzymes, E3 ligases, and E4 factors. The resulting K48-linked and K11-linked ubiquitin chains do not only represent a signal for degradation by the proteasome but are also recognized by the AAA+ ATPase Cdc48 and get in the process of retrotranslocation modified by enzymes bound to Cdc48. Lastly we discuss the conformations adopted in particular by K48-linked ubiquitin chains and their importance for degradation.
A new pseudopolymorph of perchlorinated neopentasilane: the benzene monosolvate Si(SiCl3)4·C6H6
(2020)
A new pseudopolymorph of dodecachloropentasilane, namely a benzene monosolvate, Si5Cl12·C6H6, is described. There are two half molecules of each kind in the asymmetric unit. Both Si5Cl12 molecules are completed by crystallographic twofold symmetry. One of the benzene molecules is located on a twofold rotation axis with two C—H groups located on this rotation axis. The second benzene molecule has all atoms on a general position: it is disordered over two equally occupied orientations. No directional interactions beyond normal van der Waals contacts occur in the crystal.