Detection of ubiquitin–proteasome enzymatic activities in cells: Application of activity-based probes to inhibitor development

AbstractBackground: Synthetic probes that mimic natural substrates can enable the detection of enzymatic activities in a cellular environment. One area where such activity-based probes have been applied is the ubiquitin–proteasome pathway, which is emerging as an important therapeutic target. A family of reagents has been developed that specifically label deubiquitylating enzymes (DUBs) and facilitate characterization of their inhibitors. Scope of review: Here we focus on the application of probes for intracellular DUBs, a group of specific proteases involved in the ubiquitin proteasome system. In particular, the functional characterization of the active subunits of this family of proteases that specifically recognize ubiquitin and ubiquitin-like proteins will be discussed. In addition we present the potential and design of activity-based probes targeting kinases and phosphatases to study phosphorylation. Major conclusions: Synthetic molecular probes have increased our understanding of the functional role of DUBs in living cells. In addition to the detection of enzymatic activities of known members, activity-based probes have contributed to a number of functional assignments of previously uncharacterized enzymes. This method enables cellular validation of the specificity of small molecule DUB inhibitors. General significance: Molecular probes combined with mass spectrometry-based proteomics and cellular assays represent a powerful approach for discovery and functional validation, a concept that can be expanded to other enzyme classes. This addresses a need for more informative cell-based assays that are required to accelerate the drug development process. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics

Label-free quantitative proteomics reveals regulation of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) and 5'-3'-exoribonuclease 2 (XRN2) during respiratory syncytial virus infection

A large quantitative study was carried out to compare the proteome of respiratory syncytial virus (RSV) infected versus uninfected cells in order to determine novel pathways regulated during viral infection. RSV infected and mock-infected HEp2 cells were lysed and proteins separated by preparative isoelectric focussing using offgel fractionation. Following tryptic digestion, purified peptides were characterized using label-free quantitative expression profiling by nano-ultra performance liquid chromatography coupled to electrospray ionisation mass spectrometry with collision energy ramping for all-ion fragmentation (UPLC-MSE). A total of 1352 unique cellular proteins were identified and their abundance compared between infected and non-infected cells. Ingenuity pathway analysis revealed regulation of several central cellular metabolic and signalling pathways during infection. Selected proteins that were found regulated in RSV infected cells were screened by quantitative real-time PCR for their regulation on the transcriptional level. Synthesis of interferon-induced protein with tetratricopeptide repeats 3 (IFIT3) and 5'-3'-exoribonuclease 2 (XRN2) mRNAs were found to be highly induced upon RSV infection in a time dependent manner. Accordingly, IFIT3 protein levels accumulated during the time course of infection. In contrast, little variation was observed in XRN2 protein levels, but different forms were present in infected versus non-infected cells. This suggests a role of these proteins in viral infection, and analysis of their function will shed further light on mechanisms of RNA virus replication and the host cell defence machinery

Geographical educational research

An der Schnittstelle von Bildungswissenschaften und Humangeographie eröffnet die auch als Bildungsgeographie bezeichnete geographische Bildungsforschung attraktive Perspektiven für eine wissenschaftliche Betrachtung der wechselseitigen Beziehungen zwischen Bildung und Raum. Dieser Beitrag hat zum Ziel, die Bildungsgeographie disziplinübergreifend zu verorten sowie Entwicklungslinien, theoretisch-konzeptionelle Verankerung und Forschungsthemen der geographischen Bildungsforschung zu skizzieren. (DIPF/Orig.)At the intersection of educational sciences and human geography, geographical educational research and geographies of education respectively open up attractive perspectives for academic studies on the interrelations between education and space. The aim of this article is to outline geographies of education as an interdisciplinary research field and to sketch out trends, theoretical and conceptual foundations and topics to be studied in geographical educational research. (DIPF/Orig.

Growth-rate-dependent and nutrient-specific gene expression resource allocation in fission yeast

Cellular resources are limited and their relative allocation to gene expression programmes determines physiological states and global properties such as the growth rate. Here, we determined the importance of the growth rate in explaining relative changes in protein and mRNA levels in the simple eukaryote Schizosaccharomyces pombe grown on non-limiting nitrogen sources. Although expression of half of fission yeast genes was significantly correlated with the growth rate, this came alongside wide-spread nutrient-specific regulation. Proteome and transcriptome often showed coordinated regulation but with notable exceptions, such as metabolic enzymes. Genes positively correlated with growth rate participated in every level of protein production apart from RNA polymerase II-dependent transcription. Negatively correlated genes belonged mainly to the environmental stress response programme. Critically, metabolic enzymes, which represent ∼55-70% of the proteome by mass, showed mostly condition-specific regulation. In summary, we provide a rich account of resource allocation to gene expression in a simple eukaryote, advancing our basic understanding of the interplay between growth-rate-dependent and nutrient-specific gene expression

Trimetallaborides as starting points for the syntheses of large metal-rich molecular borides and clusters

Treatment of an anionic dimanganaborylene complex ([{Cp(CO)2Mn}2B]–) with coinage metal cations stabilized by a very weakly coordinating Lewis base (SMe2) led to the coordination of the incoming metal and subsequent displacement of dimethylsulfide in the formation of hexametalladiborides featuring planar four-membered M2B2 cores (M = Cu, Au) comparable to transition metal clusters constructed around four-membered rings composed solely of coinage metals. The analogies between compounds consisting of B2M2 units and M4 (M = Cu, Au) units speak to the often overlooked metalloid nature of boron. Treatment of one of these compounds (M = Cu) with a Lewis-basic metal fragment (Pt(PCy3)2) led to the formation of a tetrametallaboride featuring two manganese, one copper and one platinum atom, all bound to boron in a geometry not yet seen for this kind of compound. Computational examination suggests that this geometry is the result of d10-d10 dispersion interactions between the copper and platinum fragments

Exhaled and nasal nitric oxide in laryngectomized patients

<p>Abstract</p> <p>Background</p> <p>Nitric oxide (NO) shows differing concentrations in lower and upper airways. Patients after total laryngectomy are the only individuals, in whom a complete separation of upper and lower airways is guaranteed. Thus the objective of our study was to assess exhaled and nasal NO in these patients.</p> <p>Methods</p> <p>Exhaled bronchial NO (FE_NO) and nasal nitric oxide (nNO) were measured in patients after total laryngectomy (n = 14) and healthy controls (n = 24). To assess lung function we additionally performed spirometry. Co-factors possibly influencing NO, such as smoking, infections, and atopy were excluded.</p> <p>Results</p> <p>There was a markedly (p < 0.001) lower FE_NOin patients after total laryngectomy (median (range): 4 (1-22) ppb) compared to healthy controls 21 (9-41) ppb). In contrast, nNO was comparable between groups (1368 <it>versus </it>1380 in controls) but showed higher variability in subjects after laryngectomy.</p> <p>Conclusions</p> <p>Our data suggest that either bronchial NO production in patients who underwent laryngectomy is very low, possibly due to alterations of the mucosa or oxidant production/inflammation, or that substantial contributions to FE_NOarise from the larynx, pharynx and mouth, raising FE_NOdespite velum closure. The data fit to those indicating a substantial contribution to FE_NOby the mouth in healthy subjects. The broader range of nNO values found in subjects after laryngectomy may indicate chronic alteration or oligo-symptomatic inflammation of nasal mucosa, as frequently found after total laryngectomy.</p

Three-state equilibrium of Escherichia coli trigger factor

Trigger Factor (TF) is the first chaperone that interacts with nascent chains of cytosolic proteins in Escherichia coli. Although its chaperone activity requires association with ribosomes, TF is present in vivo in a 2 -3 fold molar excess over ribosomes and a fraction of it is not ribosome-associated after cell lysis. Here we show that TF follows a three-state equilibrium. Size exclusion chromatography, crosslinking and analytical ultracentrifugation revealed that uncomplexed TF dimerizes with an apparent K d of 18 µM. Dimerization is mediated by the N-terminal ribosome binding domain and the C-terminal domain of TF, whereas the central peptidyl prolyl isomerase (PPIase) and substrate binding domain does not contribute to dimerization. Crosslinking experiments showed that TF is monomeric in its ribosome-associated state. Quantitative analysis of TF binding to ribosomes revealed a dissociation constant for the TF-ribosome complex of approximately 1.2 µM. From these data we estimate that in vivo most of the ribosomes are in complex with monomeric TF. Uncomplexed TF, however, is in a monomer-dimer equilibrium with approximately two thirds of TF existing in a dimeric state

Evaluation of Antimicrobial Usage in Dogs and Cats at a Veterinary Teaching Hospital in Germany in 2017 and 2018

In contrast to food-producing animals, where the documentation of the usage of antimicrobials is regulated by law, antimicrobial usage (AMU) in dogs and cats is only sparsely monitored. We collected data generated by an electronic practice management software (EPMS) between January 1, 2017 and December 31, 2018 to investigate AMU. All information was obtained from clinical routine data from the Department of Small Animal Medicine and Surgery (DSAM), University of Veterinary Medicine Hannover (TiHo). In 2017, 78,076 drug administrations were documented for 5,471 dogs and cats, of which 14,020 (17.96%) were antimicrobial drugs (AMs) specifically documented in 2,910 (51.31%) dogs and cats. In 2018, 104,481 drug administrations were documented for 5,939 dogs and cats. Of these drug administrations, 18,170 (17.39%) AM administrations were documented for 3,176 (53.48%) dogs and cats. Despite the increasing documentation of AM administrations, differences between 2017 and 2018 were not statistically significant [odds ratio (OR), 1.01; 95% confidence interval (CI), 0.98–1.03]. Prescription diversity (PD) in 2017 for dogs was 0.92 and for cats 0.89. In 2018, PD for dogs was 0.93 and for cats 0.88. As well as the documented number of AM administrations, the documented amount of active ingredients administered in 2018 (total: 17.06 kg; dogs: 16.11 kg, cats: 0.96 kg) increased compared with 2017 (total: 15.60 kg; dogs: 14.80 kg, cats: 0.80 kg). In 2017 and 2018, the most commonly administered antimicrobial groups were penicillins, nitroimidazoles, and quinolones for dogs and cats, respectively. While the in-house point-of-care administration accounts for the largest share of the documented amount of AMs administered, the highest number of documented AM administrations was assigned to inpatient care in 2017 and 2018, respectively. However, AM administration in outpatient care remained the lowest in both years. Since no statistically significant difference in AM administrations was observed between 2017 and 2018 and the most commonly used AMs at the DSAM were ranked, data can be used as a baseline to evaluate how changes in in-house guidelines and future legal requirements affect the prescribing culture. Data generated within the DSAM should be evaluated annually

Strongly phosphorescent transition metal π complexes of boron-boron triple bonds

Herein are reported the first π complexes of compounds with boron-boron triple bonds to transition metals, in this case CuI. Three different compounds were isolated that differ in the number of copper atoms bound to the BB unit. Metallation of the B-B triple bonds causes significant lengthening of the B-B and B-CNHC bonds, as well as large upfield shifts of the 11B NMR signals, suggesting greater orbital interactions between the boron and transition metal atoms than those observed with recently published diboryne / alkali metal cation complexes. In contrast to previously-reported fluorescent copper(I) π complexes of boron-boron double bonds, the Cun-π-diboryne compounds (n = 2, 3) show intense phosphorescence in the red to near-IR region from their triplet excited states, according to their microsecond lifetimes, with quantum yields of up to 58%. The bonding situation, as well as the unusual photophysical properties, has been further corroborated by DFT studies