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Single nucleotide polymorphisms (SNPs) are genetic variations which can play a vital role in the study of human health. SNP studies are often used to identify point mutations that are associated with diseases. Arkadia (RNF111) is an E3 ubiquitin ligase that enhances transforming growth factor-beta (TGF-β) signaling by targeting negative regulators for degradation. Dysregulation of the TGF-β pathway is implicated in cancer because it exhibits tumor suppressive activity in normal cells while in tumor cells it promotes invasiveness and metastasis. Τhe SNP CGT > TGT generated an amino-acid (aa) substitution of Arginine 957 to Cysteine on the enzymatic RING domain of Arkadia. This was more prevalent in a tumor than in a normal tissue sample of a patient with colorectal cancer. This prompted us to investigate the effect of this mutation in the structure and activity of Arkadia RING. We used nuclear magnetic resonance (NMR) to analyze at an atomic-level the structural and dynamic properties of the R957C Arkadia RING domain, while ubiquitination and luciferase assays provided information about its enzymatic functionality. Our study showed that the R957C mutation changed the electrostatic properties of the RING domain however, without significant effects on the structure of its core region. However, the functional studies revealed that the R957C Arkadia exhibits significantly increased enzymatic activity supporting literature data that Arkadia within tumor cells promotes aggressive and metastatic behavior.

The highly infectious disease COVID-19 caused by the Betacoronavirus SARS-CoV-2 poses a severe threat to humanity and demands the redirection of scientific efforts and criteria to organized research projects. The international COVID19-NMR consortium seeks to provide such new approaches by gathering scientific expertise worldwide. In particular, making available viral proteins and RNAs will pave the way to understanding the SARS-CoV-2 molecular components in detail. The research in COVID19-NMR and the resources provided through the consortium are fully disclosed to accelerate access and exploitation. NMR investigations of the viral molecular components are designated to provide the essential basis for further work, including macromolecular interaction studies and high-throughput drug screening. Here, we present the extensive catalog of a holistic SARS-CoV-2 protein preparation approach based on the consortium’s collective efforts. We provide protocols for the large-scale production of more than 80% of all SARS-CoV-2 proteins or essential parts of them. Several of the proteins were produced in more than one laboratory, demonstrating the high interoperability between NMR groups worldwide. For the majority of proteins, we can produce isotope-labeled samples of HSQC-grade. Together with several NMR chemical shift assignments made publicly available on covid19-nmr.com, we here provide highly valuable resources for the production of SARS-CoV-2 proteins in isotope-labeled form.