Structural basis for the cyclophilin A binding affinity and immunosuppressive potency of E-­ISA247 (voclosporin)

X-ray crystal structures of the cyclosporin A analogue E-ISA247 (voclosporin) and its stereoisomer Z-ISA247 bound to cyclophilin A suggest the molecular basis for the differences in their binding affinities and immunosuppressive efficacies

MultiBacMam Bimolecular Fluorescence Complementation (BiFC) tool-kit identifies new small-molecule inhibitors of the CDK5-p25 protein-protein interaction (PPI)

Abstract Protein-protein interactions (PPIs) are at the core of virtually all biological processes in cells. Consequently, targeting PPIs is emerging at the forefront of drug discovery. Cellular assays which closely recapitulate native conditions in vivo are instrumental to understand how small molecule drugs can modulate such interactions. We have integrated MultiBacMam, a baculovirus-based mammalian gene delivery tool we developed, with bimolecular fluorescence complementation (BiFC), giving rise to a highly efficient system for assay development, identification and characterization of PPI modulators. We used our system to analyze compounds impacting on CDK5-p25 PPI, which is implicated in numerous diseases including Alzheimer’s. We evaluated our tool-kit with the known inhibitor p5T, and we established a mini-screen to identify compounds that modulate this PPI in dose-response experiments. Finally, we discovered several compounds disrupting CDK5-p25 PPI, which had not been identified by other screening or structure-based methods before

MOSAiC goes O2A - Arctic Expedition Data Flow from Observations to Archives

During the largest polar expedition in history starting in September 2019, the German research icebreaker Polarstern spends a whole year drifting with the ice through the Arctic Ocean. The MOSAiC expedition takes the closest look ever at the Arctic even throughout the polar winter to gain fundamental insights and most unique on-site data for a better understanding of global climate change. Hundreds of researchers from 20 countries are involved. Scientists will use the in situ gathered data instantaneously in near-real time modus as well as long afterwards all around the globe taking climate research to a completely new level. Hence, proper data management, sampling strategies beforehand, and monitoring actual data flow as well as processing, analysis and sharing of data during and long after the MOSAiC expedition are the most essential tools for scientific gain and progress. To prepare for that challenge we adapted and integrated the research data management framework O2A “Data flow from Observations to Archives” to the needs of the MOSAiC expedition on board Polarstern as well as on land for data storage and access at the Alfred Wegener Institute Computing and Data Center in Bremerhaven, Germany. Our O2A-framework assembles a modular research infrastructure comprising a collection of tools and services. These components allow researchers to register all necessary sensor metadata beforehand linked to automatized data ingestion and to ensure and monitor data flow as well as to process, analyze, and publish data to turn the most valuable and uniquely gained arctic data into scientific outcomes. The framework further allows for the integration of data obtained with discrete sampling devices into the data flow. These requirements have led us to adapt the generic and cost-effective framework O2A to enable, control, and access the flow of sensor observations to archives in a cloud-like infrastructure on board Polarstern and later on to land based repositories for international availability. Major roadblocks of the MOSAiC-O2A data flow framework are (i) the increasing number and complexity of research platforms, devices, and sensors, (ii) the heterogeneous interdisciplinary driven requirements towards, e. g., satellite data, sensor monitoring, in situ sample collection, quality assessment and control, processing, analysis and visualization, and (iii) the demand for near real time analyses on board as well as on land with limited satellite bandwidth. The key modules of O2A's digital research infrastructure established by AWI are implementing the FAIR principles: SENSORWeb, to register sensor applications and sampling devices and capture controlled meta data before and alongside any measurements in the field Data ingest, allowing researchers to feed data into storage systems and processing pipelines in a prepared and documented way, at best in controlled near real-time data streams Dashboards allowing researchers to find and access data and share and collaborate among partners Workspace enabling researchers to access and use data with research software utilizing a cloud-based virtualized infrastructure that allows researchers to analyze massive amounts of data on the spot Archiving and publishing data via repositories and Digital Object Identifiers (DOI

Cooperation of RAD51 and RAD54 in regression of a model replication fork

DNA lesions cause stalling of DNA replication forks, which can be lethal for the cell. Homologous recombination (HR) plays an important role in DNA lesion bypass. It is thought that Rad51, a key protein of HR, contributes to the DNA lesion bypass through its DNA strand invasion activity. Here, using model stalled replication forks we found that RAD51 and RAD54 by acting together can promote DNA lesion bypass in vitro through the ‘template-strand switch’ mechanism. This mechanism involves replication fork regression into a Holliday junction (‘chicken foot structure’), DNA synthesis using the nascent lagging DNA strand as a template and fork restoration. Our results demonstrate that RAD54 can catalyze both regression and restoration of model replication forks through its branch migration activity, but shows strong bias toward fork restoration. We find that RAD51 modulates this reaction; by inhibiting fork restoration and stimulating fork regression it promotes accumulation of the chicken foot structure, which we show is essential for DNA lesion bypass by DNA polymerase in vitro. These results indicate that RAD51 in cooperation with RAD54 may have a new role in DNA lesion bypass that is distinct from DNA strand invasion

Evolutionarily Conserved Herpesviral Protein Interaction Networks

Herpesviruses constitute a family of large DNA viruses widely spread in vertebrates and causing a variety of different diseases. They possess dsDNA genomes ranging from 120 to 240 kbp encoding between 70 to 170 open reading frames. We previously reported the protein interaction networks of two herpesviruses, varicella-zoster virus (VZV) and Kaposi's sarcoma-associated herpesvirus (KSHV). In this study, we systematically tested three additional herpesvirus species, herpes simplex virus 1 (HSV-1), murine cytomegalovirus and Epstein-Barr virus, for protein interactions in order to be able to perform a comparative analysis of all three herpesvirus subfamilies. We identified 735 interactions by genome-wide yeast-two-hybrid screens (Y2H), and, together with the interactomes of VZV and KSHV, included a total of 1,007 intraviral protein interactions in the analysis. Whereas a large number of interactions have not been reported previously, we were able to identify a core set of highly conserved protein interactions, like the interaction between HSV-1 UL33 with the nuclear egress proteins UL31/UL34. Interactions were conserved between orthologous proteins despite generally low sequence similarity, suggesting that function may be more conserved than sequence. By combining interactomes of different species we were able to systematically address the low coverage of the Y2H system and to extract biologically relevant interactions which were not evident from single species