Characterization of human hect domain family members and their interaction with UbcH5 and UbcH7

The hect domain protein family was originally identified by sequence similarity of its members to the C-terminal region of E6-AP, an E3 ubiquitin-protein ligase. Since the C terminus of E6-AP mediates thioester complex formation with ubiquitin, a necessary intermediate step in E6-AP-dependent ubiquitination, it was proposed that members of the hect domain family in general have E3 activity. The hect domain is approximately 350 amino acids in length, and we show here that the hect domain of E6-AP is necessary and sufficient for ubiquitin thioester adduct formation. Furthermore, the human genome encodes at least 20 different hect domain proteins, and in further support of the hypothesis that hect domain proteins represent a family of E3s, several of these are shown to form thioester complexes with ubiquitin. In addition, some hect domain proteins interact preferentially with UbcH5, whereas others interact with UbcH7, indicating that human hect domain proteins can be grouped into at least two classes based on their E2 specificity. Since E3s are thought to play a major role in substrate recognition, the presence of a large family of E3s should contribute to ensure the specificity and selectivity of ubiquitin-dependent proteolytic pathways

Die Bedeutung regionaler Wertschöpfungscluster der Automobilindustrie im Prozess fortschreitender Globalisierung und der Transformation zur Elektromobilität

Anhaltende Globalisierung und die Transformation zu elektrischen Antriebssträngen sind neben der Digitalisierung die wichtigsten Treiber einer gravierenden Veränderung in der Automobilindustrie Deutschlands. Bisher stützte sich diese Branche auf ein Netzwerk regional spezialisierter Wertschöpfungscluster. In Deutschland nutzen die verschiedenen Clusterunternehmen der Endhersteller und Zulieferer ihre räumliche und kulturelle Nähe vorrangig als Basis hoher Innovationsleistungen. Am Beispiel der Wirtschaftsregion Baden-Württemberg wird hier untersucht, wie sich der strukturelle Wandel in der Branche auf diese regionalen Wertschöpfungscluster auswirkt und ob diese ihre Rolle als bedeutender Erfolgsfaktor für die Branche unter den neuen Bedingungen noch erfüllen können. Im Ergebnis lässt sich zeigen, dass auch in Zukunft die Bedeutung regionaler Wertschöpfungscluster ungebrochen sein wird. Allerdings stellt sich dieser Zusammenhang nicht mehr automatisch ein, weil in dem vielfältigen Transformationsprozess die Produktionsbasis im Cluster gefährdet erscheint. Deshalb braucht es die aktive Unterstützung der Akteure aus Wirtschaft und Politik, damit das vollständige regionale Wertschöpfungscluster als Basis des Branchenerfolgs erhalten bleibt.Continuing globalization and the transformation into electric powertrains are, alongside digitization, the main drivers of a major change in Germany's automotive industry. So far, this industry has relied on a variety of regional value-added clusters, where different original equipment manufacturer and suppliers used spatial and and cultural proximity as a basis for high innovation performance. Using the example of the economic region of Baden-Württemberg, it examines how the structural change in the industry affects these regional value-added clusters and whether they can still fulfill their role as a significant success factor for the industry under the new conditions. As a result, it can be shown that the importance of regional value-added clusters will continue in the future as well. However, this relationship no longer adjusts automatically, because in the multifaceted transformation process, the production base in the cluster appears endangered. Therefore, the active support of economic and political actors is needed to maintain the full regional value chain as an important industry success base

Prevalence and Epidemiology of Multidrug-Resistant Pathogens in the Food Chain and the Urban Environment in Northwestern Germany

The surveillance of antimicrobial resistance among humans and food-producing animals is important to monitor the zoonotic transmission of multidrug-resistant bacteria (MDRB). We assessed the prevalence of four MDRB within the meat production chain, including extended-spectrum β-lactamase (ESBL)-producing, carbapenemase-producing Enterobacterales (CPE) and colistin-resistant Enterobacterales (Col-E), as well as vancomycin-resistant enterococci (VRE). In total, 505 samples from four stages of meat production, i.e., slaughterhouses, meat-processing plants, fresh food products and the urban environment, were collected in northwestern Germany in 2018/2019 and screened for the presence of MDRB using both culture-based and PCR-based techniques. We detected genes encoding for carbapenemases in 9–56% (blaOXA-48, blaKPC, blaNDM, blaVIM) and colistin resistance-encoding mcr genes in 9–26% of the samples from all stages. Culture-based analysis found CPE and VRE only in environmental samples (11% and 7%, respectively), but Col-E and ESBL-producers in 1–7% and 12–46% of samples from all stages, respectively. Overall, our results showed that ESBL-producers and mcr-carrying Col-E were common in food-producing animals at slaughterhouses, in meat-processing plants and in food items at retail, while CPE and VRE were only found in the environment. The discrepancy between detected carbapenemase genes and isolated CPE emphasizes the need for more sensitive detection methods for CPE monitoring

Synthesis and in vitro evaluation of cyclodextrin hyaluronic acid conjugates as a new candidate for intestinal drug carrier for steroid hormones

Steroid hormones became increasingly interesting as active pharmaceutical ingredients for the treatment of endocrine disorders. However, medical applications of many steroidal drugs are inhibited by their very low aqueous solubilities giving rise to low bioavailabilities. Therefore, the prioritized oral administration of steroidal drugs remains problematic. Cyclodextrins are promising candidates for the development of drug delivery systems for oral route applications, since they solubilize hydrophobic steroids and increase their rate of transport in aqueous environments. In this study, the synthesis and characterization of polymeric β-cyclodextrin derivates is described, which result from the attachment of a hydrophilic β-CD-thioether to hyaluronic acid. Host-guest complexes of the synthesized β-cyclodextrin hyaluronic acid conjugates were formed with two poorly soluble model steroids (β-estradiol, dexamethasone) and compared to monomeric β-cyclodextrin derivates regarding solubilization and complexation efficiency. The β-cyclodextrin-drug (host-guest) complexes were evaluated in vitro for their suitability (cytotoxicity and transport rate) as intestinal drug carriers for steroid hormones. In case of β-estradiol, higher solubilities could be achieved by complexation with both synthesized β-cyclodextrin derivates, leading to significantly higher intestinal transport rates in vitro. However, this success could not be shown for dexamethasone, which namely solubilized better, but could not enhance the transport rate significantly. Thus, this study demonstrates the biocompatibility of the synthesized and characterized β-cyclodextrin derivates and shows their potential as new candidate for intestinal drug carrier for steroid hormones like β-estradiol

Role of Predicted Metalloprotease Motif of Jab1/Csn5 in Cleavage of Nedd8 from Cul1

COP9 signalosome (CSN) cleaves the ubiquitin-like protein Nedd8 from the Cul1 subunit of SCF ubiquitin ligases. The Jab1/MPN domain metalloenzyme (JAMM) motif in the Jab1/Csn5 subunit was found to underlie CSN's Nedd8 isopeptidase activity. JAMM is found in proteins from archaea, bacteria, and eukaryotes, including the Rpn11 subunit of the 26S proteasome. Metal chelators and point mutations within JAMM abolished CSN-dependent cleavage of Nedd8 from Cul1, yet had little effect on CSN complex assembly. Optimal SCF activity in yeast and both viability and proper photoreceptor cell (R cell) development in Drosophila melanogaster required an intact Csn5 JAMM domain. We propose that JAMM isopeptidases play important roles in a variety of physiological pathways

Identification of molecular candidates which regulate calcium-dependent CD8+ T-cell cytotoxicity

Cytotoxic CD8+ T lymphocytes (CTL) eliminate infected cells or transformed tumor cells by releasing perforincontaining cytotoxic granules at the immunological synapse. The secretion of such granules depends on Ca2+- influx through store operated Ca2+ channels, formed by STIM (stromal interaction molecule)-activated Orai proteins. Whereas molecular mechanisms of the secretion machinery are well understood, much less is known about the molecular machinery that regulates the efficiency of Ca2+-dependent target cell killing. CTL killing efficiency is of high interest considering the number of studies on CD8+ T lymphocytes modified for clinical use. Here, we isolated total RNA from primary human cells: natural killer (NK) cells, non-stimulated CD8+ T-cells, and from Staphylococcus aureus enterotoxin A (SEA) stimulated CD8+ T-cells (SEA-CTL) and conducted whole genome expression profiling by microarray experiments. Based on differential expression analysis of the transcriptome data and analysis of master regulator genes, we identified 31 candidates which potentially regulate Ca2+-homeostasis in CTL. To investigate a putative function of these candidates in CTL cytotoxicity, we transfected either SEA-stimulated CTL (SEA-CTL) or antigen specific CD8+ T-cell clones (CTL-MART-1) with siRNAs specific against the identified candidates and analyzed the killing capacity using a real-time killing assay. In addition, we complemented the analysis by studying the effect of inhibitory substances acting on the candidate proteins if available. Finally, to unmask their involvement in Ca2+ dependent cytotoxicity, candidates were also analyzed under Ca2+-limiting conditions. Overall, we identified four hits, CCR5 (C-C chemokine receptor type five), KCNN4 (potassium calcium-activated channel subfamily N), RCAN3 (regulator of calcineurin) and BCL (Bcell lymphoma) 2 which clearly affect the efficiency of Ca2+ dependent cytotoxicity in CTL-MART-1 cells, CCR5, BCL2, and KCNN4 in a positive manner, and RCAN3 in a negative way

Investigating Alkylated Prodigiosenes and Their Cu(II)-Dependent Biological Activity: Interactions with DNA, Antimicrobial and Photoinduced Anticancer Activity

Prodigiosenes are a family of red pigments with versatile biological activity. Their tripyrrolic core structure has been modified many times in order to manipulate the spectrum of activity. We have been looking systematically at prodigiosenes substituted at the C ring with alkyl chains of different lengths, in order to assess the relevance of this substituent in a context that has not been investigated before for these derivatives: Cu(II) complexation, DNA binding, self-activated DNA cleavage, photoinduced cytotoxicity and antimicrobial activity. Our results indicate that the hydrophobic substituent has a clear influence on the different aspects of their biological activity. The cytotoxicity study of the Cu(II) complexes of these prodigiosenes shows that they exhibit a strong cytotoxic effect towards the tested tumor cell lines. The Cu(II) complex of a prodigiosene lacking any alkyl chain excelled in its photoinduced anticancer activity, thus demonstrating the potential of prodigiosenes and their metal complexes for an application in photodynamic therapy (PDT). Two derivatives along with their Cu(II) complexes showed also antimicrobial activity against Staphylococcus aureus strains

A phase 1b study of AFM13 in combination with pembrolizumab in patients with relapsed or refractory Hodgkin lymphoma

In relapsed/refractory Hodgkin lymphoma (R/R HL), immunotherapies such as the anti-programmed death-1 inhibitor pembrolizumab have demonstrated efficacy as monotherapy and are playing an increasingly prominent role in treatment. The CD30/CD16A-bispecific antibody AFM13 is an innate immune cell engager, a first-in-class, tetravalent antibody, designed to create a bridge between CD30 on HL cells and the CD16A receptor on natural killer cells and macrophages, to induce tumor cell killing. Early studies of AFM13 have demonstrated signs of efficacy as monotherapy for patients with R/RHL and the combination of AFM13 with pembrolizumab represents a rational new treatment modality. Here, we describe a phase 1b, dose-escalation study to assess the safety and preliminary efficacy of AFM13 in combination with pembrolizumab in patients with R/R HL. The primary objective was estimating the maximum tolerated dose; the secondary objectives were to assess safety, tolerability, antitumor efficacy, pharmacokinetics, and pharmacodynamics. In this heavily pretreated patient population, treatment with the combination of AFM13 and pembrolizumab was generally well tolerated, with similar safety profiles compared to the known profiles of each agent alone. The combination of AFM13 with pembrolizumab demonstrated an objective response rate of 88% at the highest treatment dose, with an 83% overall response rate for the overall population. Pharmacokinetic assessment of AFM13 in the combination setting revealed a half-life of up to 20.6 hours. This proof-of-concept study holds promise as a novel immunotherapy combination worthy of further investigation

Chicken lung lectin is a functional C-type lectin and inhibits haemagglutination by influenza A virus

Many proteins of the calcium-dependent (C-type) lectin family have been shown to play an important role in innate immunity. They can bind to a broad range of carbohydrates, which enables them to interact with ligands present on the surface of micro-organisms.We previously reported the finding of a new putative chicken lectin, which was predominantly localized to the respiratory tract, and thus termed chicken lung lectin (cLL). In order to investigate the biochemical and biophysical properties of cLL, the recombinant protein was expressed, affinity purified and characterized. Recombinant cLL was expressed as four differently sized peptides, which is most likely due to post-translational modification. Crosslinking of the protein led to the formation of two high-molecular weight products, indicating that cLL forms trimeric and possibly even multimeric subunits. cLL was shown to have lectin activity, preferentially binding to a-mannose in a calcium-dependent manner. Furthermore, cLL was shown to inhibit the haemagglutination-activity of human isolates of influenza A virus, subtype H3N2 and H1N1. These result show that cLL is a true C-type lectin with a very distinct sugar specificity, and that this chicken lectin could play an important role in innate immunity