Dissecting Ubiquitin Signaling with Linkage‐Defined and Protease Resistant Ubiquitin Chains

Ubiquitylation is a complex posttranslational protein modification and deregulation of this pathway has been associated with different human disorders. Ubiquitylation comes in different flavors: Besides mono‐ubiquitylation, ubiquitin chains of various topologies are formed on substrate proteins. The fate of ubiquitylated proteins is determined by the linkage‐type of the attached ubiquitin chains, however, the underlying mechanism is poorly characterized. Herein, we describe a new method based on codon expansion and click‐chemistry‐based polymerization to generate linkage‐defined ubiquitin chains that are resistant to ubiquitin‐specific proteases and adopt native‐like functions. The potential of these artificial chains for analyzing ubiquitin signaling is demonstrated by linkage‐specific effects on cell‐cycle progression.     

The Lysine48‐Based Polyubiquitin Chain Proteasomal Signal: Not a Single Child Anymore

The conjugation of ubiquitin (Ub) to proteins is involved in the regulation of many processes. The modification serves as a recognition element in trans, in which downstream effectors bind to the modified protein and determine its fate and/or function. A polyUb chain that is linked through internal lysine (Lys)‐48 of Ub and anchored to an internal Lys residue of the substrate has become the accepted “canonical” signal for proteasomal targeting and degradation. However, recent studies show that the signal is far more diverse and that chains based on other internal linkages, as well as linear or heterologous chains made of Ub and Ub‐like proteins and even monoUb, are recognized by the proteasome. In addition, chains linked to residues other than internal Lys were described, all challenging the current paradigm.     

Expanded Click Conjugation of Recombinant Proteins with Ubiquitin-Like Modifiers Reveals Altered Substrate Preference of SUMO2-Modified Ubc9

Wrestling with SUMO: The chemical conjugation of proteins with small ubiquitin-like modifiers (SUMO) can be achieved by a copper(I)-catalyzed cycloaddition and unnatural amino acid mutagenesis. This approach overcomes previous restrictions related to the primary sequence of proteins and coupling conditions. Moreover, biochemical data suggests that this triazole linkage presents the modifier in a proper distance and orientation relative to the target protein.     

Proteins, lipids, and water in the gas phase

Evidence from mass‐spectrometry experiments and molecular dynamics simulations suggests that it is possible to transfer proteins, or in general biomolecular aggregates, from solution to the gas‐phase without grave impact on the structure. If correct, this allows interpretation of such experiments as a probe of physiological behavior. Here, we survey recent experimental results from mass spectrometry and ion‐mobility spectroscopy and combine this with observations based on molecular dynamics simulation, in order to give a comprehensive overview of the state of the art in gas‐phase studies. We introduce a new concept in protein structure analysis by determining the fraction of the theoretical possible numbers of hydrogen bonds that are formed in solution and in the gas‐phase. In solution on average 43% of the hydrogen bonds is realized, while in vacuo this fraction increases to 56%. The hydrogen bonds stabilizing the secondary structure (α‐helices, β‐sheets) are maintained to a large degree, with additional hydrogen bonds occurring when side chains make new hydrogen bonds to rest of the protein rather than to solvent. This indicates that proteins that are transported to the gas phase in a native‐like manner in many cases will be kinetically trapped in near‐physiological structures. Simulation results for lipid‐ and detergent‐aggregates and lipid‐coated (membrane) proteins in the gas phase are discussed, which in general point to the conclusion that encapsulating proteins in “something” aids in the conservation of native‐like structure. Isolated solvated micelles of cetyl‐tetraammonium bromide quickly turn into reverse micelles whereas dodecyl phosphocholine micelles undergo much slower conversions, and do not quite reach a reverse micelle conformation within 100 ns.

     

Diverse ubiquitin codes in the regulation of inflammatory signaling

Ubiquitin is a small protein used for posttranslational modification and it regulates every aspect of biological functions. Through a three-step cascade of enzymatic action, ubiquitin is conjugated to a substrate. Because ubiquitin itself can be post-translationally modified, this small protein generates various ubiquitin codes and triggers differing regulation of biological functions. For example, ubiquitin itself can be ubiquitinated, phosphorylated, acetylated, or SUMOylated. Via the type three secretion system, some bacterial effectors also modify the ubiquitin system in host cells. This review describes the general concept of the ubiquitin system as well as the fundamental functions of ubiquitin in the regulation of cellular responses during inflammation and bacterial infection.     

Synthesis and characterization of novel chromone Schiff base complexes as p53 activators

A series of chromone Schiff base complexes were prepared and analytically as well as spectroscopically characterized. The ligand was found to act as a monobasic tridentate ligand bonded covalently or coordinatively to the metal ion via deprotonated hydroxyl group, azomethine nitrogen atom and carbonyl oxygen atom of antipyrine moiety. Both electronic spectra and magnetic measurements indicated an octahedral or a distorted octahedral geometry around the metal ions for all metal complexes except the nickel complex, which had a tetrahedral geometry. In addition, the ability of the newly prepared compounds to activate the tumour suppressor p53 in cancer cells was studied, with zinc and copper complexes showing promising activities for p53 ubiquitination compared with diphenylimidazole (reference drug).